Tabla espec. clave
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|M, R||ICC, IHC, IH(P), WB||M||Purified||Monoclonal Antibody|
|Presentation||Purified mouse monoclonal IgG1 in buffer containing 0.09% sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at 2-8ºC from date of receipt.|
|Material Size||100 µg|
|IHC Select Paraffin Protocol|
|MAB353 IH(P) Summary|
Ficha datos de seguridad (MSDS)
Referencias bibliográficas | 174 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Especie||Pub Med ID|
|An integrated assessment of histopathological changes of the enteric neuromuscular compartment in experimental colitis. |
Ippolito, C; Segnani, C; Errede, M; Virgintino, D; Colucci, R; Fornai, M; Antonioli, L; Blandizzi, C; Dolfi, A; Bernardini, N
Journal of cellular and molecular medicine 19 485-500 2015
Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4-dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c-Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS-treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA-positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c-Kit- and TMEM16A/ANO1-positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS-induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.
|Neuroprotective effects of oligodendrocyte progenitor cell transplantation in premature rat brain following hypoxic-ischemic injury. |
Chen, LX; Ma, SM; Zhang, P; Fan, ZC; Xiong, M; Cheng, GQ; Yang, Y; Qiu, ZL; Zhou, WH; Li, J
PloS one 10 e0115997 2015
Periventricular leukomalacia (PVL) is a common ischemic brain injury in premature infants for which there is no effective treatment. The objective of this study was to determine whether transplanted mouse oligodendrocyte progenitor cells (OPCs) have neuroprotective effects in a rat model of PVL. Hypoxia-ischemia (HI) was induced in 3-day-old rat pups by left carotid artery ligation, followed by exposure to 6% oxygen for 2.5 h. Animals were assigned to OPC transplantation or sham control groups and injected with OPCs or PBS, respectively, and sacrificed up to 6 weeks later for immunohistochemical analysis to investigate the survival and differentiation of transplanted OPCs. Apoptosis was evaluated by double immunolabeling of brain sections for caspase-3 and neuronal nuclei (NeuN), while proliferation was assessed using a combination of anti-Nestin and -bromodeoxyuridine antibodies. The expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 was examined 7 days after OPC transplantation. The Morris water maze was used to test spatial learning and memory. The results showed that transplanted OPCs survived and formed a myelin sheath, and stimulated BDNF and Bcl-2 expression and the proliferation of neural stem cells (NSC), while inhibiting HI-induced neuronal apoptosis relative to control animals. Moreover, deficits in spatial learning and memory resulting from HI were improved by OPC transplantation. These results demonstrate an important neuroprotective role for OPCs that can potentially be exploited in cell-based therapeutic approaches to minimize HI-induced brain injury.
|Skin regeneration in deep second-degree scald injuries either by infusion pumping or topical application of recombinant human erythropoietin gel. |
Giri, P; Ebert, S; Braumann, UD; Kremer, M; Giri, S; Machens, HG; Bader, A
Drug design, development and therapy 9 2565-79 2015
Large doses of recombinant growth factors formulated in solution form directly injected into the body is usual clinical practice in treating second-degree scald injuries, with promising results, but this approach creates side effects; furthermore, it may not allow appropriate levels of the factor to be sensed by the target injured tissue/organ in the specific time frame, owing to complications arising from regeneration. In this research, two delivery methods (infusion pumping and local topical application) were applied to deliver recombinant human erythropoietin (rHuEPO) for skin regeneration. First, rHuEPO was given in deep second-degree scald injury sites in mice by infusion pump. Vascularization was remarkably higher in the rHuEPO pumping group than in controls. Second, local topical application of rHuEPO gel was given in deep second-degree scald injury sites in rats. Histological analysis showed that epithelialization rate was significantly higher in the rHuEPO gel-treated group than in controls. Immunohistochemical studies showed that the rHuEPO gel-treated group showed remarkably higher expression of skin regeneration makers than the control group. An accurate method for visualization and quantification of blood vessel networks in target areas has still not been developed up to this point, because of technical difficulties in detecting such thin blood vessels. A method which utilizes a series of steps to enhance the image, removes noise from image background, and tracks the vessels edges for vessel segmentation and quantification has been used in this study. Using image analysis methods, we were able to detect the microvascular networks of newly formed blood vessels (less than 500 μm thickness), which participate in the healing process, providing not only nutrition and oxygen to grow tissues but also necessary growth factors to grow tissue cells for complete skin regeneration. The rHuEPO-treated group showed higher expression of stem cell markers (CD 31, CD 90, CD 71, and nestin), which actively contribute to in-wound-healing processes for new hair follicle generation as well as skin regeneration. Collectively, both rHuEPO group pumping into the systemic circulation system, and injection into the local injury area, prompted mice and rats to form new blood vessel networks in scald injury sites, which significantly participate in the scald healing process. These results may lead to the development of novel treatments for scald wounds.
|Subset of early radial glial progenitors that contribute to the development of callosal neurons is absent from avian brain. |
García-Moreno, F; Molnár, Z
Proceedings of the National Academy of Sciences of the United States of America 112 E5058-67 2015
The classical view of mammalian cortical development suggests that pyramidal neurons are generated in a temporal sequence, with all radial glial cells (RGCs) contributing to both lower and upper neocortical layers. A recent opposing proposal suggests there is a subgroup of fate-restricted RGCs in the early neocortex, which generates only upper-layer neurons. Little is known about the existence of fate restriction of homologous progenitors in other vertebrate species. We investigated the lineage of selected Emx2+ [vertebrate homeobox gene related to Drosophila empty spiracles (ems)] RGCs in mouse neocortex and chick forebrain and found evidence for both sequential and fate-restricted programs only in mouse, indicating that these complementary populations coexist in the developing mammalian but not avian brain. Among a large population of sequentially programmed RGCs in the mouse brain, a subset of self-renewing progenitors lack neurogenic potential during the earliest phase of corticogenesis. After a considerable delay, these progenitors generate callosal upper-layer neurons and glia. On the other hand, we found no homologous delayed population in any sectors of the chick forebrain. This finding suggests that neurogenic delay of selected RGCs may be unique to mammals and possibly associated with the evolution of the corpus callosum.
|Double minute amplification of mutant PDGF receptor α in a mouse glioma model. |
Zou, H; Feng, R; Huang, Y; Tripodi, J; Najfeld, V; Tsankova, NM; Jahanshahi, M; Olson, LE; Soriano, P; Friedel, RH
Scientific reports 5 8468 2015
In primary brain tumors, oncogenes are frequently amplified and maintained on extrachromosomal DNA as double minutes (DM), but the underlying mechanisms remain poorly understood. We have generated a mouse model of malignant glioma based on knock-in of a mutant PDGF receptor α (PDGFRα) that is expressed in oligodendrocyte precursor cells (OPCs) after activation by a Cre recombinase. In the tumor suppressor INK4/Arf(-/-) background, mutant animals frequently developed brain tumors resembling anaplastic human gliomas (WHO grade III). Besides brain tumors, most animals also developed aggressive fibrosarcomas, likely triggered by Cre activation of mutant PDGFRα in fibroblastic cell lineages. Importantly, in the brain tumors and cell lines derived from brain tumor tissues, we identified a high prevalence of DM Pdgfra gene amplification, suggesting its occurrence as an early mutational event contributing to the malignant transformation of OPCs. Amplicons extended beyond the Pdgfra locus and included in some cases neighboring genes Kit and Kdr. Our genetically defined mouse brain tumor model therefore supports OPC as a cell of origin for malignant glioma and offers an example of a defined temporal sequence of mutational events, thus providing an entry point for a mechanistic understanding of DM gene amplification and its functionality in gliomagenesis.
|Periostin Promotes Neural Stem Cell Proliferation and Differentiation following Hypoxic-Ischemic Injury. |
Ma, SM; Chen, LX; Lin, YF; Yan, H; Lv, JW; Xiong, M; Li, J; Cheng, GQ; Yang, Y; Qiu, ZL; Zhou, WH
PloS one 10 e0123585 2015
Neural stem cell (NSC) proliferation and differentiation are required to replace neurons damaged or lost after hypoxic-ischemic events and recover brain function. Periostin (POSTN), a novel matricellular protein, plays pivotal roles in the survival, migration, and regeneration of various cell types, but its function in NSCs of neonatal rodent brain is still unknown. The purpose of this study was to investigate the role of POSTN in NSCs following hypoxia-ischemia (HI). We found that POSTN mRNA levels significantly increased in differentiating NSCs. The proliferation and differentiation of NSCs in the hippocampus is compromised in POSTN knockout mice. Moreover, NSC proliferation and differentiation into neurons and astrocytes significantly increased in cultured NSCs treated with recombinant POSTN. Consistently, injection of POSTN into neonatal hypoxic-ischemic rat brains stimulated NSC proliferation and differentiation in the subventricular and subgranular zones after 7 and 14 days of brain injury. Lastly, POSTN treatment significantly improved the spatial learning deficits of rats subjected to HI. These results suggest that POSTN significantly enhances NSC proliferation and differentiation after HI, and provides new insights into therapeutic strategies for the treatment of hypoxic-ischemic encephalopathy.
|Wild-type ALK and activating ALK-R1275Q and ALK-F1174L mutations upregulate Myc and initiate tumor formation in murine neural crest progenitor cells. |
Montavon, G; Jauquier, N; Coulon, A; Peuchmaur, M; Flahaut, M; Bourloud, KB; Yan, P; Delattre, O; Sommer, L; Joseph, JM; Janoueix-Lerosey, I; Gross, N; Mühlethaler-Mottet, A
Oncotarget 5 4452-66 2014
The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC- 1 parental cells in nude mice generated various tumor types, such as NB, osteo/ chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.
|Maintenance of the enteric stem cell niche by bacterial lipopolysaccharides? Evidence and perspectives. |
Schuster, A; Klotz, M; Schwab, T; Di Liddo, R; Bertalot, T; Schrenk, S; Martin, M; Nguyen, TD; Nguyen, TN; Gries, M; Faßbender, K; Conconi, MT; Parnigotto, PP; Schäfer, KH
Journal of cellular and molecular medicine 18 1429-43 2014
The enteric nervous system (ENS) has to respond to continuously changing microenvironmental challenges within the gut and is therefore dependent on a neural stem cell niche to keep the ENS functional throughout life. In this study, we hypothesize that this stem cell niche is also affected during inflammation and therefore investigated lipopolysaccharides (LPS) effects on enteric neural stem/progenitor cells (NSPCs). NSPCs were derived from the ENS and cultured under the influence of different LPS concentrations. LPS effects upon proliferation and differentiation of enteric NSPC cultures were assessed using immunochemistry, flow cytometry, western blot, Multiplex ELISA and real-time PCR. LPS enhances the proliferation of enteric NSPCs in a dose-dependent manner. It delays and modifies the differentiation of these cells. The expression of the LPS receptor toll-like receptor 4 on NSPCs could be demonstrated. Moreover, LPS induces the secretion of several cytokines. Flow cytometry data gives evidence for individual subgroups within the NSPC population. ENS-derived NSPCs respond to LPS in maintaining at least partially their stem cell character. In the case of inflammatory disease or trauma where the liberation and exposure to LPS will be increased, the expansion of NSPCs could be a first step towards regeneration of the ENS. The reduced and altered differentiation, as well as the induction of cytokine signalling, demonstrates that the stem cell niche may take part in the LPS-transmitted inflammatory processes in a direct and defined way.
|Dysbindin-1C is required for the survival of hilar mossy cells and the maturation of adult newborn neurons in dentate gyrus. |
Wang, H; Yuan, Y; Zhang, Z; Yan, H; Feng, Y; Li, W
The Journal of biological chemistry 289 29060-72 2014
DTNBP1 (dystrobrevin-binding protein 1), which encodes dysbindin-1, is one of the leading susceptibility genes for schizophrenia. Both dysbindin-1B and -1C isoforms are decreased, but the dysbindin-1A isoform is unchanged in schizophrenic hippocampal formation, suggesting dysbindin-1 isoforms may have distinct roles in schizophrenia. We found that mouse dysbindin-1C, but not dysbindin-1A, is localized in the hilar glutamatergic mossy cells of the dentate gyrus. The maturation rate of newborn neurons in sandy (sdy) mice, in which both dysbindin-1A and -1C are deleted, is significantly delayed when compared with that in wild-type mice or with that in muted (mu) mice in which dysbindin-1A is destabilized but dysbindin-1C is unaltered. Dysbindin-1C deficiency leads to a decrease in mossy cells, which causes the delayed maturation of newborn neurons. This suggests that dysbindin-1C, rather than dysbindin-1A, regulates adult hippocampal neurogenesis in a non-cell autonomous manner.
|Characterization of ectopic colonies that form in widespread areas of the nervous system with neural stem cell transplants into the site of a severe spinal cord injury. |
Steward, O; Sharp, KG; Yee, KM; Hatch, MN; Bonner, JF
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 14013-21 2014
We reported previously the formation of ectopic colonies in widespread areas of the nervous system after transplantation of fetal neural stem cells (NSCs) into spinal cord transection sites. Here, we characterize the incidence, distribution, and cellular composition of the colonies. NSCs harvested from E14 spinal cords from rats that express GFP were treated with a growth factor cocktail and grafted into the site of a complete spinal cord transection. Two months after transplant, spinal cord and brain tissue were analyzed histologically. Ectopic colonies were found at long distances from the transplant in the central canal of the spinal cord, the surface of the brainstem and spinal cord, and in the fourth ventricle. Colonies were present in 50% of the rats, and most rats had multiple colonies. Axons extended from the colonies into the host CNS. Colonies were strongly positive for nestin, a marker for neural precursors, and contained NeuN-positive cells with processes resembling dendrites, GFAP-positive astrocytes, APC/CC1-positive oligodendrocytes, and Ki-67-positive cells, indicating ongoing proliferation. Stereological analyses revealed an estimated 21,818 cells in a colony in the fourth ventricle, of which 1005 (5%) were Ki-67 positive. Immunostaining for synaptic markers (synaptophysin and VGluT-1) revealed large numbers of synaptophysin-positive puncta within the colonies but fewer VGluT-1 puncta. Continuing expansion of NSC-derived cell masses in confined spaces in the spinal cord and brain could produce symptoms attributable to compression of nearby tissue. It remains to be determined whether other cell types with self-renewing potential can also form colonies.
|Mitochondrial translocation of p53 modulates neuronal fate by preventing differentiation-induced mitochondrial stress. |
Xavier, JM; Morgado, AL; Solá, S; Rodrigues, CM
Antioxidants & redox signaling 21 1009-24 2014
Apoptosis regulatory proteins, such as p53, play a pivotal role in neural differentiation, through mechanisms independent of cell death. In addition, p53 has been identified as an important regulator of mitochondrial survival response, maintaining mitochondrial DNA (mtDNA) integrity and oxidative protection. The aim of this study was to determine the role of mitochondrial p53 in organelle damage and neural differentiation.Our results show that mitochondrial apoptotic events such as reactive oxygen species production, mitochondrial membrane permeabilization, and cytochrome c release are typical of early-stage mouse neural stem cell differentiation, which occurs 3-18 h after induction of differentiation, with no evidence of cell death. In addition, decreased mtDNA content, lipidated LC3 (LC3-II), colocalization of mitochondria and LC3-II puncta, and mitochondria-associated Parkin are consistent with activation of mitophagy. Importantly, at early stages of neural differentiation, p53 was actively translocated to mitochondria and attenuated mitochondrial oxidative stress, cytochrome c release, and mitophagy. Forced mitochondrial translocation of p53 increased neurogenic potential and neurite outgrowth.In conclusion, our results reveal a novel role for mitochondrial p53, which modulates mitochondrial damage and apoptosis-related events in the context of neural differentiation, thus enhancing neuronal fate.
|Cdk5-mediated phosphorylation of RapGEF2 controls neuronal migration in the developing cerebral cortex. |
Ye, T; Ip, JP; Fu, AK; Ip, NY
Nature communications 5 4826 2014
During cerebral cortex development, pyramidal neurons migrate through the intermediate zone and integrate into the cortical plate. These neurons undergo the multipolar-bipolar transition to initiate radial migration. While perturbation of this polarity acquisition leads to cortical malformations, how this process is initiated and regulated is largely unknown. Here we report that the specific upregulation of the Rap1 guanine nucleotide exchange factor, RapGEF2, in migrating neurons corresponds to the timing of this polarity transition. In utero electroporation and live-imaging studies reveal that RapGEF2 acts on the multipolar-bipolar transition during neuronal migration via a Rap1/N-cadherin pathway. Importantly, activation of RapGEF2 is controlled via phosphorylation by a serine/threonine kinase Cdk5, whose activity is largely restricted to the radial migration zone. Thus, the specific expression and Cdk5-dependent phosphorylation of RapGEF2 during multipolar-bipolar transition within the intermediate zone are essential for proper neuronal migration and wiring of the cerebral cortex.
|Microglia enhance neurogenesis and oligodendrogenesis in the early postnatal subventricular zone. |
Shigemoto-Mogami, Y; Hoshikawa, K; Goldman, JE; Sekino, Y; Sato, K
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 2231-43 2014
Although microglia have long been considered as brain resident immune cells, increasing evidence suggests that they also have physiological roles in the development of the normal CNS. In this study, we found large numbers of activated microglia in the forebrain subventricular zone (SVZ) of the rat from P1 to P10. Pharmacological suppression of the activation, which produces a decrease in levels of a number of proinflammatory cytokines (i.e., IL-1β, IL-6, TNF-α, and IFN-γ) significantly inhibited neurogenesis and oligodendrogenesis in the SVZ. In vitro neurosphere assays reproduced the enhancement of neurogenesis and oligodendrogenesis by activated microglia and showed that the cytokines revealed the effects complementarily. These results suggest that activated microglia accumulate in the early postnatal SVZ and that they enhance neurogenesis and oligodendrogenesis via released cytokines.
|17β-estradiol protects human eyelid-derived adipose stem cells against cytotoxicity and increases transplanted cell survival in spinal cord injury. |
Zhou, J; Lu, P; Ren, H; Zheng, Z; Ji, J; Liu, H; Jiang, F; Ling, S; Heng, BC; Hu, X; Ouyang, H
Journal of cellular and molecular medicine 18 326-43 2014
Stem cell transplantation represents a promising strategy for the repair of spinal cord injury (SCI). However, the low survival rate of the grafted cells is a major obstacle hindering clinical success because of ongoing secondary injury processes, which includes excitotoxicity, inflammation and oxidative stress. Previous studies have shown that 17b-estradiol (E2) protects several cell types against cytotoxicity. Thus, we examined the effects of E2 on the viability of human eyelid adipose-derived stem cells (hEASCs) in vitro with hydrogen peroxide (H₂O₂)-induced cell model and in vivo within a rat SCI model. Our results showed that E2 protected hEASCs against H₂O₂-induced cell death in vitro, and enhanced the survival of grafted hEASCs in vivo by reducing apoptosis. Additionally, E2 also enhanced the secretion of growth factors by hEASCs, thereby making the local microenvironment more conducive for tissue regeneration. Overall, E2 administration enhanced the therapeutic efficacy of hEASCs transplantation and facilitated motor function recovery after SCI. Hence, E2 administration may be an intervention of choice for enhancing survival of transplanted hEASCs after SCI.
|Prohibitin 2 regulates the proliferation and lineage-specific differentiation of mouse embryonic stem cells in mitochondria. |
Kowno, M; Watanabe-Susaki, K; Ishimine, H; Komazaki, S; Enomoto, K; Seki, Y; Wang, YY; Ishigaki, Y; Ninomiya, N; Noguchi, TA; Kokubu, Y; Ohnishi, K; Nakajima, Y; Kato, K; Intoh, A; Takada, H; Yamakawa, N; Wang, PC; Asashima, M; Kurisaki, A
PloS one 9 e81552 2014
The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown.In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria.Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.
|GSK3β, but not GSK3α, inhibits the neuronal differentiation of neural progenitor cells as a downstream target of mammalian target of rapamycin complex1. |
Ahn, J; Jang, J; Choi, J; Lee, J; Oh, SH; Lee, J; Yoon, K; Kim, S
Stem cells and development 23 1121-33 2014
Glycogen synthase kinase 3 (GSK3) acts as an important regulator during the proliferation and differentiation of neural progenitor cells (NPCs), but the roles of the isoforms of this molecule (GSK3α and GSK3β) have not been clearly defined. In this study, we investigated the functions of GSK3α and GSK3β in the context of neuronal differentiation of murine NPCs. Treatment of primary NPCs with a GSK3 inhibitor (SB216763) resulted in an increase in the percentage of TuJ1-positive immature neurons, suggesting an inhibitory role of GSK3 in embryonic neurogenesis. Downregulation of GSK3β expression increased the percentage of TuJ1-positive cells, while knock-down of GSK3α seemed to have no effect. When primary NPCs were engineered to stably express either isoform of GSK3 using retroviral vectors, GSK3β, but not GSK3α, inhibited neuronal differentiation and helped the cells to maintain the characteristics of NPCs. Mutant GSK3β (Y216F) failed to suppress neuronal differentiation, indicating that the kinase activity of GSK3β is important for this regulatory function. Similar results were obtained in vivo when a retroviral vector expressing GSK3β was delivered to E9.5 mouse brains using the ultrasound image-guided gene delivery technique. In addition, SB216763 was found to block the rapamycin-mediated inhibition of neuronal differentiation of NPCs. Taken together, our results demonstrate that GSK3β, but not GSK3α, negatively controls the neuronal differentiation of progenitor cells and that GSK3β may act downstream of the mammalian target of rapamycin complex1 signaling pathway.
|The miR-7 identified from collagen biomaterial-based three-dimensional cultured cells regulates neural stem cell differentiation. |
Cui, Y; Xiao, Z; Chen, T; Wei, J; Chen, L; Liu, L; Chen, B; Wang, X; Li, X; Dai, J
Stem cells and development 23 393-405 2014
Increasing evidence suggests that three-dimensional (3D) cultures provide more appropriate microenvironments to control stem cell response compared with traditional two-dimensional (2D) cultures. However, the molecular mechanism involved in 3D cultured stem cells is not well known. Several microRNAs whose target genes involved in the regulation of self-renewal and differentiation of stem cells were found to be downregulated in 3D cultured PA-1 cells. Among them, miR-7 was predicted to target Kruppel-like factor 4 (Klf4), a key gene for self-renewal of neural stem cells (NSCs). We showed that the differentiation of NSCs was inhibited in 3D collagen scaffolds compared with 2D cultured cells. The quantitative real-time PCR (qPCR) analysis indicated that the expression of miR-7 and Klf4 changed significantly in 2D cultures, whereas the expression stability of miR-7 and Klf4 was detected in 3D cultures. Using luciferase assay and western blot, Klf4 was identified as a target of miR-7 indicating that miR-7 plays a critical role in maintaining the self-renewal capacity through a Klf4-dependent mechanism in 3D cultured cells. Thus, the collagen scaffold-based 3D cell cultures may provide a platform to reveal the regulatory mechanism of cell regulators, which are difficult to find in traditional 2D cell cultures.
|The neuron regrowth is associated with the proliferation of neural precursor cells after leukemia inhibitory factor administration following spinal cord injury in mice. |
Li, Y; Zang, D
PloS one 9 e116031 2014
To explore whether LIF could promote the proliferation of neural precursor cells (NPCs) and to analyze the correlation between increased NPCs and FluoroGold (FG) labeled neurons in mice after spinal cord injury (SCI).Motor behavior was assessed using Rotarod and Platform Hang tests; neurons in the corticospinal and rubrospinal systems were labeled with FG, NPCs were immustained with nestin-FITC conjugate. The numbers of FG-labeled neurons and NPCs were estimated, and the correlation between FG-labeled neurons and NPCs was assessed.Mice in the SCI group showed negligible recovery of locomotor behavior; in contrast, mice in the LIF group showed a statically significant improvement. Both FG-labeled neurons and NPCs were significantly increased in the LIF group compared to the SCI group, and this increase in FG-labeled neurons and NPCs showed a clear association above the lesion level.LIF could promote locomotive behaviors in mice post-SCI by encouraging the proliferation of NPCs; LIF may in fact be a potential cytokine for the induction of NPCs post-SCI.
|Endogenous cerebellar neurogenesis in adult mice with progressive ataxia. |
Kumar, M; Csaba, Z; Peineau, S; Srivastava, R; Rasika, S; Mani, S; Gressens, P; El Ghouzzi, V
Annals of clinical and translational neurology 1 968-81 2014
Transplanting exogenous neuronal progenitors to replace damaged neurons in the adult brain following injury or neurodegenerative disorders and achieve functional amelioration is a realistic goal. However, studies so far have rarely taken into consideration the preexisting inflammation triggered by the disease process that could hamper the effectiveness of transplanted cells. Here, we examined the fate and long-term consequences of human cerebellar granule neuron precursors (GNP) transplanted into the cerebellum of Harlequin mice, an adult model of progressive cerebellar degeneration with early-onset microgliosis.Human embryonic stem cell-derived progenitors expressing Atoh1, a transcription factor key to GNP specification, were generated in vitro and stereotaxically transplanted into the cerebellum of preataxic Harlequin mice. The histological and functional impact of these transplants was followed using immunolabeling and Rotarod analysis.Although transplanted GNPs did not survive beyond a few weeks, they triggered the proliferation of endogenous nestin-positive precursors in the leptomeninges that crossed the molecular layer and differentiated into mature neurons. These phenomena were accompanied by the preservation of the granule and Purkinje cell layers and delayed ataxic changes. In vitro neurosphere generation confirmed the enhanced neurogenic potential of the cerebellar leptomeninges of Harlequin mice transplanted with exogenous GNPs.The cerebellar leptomeninges of adult mice contain an endogenous neurogenic niche that can be stimulated to yield mature neurons from an as-yet unidentified population of progenitors. The transplantation of human GNPs not only stimulates this neurogenesis, but, despite the potentially hostile environment, leads to neuroprotection and functional amelioration.
|Maintenance and neuronal differentiation of chicken induced pluripotent stem-like cells. |
Dai, R; Rossello, R; Chen, CC; Kessler, J; Davison, I; Hochgeschwender, U; Jarvis, ED
Stem cells international 2014 182737 2014
Pluripotent stem cells have the potential to become any cell in the adult body, including neurons and glia. Avian stem cells could be used to study questions, like vocal learning, that would be difficult to examine with traditional mouse models. Induced pluripotent stem cells (iPSCs) are differentiated cells that have been reprogrammed to a pluripotent stem cell state, usually using inducing genes or other molecules. We recently succeeded in generating avian iPSC-like cells using mammalian genes, overcoming a limitation in the generation and use of iPSCs in nonmammalian species (Rosselló et al., 2013). However, there were no established optimal cell culture conditions for avian iPSCs to establish long-term cell lines and thus to study neuronal differentiation in vitro. Here we present an efficient method of maintaining chicken iPSC-like cells and for differentiating them into action potential generating neurons.
|Pathological roles of the VEGF/SphK pathway in Niemann-Pick type C neurons. |
Lee, H; Lee, JK; Park, MH; Hong, YR; Marti, HH; Kim, H; Okada, Y; Otsu, M; Seo, EJ; Park, JH; Bae, JH; Okino, N; He, X; Schuchman, EH; Bae, JS; Jin, HK
Nature communications 5 5514 2014
Sphingosine is a major storage compound in Niemann-Pick type C disease (NP-C), although the pathological role(s) of this accumulation have not been fully characterized. Here we found that sphingosine kinase (SphK) activity is reduced in NP-C patient fibroblasts and NP-C mouse Purkinje neurons (PNs) due to defective vascular endothelial growth factor (VEGF) levels. Sphingosine accumulation due to inactivation of VEGF/SphK pathway led to PNs loss via inhibition of autophagosome-lysosome fusion in NP-C mice. VEGF activates SphK by binding to VEGFR2, resulting in decreased sphingosine storage as well as improved PNs survival and clinical outcomes in NP-C cells and mice. We also show that induced pluripotent stem cell (iPSC)-derived human NP-C neurons are generated and the abnormalities caused by VEGF/SphK inactivity in these cells are corrected by replenishment of VEGF. Overall, these results reveal a pathogenic mechanism in NP-C neurons where defective SphK activity is due to impaired VEGF levels.
|Subplate in a rat model of preterm hypoxia-ischemia. |
Okusa, C; Oeschger, F; Ginet, V; Wang, WZ; Hoerder-Suabedissen, A; Matsuyama, T; Truttmann, AC; Molnár, Z
Annals of clinical and translational neurology 1 679-91 2014
Hypoxia-ischemia (HI) in preterm infants primarily leads to injuries in the cerebral white matter. However, there is growing evidence that perinatal injury in preterms can also involve other zones including the cortical gray matter. In a neonatal rat model of HI, selective vulnerability of subplate has been suggested using BrdU birth-dating methods. In this study, we aimed to investigate the neuropathological changes of the subplate and deep layers of the cortex following cerebral HI in neonatal rats with specific cell markers.P2 rats underwent permanent occlusion of the right common carotid artery followed by a period of hypoxia. P8 rats were analyzed using immunohistochemistry; subplate and deep layers cells were quantified and compared with sham-operated case.A large variability in the extent of the cerebral injury was apparent. For the three analyzed subplate populations (Nurr1+, Cplx3+, and Ctgf+ cells), no significant cell reduction was observed in mild and moderate cases. Only in severe cases, subplate cells were strongly affected, but these injuries were always accompanied by the cell reductions in layers VI and V.We could therefore not confirm a specific vulnerability of subplate cells compared to other deep layers or the white matter in our model.
|Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors. |
Forostyak, Oksana, et al.
Stem Cells Dev., 22: 1506-21 (2013) 2013
Human embryonic stem cell-derived neural precursors (hESC NPs) are considered to be a promising tool for cell-based therapy in central nervous system injuries and neurodegenerative diseases. The Ca(2+) ion is an important intracellular messenger essential for the regulation of various cellular functions. We investigated the role and physiology of Ca(2+) signaling to characterize the functional properties of CCTL14 hESC NPs during long-term maintenance in culture (in vitro). We analyzed changes in cytoplasmic Ca(2+) concentration ([Ca(2+)]i) evoked by high K(+), adenosine-5'-triphosphate (ATP), glutamate, γ-aminobutyric acid (GABA), and caffeine in correlation with the expression of various neuronal markers in different passages (P6 through P10) during the course of hESC differentiation. We found that only differentiated NPs from P7 exhibited significant and specific [Ca(2+)]i responses to various stimuli. About 31% of neuronal-like P7 NPs exhibited spontaneous [Ca(2+)]i oscillations. Pharmacological and immunocytochemical assays revealed that P7 NPs express L- and P/Q-type Ca(2+) channels, P2X2, P2X3, P2X7, and P2Y purinoreceptors, glutamate receptors, and ryanodine (RyR1 and RyR3) receptors. The ATP- and glutamate-induced [Ca(2+)]i responses were concentration-dependent. Higher glutamate concentrations (over 100 μM) caused cell death. Responses to ATP were observed in the presence or in the absence of extracellular Ca(2+). These results emphasize the notion that with time in culture, these cells attain a transient period of operative Ca(2+) signaling that is predictive of their ability to act as stem elements.
|Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA. |
Angulo-Rojo, C; Manning-Cela, R; Aguirre, A; Ortega, A; López-Bayghen, E
ASN neuro 5 e00130 2013
The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD) and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJ(κ) pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision.
|TGF-β superfamily gene expression and induction of the Runx1 transcription factor in adult neurogenic regions after brain injury. |
Logan, TT; Villapol, S; Symes, AJ
PloS one 8 e59250 2013
Traumatic brain injury (TBI) increases neurogenesis in the forebrain subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). Transforming growth factor-β (TGF-β) superfamily cytokines are important regulators of adult neurogenesis, but their involvement in the regulation of this process after brain injury is unclear. We subjected adult mice to controlled cortical impact (CCI) injury, and isolated RNA from the SVZ and DG at different post-injury time points. qPCR array analysis showed that cortical injury caused significant alterations in the mRNA expression of components and targets of the TGF-β, BMP, and activin signaling pathways in the SVZ and DG after injury, suggesting that these pathways could regulate post-injury neurogenesis. In both neurogenic regions, the injury also induced expression of Runt-related transcription factor-1 (Runx1), which can interact with intracellular TGF-β Smad signaling pathways. CCI injury strongly induced Runx1 expression in activated and proliferating microglial cells throughout the neurogenic regions. Runx1 protein was also expressed in a subset of Nestin- and GFAP-expressing putative neural stem or progenitor cells in the DG and SVZ after injury. In the DG only, these Runx1+ progenitors proliferated. Our data suggest potential roles for Runx1 in the processes of microglial cell activation and proliferation and in neural stem cell proliferation after TBI.
|Harvest site influences the growth properties of adipose derived stem cells. |
Engels, PE; Tremp, M; Kingham, PJ; di Summa, PG; Largo, RD; Schaefer, DJ; Kalbermatten, DF
Cytotechnology 65 437-45 2013
The therapeutic potential of adult stem cells may become a relevant option in clinical care in the future. In hand and plastic surgery, cell therapy might be used to enhance nerve regeneration and help surgeons and clinicians to repair debilitating nerve injuries. Adipose-derived stem cells (ASCs) are found in abundant quantities and can be harvested with a low morbidity. In order to define the optimal fat harvest location and detect any potential differences in ASC proliferation properties, we compared biopsies from different anatomical sites (inguinal, flank, pericardiac, omentum, neck) in Sprague-Dawley rats. ASCs were expanded from each biopsy and a proliferation assay using different mitogenic factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was performed. Our results show that when compared with the pericardiac region, cells isolated from the inguinal, flank, omental and neck regions grow significantly better in growth medium alone. bFGF significantly enhanced the growth rate of ASCs isolated from all regions except the omentum. PDGF had minimal effect on ASC proliferation rate but increases the growth of ASCs from the neck region. Analysis of all the data suggests that ASCs from the neck region may be the ideal stem cell sources for tissue engineering approaches for the regeneration of nervous tissue.
|Organelle and cellular abnormalities associated with hippocampal heterotopia in neonatal doublecortin knockout mice. |
Khalaf-Nazzal, R; Bruel-Jungerman, E; Rio, JP; Bureau, J; Irinopoulou, T; Sumia, I; Roumegous, A; Martin, E; Olaso, R; Parras, C; Cifuentes-Diaz, C; Francis, F
PloS one 8 e72622 2013
Heterotopic or aberrantly positioned cortical neurons are associated with epilepsy and intellectual disability. Various mouse models exist with forms of heterotopia, but the composition and state of cells developing in heterotopic bands has been little studied. Dcx knockout (KO) mice show hippocampal CA3 pyramidal cell lamination abnormalities, appearing from the age of E17.5, and mice suffer from spontaneous epilepsy. The Dcx KO CA3 region is organized in two distinct pyramidal cell layers, resembling a heterotopic situation, and exhibits hyperexcitability. Here, we characterized the abnormally organized cells in postnatal mouse brains. Electron microscopy confirmed that the Dcx KO CA3 layers at postnatal day (P) 0 are distinct and separated by an intermediate layer devoid of neuronal somata. We found that organization and cytoplasm content of pyramidal neurons in each layer were altered compared to wild type (WT) cells. Less regular nuclei and differences in mitochondria and Golgi apparatuses were identified. Each Dcx KO CA3 layer at P0 contained pyramidal neurons but also other closely apposed cells, displaying different morphologies. Quantitative PCR and immunodetections revealed increased numbers of oligodendrocyte precursor cells (OPCs) and interneurons in close proximity to Dcx KO pyramidal cells. Immunohistochemistry experiments also showed that caspase-3 dependent cell death was increased in the CA1 and CA3 regions of Dcx KO hippocampi at P2. Thus, unsuspected ultrastructural abnormalities and cellular heterogeneity may lead to abnormal neuronal function and survival in this model, which together may contribute to the development of hyperexcitability.
|Imaging-based chemical screening reveals activity-dependent neural differentiation of pluripotent stem cells. |
Sun, Y; Dong, Z; Jin, T; Ang, KH; Huang, M; Haston, KM; Peng, J; Zhong, TP; Finkbeiner, S; Weiss, WA; Arkin, MR; Jan, LY; Guo, S
eLife 2 e00508 2013
Mammalian pluripotent stem cells (PSCs) represent an important venue for understanding basic principles regulating tissue-specific differentiation and discovering new tools that may facilitate clinical applications. Mechanisms that direct neural differentiation of PSCs involve growth factor signaling and transcription regulation. However, it is unknown whether and how electrical activity influences this process. Here we report a high throughput imaging-based screen, which uncovers that selamectin, an anti-helminthic therapeutic compound with reported activity on invertebrate glutamate-gated chloride channels, promotes neural differentiation of PSCs. We show that selamectin's pro-neurogenic activity is mediated by γ2-containing GABAA receptors in subsets of neural rosette progenitors, accompanied by increased proneural and lineage-specific transcription factor expression and cell cycle exit. In vivo, selamectin promotes neurogenesis in developing zebrafish. Our results establish a chemical screening platform that reveals activity-dependent neural differentiation from PSCs. Compounds identified in this and future screening might prove therapeutically beneficial for treating neurodevelopmental or neurodegenerative disorders. DOI:http://dx.doi.org/10.7554/eLife.00508.001.
|A network of heterochronic genes including Imp1 regulates temporal changes in stem cell properties. |
Nishino, J; Kim, S; Zhu, Y; Zhu, H; Morrison, SJ
eLife 2 e00924 2013
Stem cell properties change over time to match the changing growth and regeneration demands of tissues. We showed previously that adult forebrain stem cell function declines during aging because of increased expression of let-7 microRNAs, evolutionarily conserved heterochronic genes that reduce HMGA2 expression. Here we asked whether let-7 targets also regulate changes between fetal and adult stem cells. We found a second let-7 target, the RNA binding protein IMP1, that is expressed by fetal, but not adult, neural stem cells. IMP1 expression was promoted by Wnt signaling and Lin28a expression and opposed by let-7 microRNAs. Imp1-deficient neural stem cells were prematurely depleted in the dorsal telencephalon due to accelerated differentiation, impairing pallial expansion. IMP1 post-transcriptionally inhibited the expression of differentiation-associated genes while promoting the expression of self-renewal genes, including Hmga2. A network of heterochronic gene products including Lin28a, let-7, IMP1, and HMGA2 thus regulates temporal changes in stem cell properties. DOI: http://dx.doi.org/10.7554/eLife.00924.001.
|A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells. |
García-Parra, P; Maroto, M; Cavaliere, F; Naldaiz-Gastesi, N; Álava, JI; García, AG; López de Munain, A; Izeta, A
BMC neuroscience 14 48 2013
The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells.Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates.When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons.We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.
|An allelic series of mice reveals a role for RERE in the development of multiple organs affected in chromosome 1p36 deletions. |
Kim, BJ; Zaveri, HP; Shchelochkov, OA; Yu, Z; Hernández-García, A; Seymour, ML; Oghalai, JS; Pereira, FA; Stockton, DW; Justice, MJ; Lee, B; Scott, DA
PloS one 8 e57460 2013
Individuals with terminal and interstitial deletions of chromosome 1p36 have a spectrum of defects that includes eye anomalies, postnatal growth deficiency, structural brain anomalies, seizures, cognitive impairment, delayed motor development, behavior problems, hearing loss, cardiovascular malformations, cardiomyopathy, and renal anomalies. The proximal 1p36 genes that contribute to these defects have not been clearly delineated. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in this region and encodes a nuclear receptor coregulator that plays a critical role in embryonic development as a positive regulator of retinoic acid signaling. Rere-null mice die of cardiac failure between E9.5 and E11.5. This limits their usefulness in studying the role of RERE in the latter stages of development and into adulthood. To overcome this limitation, we created an allelic series of RERE-deficient mice using an Rere-null allele, om, and a novel hypomorphic Rere allele, eyes3 (c.578Tgreater than C, p.Val193Ala), which we identified in an N-ethyl-N-nitrosourea (ENU)-based screen for autosomal recessive phenotypes. Analyses of these mice revealed microphthalmia, postnatal growth deficiency, brain hypoplasia, decreased numbers of neuronal nuclear antigen (NeuN)-positive hippocampal neurons, hearing loss, cardiovascular malformations-aortic arch anomalies, double outlet right ventricle, and transposition of the great arteries, and perimembranous ventricular septal defects-spontaneous development of cardiac fibrosis and renal agenesis. These findings suggest that RERE plays a critical role in the development and function of multiple organs including the eye, brain, inner ear, heart and kidney. It follows that haploinsufficiency of RERE may contribute-alone or in conjunction with other genetic, environmental, or stochastic factors-to the development of many of the phenotypes seen in individuals with terminal and interstitial deletions that include the proximal region of chromosome 1p36.
|From the Cover: Neutralization of terminal differentiation in gliomagenesis. |
Hu, J; Ho, AL; Yuan, L; Hu, B; Hua, S; Hwang, SS; Zhang, J; Hu, T; Zheng, H; Gan, B; Wu, G; Wang, YA; Chin, L; DePinho, RA
Proceedings of the National Academy of Sciences of the United States of America 110 14520-7 2013
An immature state of cellular differentiation--characterized by stem cell-like tendencies and impaired differentiation--is a hallmark of cancer. Using glioblastoma multiforme (GBM) as a model system, we sought to determine whether molecular determinants that drive cells toward terminal differentiation are also genetically targeted in carcinogenesis and whether neutralizing such genes also plays an active role to reinforce the impaired differentiation state and promote malignancy. To that end, we screened 71 genes with known roles in promoting nervous system development that also sustain copy number loss in GBM through antineoplastic assay and identified A2BP1 (ataxin 2 binding protein 1, Rbfox1), an RNA-binding and splicing regulator that is deleted in 10% of GBM cases. Integrated in silico analysis of GBM profiles to elucidate the A2BP1 pathway and its role in glioma identified myelin transcription factor 1-like (Myt1L) as a direct transcriptional regulator of A2BP1. Reintroduction of A2BP1 or Myt1L in GBM cell lines and glioma stem cells profoundly inhibited tumorigenesis in multiple assays, and conversely, shRNA-mediated knockdown of A2BP1 or Myt1L in premalignant neural stem cells compromised neuronal lineage differentiation and promoted orthotopic tumor formation. On the mechanistic level, with the top-represented downstream target TPM1 as an illustrative example, we demonstrated that, among its multiple functions, A2BP1 serves to regulate TPM1's alternative splicing to promote cytoskeletal organization and terminal differentiation and suppress malignancy. Thus, in addition to the activation of self-renewal pathways, the neutralization of genetic programs that drive cells toward terminal differentiation may also promote immature and highly plastic developmental states that contribute to the aggressive malignant properties of GBM.
|Overexpression of CD44 in neural precursor cells improves trans-endothelial migration and facilitates their invasion of perivascular tissues in vivo. |
Deboux, C; Ladraa, S; Cazaubon, S; Ghribi-Mallah, S; Weiss, N; Chaverot, N; Couraud, PO; Baron-Van Evercooren, A
PloS one 8 e57430 2013
Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional invivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues.
|Association between tumorigenic potential and the fate of cancer cells in a syngeneic melanoma model. |
Krelin, Y; Berkovich, L; Amit, M; Gil, Z
PloS one 8 e62124 2013
The self-renewal potential of a cancer cell can be estimated by using particular assays, which include xenotransplantation in immunocompromised animals or culturing in non-adherent serum-free stem-cells media (SCM). However, whether cells with self-renewal potential actually contribute to disease is unknown. Here we investigated the tumorigenic potential and fate of cancer cells in an in-vivo melanoma model. We examined cell lines which were derived from the same parental line: a non-metastatic cell line (K1735/16), a metastatic cell line (K1735/M4) and a cell line which was selected in non-adherent conditions (K1735/16S). All cell lines exhibited similar proliferation kinetics when grown on culture plates. K1735/16 cells grown in soft agar or in suspension non-adherent conditions failed to form colonies or spheroids, whereas the other cell lines showed prominent colonogenicity and spheroid formation capacity. By using sphere limiting dilution analysis (SLDA) in serum-free media, K1735/16S and K1735/M4 cells grown in suspension were capable of forming spheroids even in low frequencies of concentrations, as opposed to K1735/16 cells. The tumorigenic potential of the cell lines was determined in SCID mice using intra footpad injections. Palpable tumors were evident in all mice. In agreement with the in-vitro studies, the K1735/M4 cell line exhibited the highest growth kinetics, followed by the K1735/16S cell line, whereas the K1735/16 cell line had the lowest tumor growth potential (Pless than 0.001). In contrast, when we repeated the experiments in syngeneic C3H/HeN mice, the K1735/16 cell line produced macroscopic tumors 30-100 days after injection, whereas K1735/M4 and K1735/16S derived tumors regressed spontaneously in 90-100% of mice. TUNEL analysis revealed significantly higher number of apoptotic cells in K1735/16S and K1735/M4 cell line-derived tumors compared to K1735/16 tumors (Pless than 0.001). The models we have examined here raised the possibility, that cells with high-tumorigenic activity may be more immunogenic and hence are more susceptible to immune-regulation.
|Cells with intense EGFR staining and a high nuclear to cytoplasmic ratio are specific for infiltrative glioma: a useful marker in neuropathological practice. |
Burel-Vandenbos, F; Turchi, L; Benchetrit, M; Fontas, E; Pedeutour, Z; Rigau, V; Almairac, F; Ambrosetti, D; Michiels, JF; Virolle, T
Neuro-oncology 15 1278-88 2013
The differential diagnosis between infiltrative glioma (IG) and benign or curable glial lesions, such as gliosis, pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, ganglioglioma, or demyelinating disease, may be challenging for the pathologist because specific markers are lacking. Recently, we described a strong EGFR immunolabelling pattern in cells with a high nuclear to cytoplasmic ratio that enables the discrimination of low-grade IG from gliosis. The aim of this study was to extend our observation to high-grade glioma to assess whether EGFR expression pattern is of value in the discrimination of all IG from noninfiltrative glial lesions (NIG), including gliosis, benign tumors, and demyelinating disease.One hundred one IG and 58 NIG were compared for immunohistochemical expression of EGFR with use of an antibody that recognizes an epitope in the extracellular domain of both EGFRwt and EGFRvIII. Highly EGFR-positive cells with a high nuclear to cytoplasmic ratio were isolated and further characterized.Cells with intense EGFR staining and a high nuclear to cytoplasmic ratio were significantly associated with the diagnosis of IG (P less than .0001). The sensitivity and specificity of this staining pattern for the diagnosis of IG were 95% and 100%, respectively. EGFR expression was independent of IDH1 mutations and EGFR amplification. Finally, we showed that these particular cells displayed the phenotype and properties of glial progenitors and coexpressed CXCR4, a marker of invasiveness.We demonstrate that cells with intense EGFR staining and a high nuclear to cytoplasmic ratio are specific criteria for the diagnosis of IG, irrespective of grade, histological subtype, and progression pathway, and their identification represents a tool to discriminate IG from benign or curable glial lesions.
|BMP induces cochlin expression to facilitate self-renewal and suppress neural differentiation of mouse embryonic stem cells. |
Zhang, J; Fei, T; Li, Z; Zhu, G; Wang, L; Chen, YG
The Journal of biological chemistry 288 8053-60 2013
BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.
|Zona occludens-2 protects against podocyte dysfunction induced by ADR in mice. |
Bautista-García, P; Reyes, JL; Martín, D; Namorado, MC; Chavez-Munguía, B; Soria-Castro, E; Huber, O; González-Mariscal, L
American journal of physiology. Renal physiology 304 F77-87 2013
Zona occludens-2 (ZO-2) is a protein present at the tight junction and nucleus of epithelial cells. ZO-2 represses the transcription of genes regulated by the Wnt/β-catenin pathway. This pathway plays a critical role in podocyte injury and proteinuria. Here, we analyze whether the overexpression of ZO-2 in the glomerulus, by hydrodynamics transfection, prevents podocyte injury mediated by the Wnt/β-catenin pathway in the mouse model of adriamycin (ADR) nephrosis. By immunofluorescence and immunogold electron microscopy, we show that ZO-2 is present in mice glomerulus, not at the slit diaphragms where nephrin concentrates, but in the cytoplasm and at processes of podocytes. Our results indicate that in the glomeruli of mice treated with ADR, ZO-2 overexpression increases the amount of phosphorylated β-catenin, inhibits the expression of the transcription factor snail, prevents nephrin and podocalyxin loss, reduces podocyte effacement and massive fusions, restrains proteinuria, and supports urea and creatinine clearance. These results suggest that ZO-2 could be a new target for the regulation of hyperactive Wnt/β-catenin signaling in proteinuric kidney diseases.
|Inhibition of the Rho signaling pathway improves neurite outgrowth and neuronal differentiation of mouse neural stem cells. |
Gu, H; Yu, SP; Gutekunst, CA; Gross, RE; Wei, L
International journal of physiology, pathophysiology and pharmacology 5 11-20 2013
Neurons in the adult mammalian CNS do not spontaneously regenerate axons after injury due to CNS myelin and other inhibitory factors. Previous studies have showed that inhibition of the Rho-ROCK pathway promotes axonal outgrowth in primary neurons or in spinal cord injury models. Furthermore, RhoA inhibitor C3 transferase has a potential effect to induce neural differentiation in primary cultured neurons and cell lines. As stem cells and stem cell-derived neural progenitor cells have emerged as a regenerative medicine for stroke, Parkinson's disease and other neurological disorders, strategies that can promote axonal outgrowth and neuronal differentiation appear to have promising benefits in the cell-based therapy. Currently, how changes in the Rho-ROCK pathway may affect the neurite outgrowth and neuronal differentiation of stem cells has been poorly understood. The present investigation examined the effects of RhoA inhibition on neurite outgrowth and neuronal differentiation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of the mouse. Our results show that inhibition of RhoA leads to neurite outgrowth of NSCs not only on normal culture substrate, poly-D-lysine (PDL), but also on myelin substrate. Moreover, inhibition of RhoA improves neuronal differentiation of NSCs and up-regulates biomarkers of neuronal gene expression. These results support that the Rho signaling pathway plays an important role in neurite development and neuronal differentiation of NSCs.
|A novel, diffusely infiltrative xenograft model of human anaplastic oligodendroglioma with mutations in FUBP1, CIC, and IDH1. |
Klink, B; Miletic, H; Stieber, D; Huszthy, PC; Campos Valenzuela, JA; Valenzuela, JA; Balss, J; Wang, J; Schubert, M; Sakariassen, PØ; Sundstrøm, T; Torsvik, A; Aarhus, M; Mahesparan, R; von Deimling, A; Kaderali, L; Niclou, SP; Schröck, E; Bjerkvig, R; Nigro, JM
PloS one 8 e59773 2013
Oligodendroglioma poses a biological conundrum for malignant adult human gliomas: it is a tumor type that is universally incurable for patients, and yet, only a few of the human tumors have been established as cell populations in vitro or as intracranial xenografts in vivo. Their survival, thus, may emerge only within a specific environmental context. To determine the fate of human oligodendroglioma in an experimental model, we studied the development of an anaplastic tumor after intracranial implantation into enhanced green fluorescent protein (eGFP) positive NOD/SCID mice. Remarkably after nearly nine months, the tumor not only engrafted, but it also retained classic histological and genetic features of human oligodendroglioma, in particular cells with a clear cytoplasm, showing an infiltrative growth pattern, and harboring mutations of IDH1 (R132H) and of the tumor suppressor genes, FUBP1 and CIC. The xenografts were highly invasive, exhibiting a distinct migration and growth pattern around neurons, especially in the hippocampus, and following white matter tracts of the corpus callosum with tumor cells accumulating around established vasculature. Although tumors exhibited a high growth fraction in vivo, neither cells from the original patient tumor nor the xenograft exhibited significant growth in vitro over a six-month period. This glioma xenograft is the first to display a pure oligodendroglioma histology and expression of R132H. The unexpected property, that the cells fail to grow in vitro even after passage through the mouse, allows us to uniquely investigate the relationship of this oligodendroglioma with the in vivo microenvironment.
|Neural precursor cells cultured at physiologically relevant oxygen tensions have a survival advantage following transplantation. |
Stacpoole, SR; Webber, DJ; Bilican, B; Compston, A; Chandran, S; Franklin, RJ
Stem cells translational medicine 2 464-72 2013
Traditionally, in vitro stem cell systems have used oxygen tensions that are far removed from the in vivo situation. This is particularly true for the central nervous system, where oxygen (O2) levels range from 8% at the pia to 0.5% in the midbrain, whereas cells are usually cultured in a 20% O2 environment. Cell transplantation strategies therefore typically introduce a stress challenge at the time of transplantation as the cells are switched from 20% to 3% O2 (the average in adult organs). We have modeled the oxygen stress that occurs during transplantation, demonstrating that in vitro transfer of neonatal rat cortical neural precursor cells (NPCs) from a 20% to a 3% O2 environment results in significant cell death, whereas maintenance at 3% O2 is protective. This survival benefit translates to the in vivo environment, where culture of NPCs at 3% rather than 20% O2 approximately doubles survival in the immediate post-transplantation phase. Furthermore, NPC fate is affected by culture at low, physiological O2 tensions (3%), with particularly marked effects on the oligodendrocyte lineage, both in vitro and in vivo. We propose that careful consideration of physiological oxygen environments, and particularly changes in oxygen tension, has relevance for the practical approaches to cellular therapies.
|Differential regulation of proliferation and neuronal differentiation in adult rat spinal cord neural stem/progenitors by ERK1/2, Akt, and PLCγ. |
Chan, WS; Sideris, A; Sutachan, JJ; Montoya G, JV; Blanck, TJ; Recio-Pinto, E
Frontiers in molecular neuroscience 6 23 2013
Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2). We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2) over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2) or the phosphoinositide 3-kinase (PI3K)/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase Cγ (PLCγ) pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCγ signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs.
|Alternative mRNA splicing from the glial fibrillary acidic protein (GFAP) gene generates isoforms with distinct subcellular mRNA localization patterns in astrocytes. |
Thomsen, R; Daugaard, TF; Holm, IE; Nielsen, AL
PloS one 8 e72110 2013
The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3'-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease.
|β-Catenin is critical for cerebellar foliation and lamination. |
Wen, J; Yang, HB; Zhou, B; Lou, HF; Duan, S
PloS one 8 e64451 2013
The cerebellum has a conserved foliation pattern and a well-organized layered structure. The process of foliation and lamination begins around birth. β-catenin is a downstream molecule of Wnt signaling pathway, which plays a critical role in tissue organization. Lack of β-catenin at early embryonic stages leads to either prenatal or neonatal death, therefore it has been difficult to resolve its role in cerebellar foliation and lamination. Here we used GFAP-Cre to ablate β-catenin in neuronal cells of the cerebellum after embryonic day 12.5, and found an unexpected role of β-catenin in determination of the foliation pattern. In the mutant mice, the positions of fissure formation were changed, and the meninges were improperly incorporated into fissures. At later stages, some lobules were formed by Purkinje cells remaining in deep regions of the cerebellum and the laminar structure was dramatically altered. Our results suggest that β-catenin is critical for cerebellar foliation and lamination. We also found a non cell-autonomous role of β-catenin in some developmental properties of major cerebellar cell types during specific stages.
|Brain tissue interaction with three-dimensional, honeycomb polycaprolactone-based scaffolds designed for cranial reconstruction following traumatic brain injury. |
Choy, DK; Nga, VD; Lim, J; Lu, J; Chou, N; Yeo, TT; Teoh, SH
Tissue engineering. Part A 19 2382-9 2013
Following traumatic brain injury (TBI), resultant voids are unable to support injections of suspension treatments, leading to ineffective healing. Moreover, without a structure to support the large defect, the defect site suffers from mechanical instability, which may impair the healing process. Therefore, having a delivery vehicle that can temporarily fill and provide mechanical support to the defect site may alleviate the healing process. In this work, we reported for the first time, the inflammatory response of brain tissue with polycaprolactone (PCL) and PCL-tricalcium phosphate (TCP) scaffolds designed and fabricated for cranial reconstruction. After cranial defects were created in Sprague-Dawley rats, PCL and PCL-TCP scaffolds were implanted for a period of 1 week and 1 month. Following histology and immunofluorescence staining with the ionized calcium binding adaptor molecule-1 (IBA-1), glial fibrillary acidic protein (GFAP), nestin, and neuronal nuclei (NeuN), results indicated that IBA-1-positive activated microglia were observed across all groups, and declined significantly by 1 month (pless than 0.05). Interestingly, IBA-1-positive microglia were significantly fewer in the PCL-TCP group (pless than 0.05), suggesting a relatively milder inflammatory response. A decrease in the number of GFAP-positive cells among all groups over time (greater than 29%) was also observed. Initially, astrocyte hypertrophy was observed proximal to the TBI site (55% in PCL and PCL-TCP groups, 75% in control groups), but it subsided by 1 month. Proximal to the TBI site, nestin immunoreactivity was intense during week 1, and which reduced by 1 month across all groups. NeuN-positive neurons were shrunken proximal to the TBI site (less than 0.9 mm), 32% smaller in the PCL-TCP group and 27% smaller in the PCL group. Based on above data indicating the comparatively milder, initial inflammatory response of brain tissue to PCL-TCP scaffolds, it is suggested that PCL-TCP scaffolds have notable clinical advantages as compared to PCL scaffolds.
|Nestin expression is lost in ventricular fibroblasts during postnatal development of the rat heart and re-expressed in scar myofibroblasts. |
Pauline C Béguin,Hugues Gosselin,Maya Mamarbachi,Angelino Calderone
Journal of cellular physiology 227 2012
Studies have reported that the intermediate filament protein nestin was expressed in various non-stem/progenitor cells during development, downregulated during postnatal growth and re-expressed following injury. The present study tested the hypothesis that an analogous paradigm was prevalent for ventricular fibroblasts. In the neonatal rat heart, nestin protein levels were significantly higher than the adult heart and the isolation of cardiac cells revealed a selective expression in ventricular fibroblasts. In adult ventricular fibroblasts, nestin protein expression was markedly lower compared to neonatal ventricular fibroblasts. Following ischemic damage to the rat heart, nestin staining was detected in a subpopulation of scar myofibroblasts (37%) and the percentage of immunoreactive cells was greater than adult ventricular fibroblasts (7%) but significantly lower than neonatal ventricular fibroblasts (86%). Moreover, dissimilar rates of (3)H-thymidine uptake were observed among the fibroblast populations and may be related in part to the disparate percentage of nestin(+) cells. To assess the role of nestin in DNA synthesis, neonatal ventricular fibroblasts were infected with a lentivirus containing a shRNAmir directed against the intermediate filament protein. The partial depletion of nestin expression in neonatal ventricular fibroblasts significantly reduced basal DNA synthesis, in the absence of an apoptotic response. Thus, postnatal development of the rat heart was associated with a selective loss of nestin expression in ventricular fibroblasts and subsequent induction in a subpopulation of myofibroblasts following ischemic injury. The re-expression of nestin in scar myofibroblasts may represent an adaptive response to enhance their proliferative rate and accelerate the healing process.
|Chondroitinase and growth factors enhance activation and oligodendrocyte differentiation of endogenous neural precursor cells after spinal cord injury. |
Karimi-Abdolrezaee, S; Schut, D; Wang, J; Fehlings, MG
PloS one 7 e37589 2012
The adult spinal cord harbours a population of multipotent neural precursor cells (NPCs) with the ability to replace oligodendrocytes. However, despite this capacity, proliferation and endogenous remyelination is severely limited after spinal cord injury (SCI). In the post-traumatic microenvironment following SCI, endogenous spinal NPCs mainly differentiate into astrocytes which could contribute to astrogliosis that exacerbate the outcomes of SCI. These findings emphasize a key role for the post-SCI niche in modulating the behaviour of spinal NPCs after SCI. We recently reported that chondroitin sulphate proteoglycans (CSPGs) in the glial scar restrict the outcomes of NPC transplantation in SCI by reducing the survival, migration and integration of engrafted NPCs within the injured spinal cord. These inhibitory effects were attenuated by administration of chondroitinase (ChABC) prior to NPC transplantation. Here, in a rat model of compressive SCI, we show that perturbing CSPGs by ChABC in combination with sustained infusion of growth factors (EGF, bFGF and PDGF-AA) optimize the activation and oligodendroglial differentiation of spinal NPCs after injury. Four days following SCI, we intrathecally delivered ChABC and/or GFs for seven days. We performed BrdU incorporation to label proliferating cells during the treatment period after SCI. This strategy increased the proliferation of spinal NPCs, reduced the generation of new astrocytes and promoted their differentiation along an oligodendroglial lineage, a prerequisite for remyelination. Furthermore, ChABC and GF treatments enhanced the response of non-neural cells by increasing the generation of new vascular endothelial cells and decreasing the number of proliferating macrophages/microglia after SCI. In conclusions, our data strongly suggest that optimization of the behaviour of endogenous spinal NPCs after SCI is critical not only to promote endogenous oligodendrocyte replacement, but also to reverse the otherwise detrimental effects of their activation into astrocytes which could negatively influence the repair process after SCI.
|Btg1 is Required to Maintain the Pool of Stem and Progenitor Cells of the Dentate Gyrus and Subventricular Zone. |
Farioli-Vecchioli, S; Micheli, L; Saraulli, D; Ceccarelli, M; Cannas, S; Scardigli, R; Leonardi, L; Cinà, I; Costanzi, M; Ciotti, MT; Moreira, P; Rouault, JP; Cestari, V; Tirone, F
Frontiers in neuroscience 6 124 2012
Btg1 belongs to a family of cell cycle inhibitory genes. We observed that Btg1 is highly expressed in adult neurogenic niches, i.e., the dentate gyrus and subventricular zone (SVZ). Thus, we generated Btg1 knockout mice to analyze the role of Btg1 in the process of generation of adult new neurons. Ablation of Btg1 causes a transient increase of the proliferating dentate gyrus stem and progenitor cells at post-natal day 7; however, at 2 months of age the number of these proliferating cells, as well as of mature neurons, greatly decreases compared to wild-type controls. Remarkably, adult dentate gyrus stem and progenitor cells of Btg1-null mice exit the cell cycle after completing the S phase, express p53 and p21 at high levels and undergo apoptosis within 5 days. In the SVZ of adult (two-month-old) Btg1-null mice we observed an equivalent decrease, associated to apoptosis, of stem cells, neuroblasts, and neurons; furthermore, neurospheres derived from SVZ stem cells showed an age-dependent decrease of the self-renewal and expansion capacity. We conclude that ablation of Btg1 reduces the pool of dividing adult stem and progenitor cells in the dentate gyrus and SVZ by decreasing their proliferative capacity and inducing apoptosis, probably reflecting impairment of the control of the cell cycle transition from G1 to S phase. As a result, the ability of Btg1-null mice to discriminate among overlapping contextual memories was affected. Btg1 appears, therefore, to be required for maintaining adult stem and progenitor cells quiescence and self-renewal.
|Cell of origin determines tumor phenotype in an oncogenic Ras/p53 knockout transgenic model of high-grade glioma. |
Ghazi, Sabah O, et al.
J. Neuropathol. Exp. Neurol., 71: 729-40 (2012) 2012
Human high-grade gliomas (HGGs) are known for their histologic diversity. To address the role of cell of origin in glioma phenotype, transgenic mice were generated in which oncogenic Ras and p53 deletion were targeted to neural stem/progenitor cells (NSPCs) and mature astrocytes. The hGFAP-Cre/Kras/p53 mice develop multifocal HGGs that vary histopathologically and with respect to the expression of markers associated with NSPCs. One HGG pattern strongly expressed markers of NSPCs and arose near the subventricular zone. Additional nonoverlapping patterns that recapitulate human HGG variants were present simultaneously in the same brain. These neoplastic foci were more often cortical or leptomeningeal based, and the neoplastic cells lacked expression of NSPC markers. To determine whether cell of origin determines tumor phenotype, astrocytes and NSPCs were harvested from neonatal mutant pups. Onorthotopic transplantation, early-passage astrocytes and NSPCs formed tumors that differed in engraftment rates, latency to clinical signs, histopathology, and protein expression. Astrocyte-derivedtumors were more aggressive, had giant-cell histology, and glial fibrillary acidic protein expression. The NSPC-derived tumors retained NSPC markers and showed evidence of differentiation along astrocytic, oligodendroglial, and neuronal lineages. These results indicate that identical tumorigenic stimuli produce markedly different glioma phenotypes, depending on the differentiation status of the transformed cell.
|Transduction of E13 murine neural precursor cells by non-immunogenic recombinant adeno-associated viruses induces major changes in neuronal phenotype. |
K R Nash,Mdel C Cardenas-Aguayo,M J Berg,N Marks
Neuroscience 210 2012
Neural precursor cells (NPCs) provide a cellular model to compare transduction efficiency and toxicity for a series of recombinant adeno-associated viruses (rAAVs). Results led to the choice of rAAV9 as a preferred candidate to transduce NPCs for in vivo transplantation. Importantly, transduction promoted a neuronal phenotype characterized by neurofilament M (NFM) with a concomitant decrease in the embryonic marker, nestin, without significant change in glial fibrillary acidic protein (GFAP). In marked contrast to recent studies for induced pluripotent stem cells (iPSCs), exposure to rAAVs is non-immunogenic and these do not result in genetic abnormalities, thus bolstering the earlier use of NPCs such as those isolated from E13 murine cells for clinical applications. Mechanisms of cellular interactions were explored by treatment with genistein, a pan-specific inhibitor of protein receptor tyrosine kinases (PRTKs) that blocked the transduction and differentiation, thus implying a central role for this pathway for inducing infectivity along with observed phenotypic changes and as a method for drug design. Implantation of transduced NPCs into adult mouse hippocampus survived up to 28 days producing a time line for targeting or migration to dentate gyrus and CA3-1 compatible with future clinical applications. Furthermore, a majority showed commitment to highly differentiated neuronal phenotypes. Lack of toxicity and immune response of rAAVs plus ability for expansion of NPCs in vitro auger well for their isolation and suggest potential therapeutic applications in repair or replacement of diseased neurons in neurodegeneration.
|Ablation of vacuole protein sorting 18 (Vps18) gene leads to neurodegeneration and impaired neuronal migration by disrupting multiple vesicle transport pathways to lysosomes. |
Peng, C; Ye, J; Yan, S; Kong, S; Shen, Y; Li, C; Li, Q; Zheng, Y; Deng, K; Xu, T; Tao, W
The Journal of biological chemistry 287 32861-73 2012
Intracellular vesicle transport pathways are critical for neuronal survival and central nervous system development. The Vps-C complex regulates multiple vesicle transport pathways to the lysosome in lower organisms. However, little is known regarding its physiological function in mammals. We deleted Vps18, a central member of Vps-C core complex, in neural cells by generating Vps18(F/F); Nestin-Cre mice (Vps18 conditional knock-out mice). These mice displayed severe neurodegeneration and neuronal migration defects. Mechanistic studies revealed that Vps18 deficiency caused neurodegeneration by blocking multiple vesicle transport pathways to the lysosome, including autophagy, endocytosis, and biosynthetic pathways. Our study also showed that ablation of Vps18 resulted in up-regulation of β1 integrin in mouse brain probably due to lysosome dysfunction but had no effects on the reelin pathway, expression of N-cadherin, or activation of JNK, which are implicated in the regulation of neuronal migration. Finally, we demonstrated that knocking down β1 integrin partially rescued the migration defects, suggesting that Vps18 deficiency-mediated up-regulation of β1 integrin may contribute to the defect of neuronal migration in the Vps18-deficient brain. Our results demonstrate important roles of Vps18 in neuron survival and migration, which are disrupted in multiple neural disorders.
|Activation of specific interneurons improves V1 feature selectivity and visual perception. |
Seung-Hee Lee,Alex C Kwan,Siyu Zhang,Victoria Phoumthipphavong,John G Flannery,Sotiris C Masmanidis,Hiroki Taniguchi,Z Josh Huang,Feng Zhang,Edward S Boyden,Karl Deisseroth,Yang Dan
Nature 488 2012
Inhibitory interneurons are essential components of the neural circuits underlying various brain functions. In the neocortex, a large diversity of GABA (γ-aminobutyric acid) interneurons has been identified on the basis of their morphology, molecular markers, biophysical properties and innervation pattern. However, how the activity of each subtype of interneurons contributes to sensory processing remains unclear. Here we show that optogenetic activation of parvalbumin-positive (PV+) interneurons in the mouse primary visual cortex (V1) sharpens neuronal feature selectivity and improves perceptual discrimination. Using multichannel recording with silicon probes and channelrhodopsin-2 (ChR2)-mediated optical activation, we found that increased spiking of PV+ interneurons markedly sharpened orientation tuning and enhanced direction selectivity of nearby neurons. These effects were caused by the activation of inhibitory neurons rather than a decreased spiking of excitatory neurons, as archaerhodopsin-3 (Arch)-mediated optical silencing of calcium/calmodulin-dependent protein kinase IIα (CAMKIIα)-positive excitatory neurons caused no significant change in V1 stimulus selectivity. Moreover, the improved selectivity specifically required PV+ neuron activation, as activating somatostatin or vasointestinal peptide interneurons had no significant effect. Notably, PV+ neuron activation in awake mice caused a significant improvement in their orientation discrimination, mirroring the sharpened V1 orientation tuning. Together, these results provide the first demonstration that visual coding and perception can be improved by increased spiking of a specific subtype of cortical inhibitory interneurons.
|Biciliated ependymal cell proliferation contributes to spinal cord growth. |
Clara Alfaro-Cervello,Mario Soriano-Navarro,Zaman Mirzadeh,Arturo Alvarez-Buylla,Jose Manuel Garcia-Verdugo
The Journal of comparative neurology 520 2012
Two neurogenic regions have been described in the adult brain, the lateral ventricle subventricular zone and the dentate gyrus subgranular zone. It has been suggested that neural stem cells also line the central canal of the adult spinal cord. Using transmission and scanning electron microscopy and immunostaining, we describe here the organization and cell types of the central canal epithelium in adult mice. The identity of dividing cells was determined by 3D ultrastructural reconstructions of [(3) H]thymidine-labeled cells and confocal analysis of bromodeoxyuridine labeling. The most common cell type lining the central canal had two long motile (9+2) cilia and was vimentin+, CD24+, FoxJ1+, Sox2+, and CD133+, but nestin- and glial fibrillary acidic protein (GFAP)-. These biciliated ependymal cells of the central canal (Ecc) resembled E2 cells of the lateral ventricles, but their basal bodies were different from those of E2 or E1 cells. Interestingly, we frequently found Ecc cells with two nuclei and four cilia, suggesting they are formed by incomplete cytokinesis or cell fusion. GFAP+ astrocytes with a single cilium and an orthogonally oriented centriole were also observed. The majority of dividing cells corresponded to biciliated Ecc cells. Central canal proliferation was most common during the active period of spinal cord growth. Pairs of labeled Ecc cells were observed within the central canal in adult mice 2.5 weeks post labeling. Our work suggests that the vast majority of postnatal dividing cells in the central canal are Ecc cells and their proliferation is associated with the growth of the spinal cord. J. Comp. Neurol. 520:3528-3552, 2012. © 2012 Wiley Periodicals, Inc.
|Modeling neural differentiation on micropatterned substrates coated with neural matrix components. |
García-Parra, P; Cavaliere, F; Maroto, M; Bilbao, L; Obieta, I; López de Munain, A; Alava, JI; Izeta, A
Frontiers in cellular neuroscience 6 10 2012
Topographical and biochemical characteristics of the substrate are critical for neuronal differentiation including axonal outgrowth and regeneration of neural circuits in vivo. Contact stimuli and signaling molecules allow neurons to develop and stabilize synaptic contacts. Here we present the development, characterization and functional validation of a new polymeric support able to induce neuronal differentiation in both PC12 cell line and adult primary skin-derived precursor cells (SKPs) in vitro. By combining a photolithographic technique with use of neural extracellular matrix (ECM) as a substrate, a biocompatible and efficient microenvironment for neuronal differentiation was developed.
|A Role for the Cannabinoid 1 Receptor in Neuronal Differentiation of Adult Spinal Cord Progenitors in vitro is Revealed through Pharmacological Inhibition and Genetic Deletion. |
Sideris, A; Bekker, T; Chan, WS; Montoya-Gacharna, JV; Blanck, TJ; Recio-Pinto, E
Frontiers in neuroscience 6 4 2012
In contrast to the adult brain, the adult spinal cord is a non-neurogenic environment. Understanding how to manipulate the spinal cord environment to promote the formation of new neurons is an attractive therapeutic strategy for spinal cord injury and disease. The cannabinoid 1 receptor (CB1R) has been implicated as a modulator of neural progenitor cell proliferation and fate specification in the brain; however, no evidence exists for modulation of adult spinal cord progenitor cells. Using adult rat spinal cord primary cultures, we demonstrated that CB1R antagonism with AM251 significantly decreased the number of Nestin(+) cells, and increased the number of βIII tubulin(+) and DCX(+) cells, indicative of neuronal differentiation. AM251's effect was blocked by co-application of the CB1R agonists, WIN 55, 212-2, or ACEA. Consistent with our hypothesis, cultures, and spinal cord slices derived from CB1R knock-out (CB1-/-) mice had significantly higher levels of DCX(+) cells compared to those derived from wild type (CB1+/+) mice, indicative of enhanced neuronal differentiation in CB1-/- spinal cords. Moreover, AM251 promoted neuronal differentiation in CB1+/+, but not in CB1-/- cultures. Since CB1R modulates synaptic transmission, and synaptic transmission has been shown to influence progenitor cell fate, we evaluated whether AM251-induced neuronal differentiation was affected by chronic inactivity. Either the presence of the voltage-dependent sodium channel blocker tetrodotoxin (TTX), or the removal of mature neurons, inhibited the AM251-induced increase in DCX(+) cells. In summary, antagonism or absence of CB1R promotes neuronal differentiation in adult spinal cords, and this action appears to require TTX-sensitive neuronal activity. Our data suggest that the previously detected elevated levels of endocannabinoids in the injured adult spinal cord could contribute to the non-neurogenic environment and CB1R antagonists could potentially be used to enhance replacement of damaged neurons.
|The novel function of Oct3/4 in mouse tooth development. |
Eizo Nakagawa,Li Zhang,Eun-Jung Kim,Jeong-Oh Shin,Sung-Won Cho,Hayato Ohshima,Han-Sung Jung
Histochemistry and cell biology 137 2012
Octamer-binding factor 3/4 (Oct3/4) is one of the key regulators maintaining the pluripotency and self-renewal in embryonic stem cells and is involved in the developmental events. However, the functional significance of Oct3/4 remains to be clarified during tooth morphogenesis. This study aimed to examine the functional role of Oct3/4 in mouse. During tooth morphogenesis (E11-E16.5), Oct3/4-positive cells, detected by nuclear immunoreaction, increased in number, and subsequently, their immunoreaction shifted from the nucleus to the cytoplasm at the stage of cell differentiation (E18.5). Quantitative real-time PCR clearly demonstrated the relationship between isoforms of Oct3/4 and the in vivo cellular localization of Oct3/4, suggesting that the Oct3/4 expressed in nucleus was Oct3/4A, whereas that expressed in the cytoplasm was Oct3/4B. RNAi knockdown of Oct3/4 induced apoptosis and arrested tooth morphogenesis. Our results suggest that (1) the increased number of Oct3/4-positive cells with nuclear immunoreaction correlate with active cell proliferation during tooth morphogenesis and (2) the shift of Oct3/4 from the nucleus to the cytoplasm plays a crucial role in cell differentiation.
|Plasma membrane-associated glycohydrolases along differentiation of murine neural stem cells. |
Massimo Aureli,Angela Gritti,Rosaria Bassi,Nicoletta Loberto,Alessandra Ricca,Vanna Chigorno,Alessandro Prinetti,Sandro Sonnino
Neurochemical research 37 2012
The activities of plasma membrane associated sialidase Neu3, total β-glucosidase, CBE-sensitive β-glucosidase, non-lysosomal β-glucosyl ceramidase GBA2, β-galactosidase, β-hexosaminidase and sphingomyelinase were determined at three different stages of differentiation of murine neural stem cell cultures, corresponding to precursors, commited progenitors, and differentiated cells. Cell immunostaining for specific markers of the differentiation process, performed after 7 days in culture in presence of differentiating agents, clearly showed the presence of oligodendrocytes, astrocytes and neurons. Glial cells were the most abundant. Sialidase Neu3 after a decrease from progenitors to precursors, showed an increase parallel to the differentiation process. All the other glycosidases increased their activity along differentiation. The activity of CBE-sensitive β-glucosidase and GBA2 were very similar at the precursor stage, but CBE-sensitive β-glucosidase increased 7 times while GBA2 only two in the differentiated cells. In addition, we analysed also sphingomyelinase as enzyme specifically associated to sphingolipids. The activity of this enzyme increased from precursors to differentiated cells.
|Mcl-1 regulates the survival of adult neural precursor cells. |
Craig D Malone,S M Mahmudul Hasan,R Brian Roome,Jieying Xiong,Michael Furlong,Joseph T Opferman,Jacqueline L Vanderluit
Molecular and cellular neurosciences 49 2012
Since the discovery of neural precursor cells (NPCs) in the adult mammalian brain, there has been a lot of excitement surrounding the potential for regeneration in the adult brain. For instance, many studies have shown that a significant number of NPCs will migrate to a site of injury and differentiate into all of the neural lineages. However, one of the main challenges affecting endogenous neural regeneration is that many of the NPCs that migrate to the injury site ultimately undergo apoptosis. Therefore, we sought to determine whether myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 protein, would promote the survival of adult NPCs by impeding apoptosis. To do this, we first confirmed that Mcl-1 is endogenously expressed within the adult NPC population using BrdU labeling assays. Next, we conditionally deleted Mcl-1 in adult NPCs using cre/lox technology and expressed Cre from the NPC-specific promoter Nestin. In vitro, cells that had Mcl-1 conditionally deleted had a 2-fold increase in apoptosis when compared to controls. In vivo, we used electroporation to conditionally delete Mcl-1 in adult NPCs and assessed apoptosis at 72h. after electroporation. As in our in vitro results, there was a 2-fold increase in apoptosis when Mcl-1 was conditionally deleted. Finally, we found that Mcl-1 over-expression reduced the endogenous rate of adult NPC apoptosis 2-fold in vitro. Collectively, these results demonstrate that Mcl-1 is crucial for the survival of adult NPCs and may be a promising target for future neural regeneration therapies.
|The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling. |
Yuko Ishikawa,Hiroko Ida-Yonemochi,Kuniko Nakakura-Ohshima,Hayato Ohshima
Cell and tissue research 348 2012
Human dental pulp contains adult stem cells. Our recent study demonstrated the localization of putative dental pulp stem/progenitor cells in the rat developing molar by chasing 5-bromo-2'-deoxyuridine (BrdU)-labeling. However, there are no available data on the localization of putative dental pulp stem/progenitor cells in the mouse molar. This study focuses on the mapping of putative dental pulp stem/progenitor cells in addition to the relationship between cell proliferation and differentiation in the developing molar using BrdU-labeling. Numerous proliferating cells appeared in the tooth germ and the most active cell proliferation in the mesenchymal cells occurred in the prenatal stages, especially on embryonic Day 15 (E15). Cell proliferation in the pulp tissue dramatically decreased in number by postnatal Day 3 (P3) when nestin-positive odontoblasts were arranged in the cusped areas and disappeared after postnatal Week 1 (P1W). Root dental papilla included numerous proliferating cells during P5 to P2W. Three to four intraperitoneal injections of BrdU were given to pregnant ICR mice and revealed slow-cycling long-term label-retaining cells (LRCs) in the mature tissues of postnatal animals. Numerous dense LRCs postnatally decreased in number and reached a plateau after P1W when they mainly resided in the center of the dental pulp, associating with blood vessels. Furthermore, numerous dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 and CD146. Thus, dense LRCs in mature pulp tissues were believed to be dental pulp stem/progenitor cells harboring in the perivascular niche surrounding the endothelium.
|Neural stem cells exposed to BrdU lose their global DNA methylation and undergo astrocytic differentiation. |
Schneider, L; d'Adda di Fagagna, F
Nucleic acids research 40 5332-42 2012
Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a halogenated nucleotide of low toxicity commonly used to monitor DNA replication. It is considered a valuable tool for in vitro and in vivo studies, including the detection of the small population of neural stem cells (NSC) in the mammalian brain. Here, we show that NSC grown in self-renewing conditions in vitro, when exposed to BrdU, lose the expression of stem cell markers like Nestin, Sox2 and Pax6 and undergo glial differentiation, strongly up-regulating the astrocytic marker GFAP. The onset of GFAP expression in BrdU exposed NSC was paralleled by a reduced expression of key DNA methyltransferases (DNMT) and a rapid loss of global DNA CpG methylation, as we determined by our specially developed analytic assay. Remarkably, a known DNA demethylating compound, 5-aza-2'-deoxycytidine (Decitabine), had similar effect on demethylation and differentiation of NSC. Since our key findings apply also to NSC derived from murine forebrain, our observations strongly suggest more caution in BrdU uses in stem cells research. We also propose that BrdU and its related substances may also open new opportunities for differentiation therapy in oncology.
|Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics. |
Huang, Xi, et al.
Genes Dev., 26: 1780-96 (2012) 2012
Medulloblastoma (MB) is the most common pediatric CNS malignancy. We identify EAG2 as an overexpressed potassium channel in MBs across different molecular and histological subgroups. EAG2 knockdown not only impairs MB cell growth in vitro, but also reduces tumor burden in vivo and enhances survival in xenograft studies. Mechanistically, we demonstrate that EAG2 protein is confined intracellularly during interphase but is enriched in the plasma membrane during late G2 phase and mitosis. Disruption of EAG2 expression results in G2 arrest and mitotic catastrophe associated with failure of premitotic cytoplasmic condensation. While the tumor suppression function of EAG2 knockdown is independent of p53 activation, DNA damage checkpoint activation, or changes in the AKT pathway, this defective cell volume control is specifically associated with hyperactivation of the p38 MAPK pathway. Inhibition of the p38 pathway significantly rescues the growth defect and G2 arrest. Strikingly, ectopic membrane expression of EAG2 in cells at interphase results in cell volume reduction and mitotic-like morphology. Our study establishes the functional significance of EAG2 in promoting MB tumor progression via regulating cell volume dynamics, the perturbation of which activates the tumor suppressor p38 MAPK pathway, and provides clinical relevance for targeting this ion channel in human MBs.
|Endogenous repair by the activation of cell survival signalling cascades during the early stages of rat Parkinsonism. |
Lui, NP; Chen, LW; Yung, WH; Chan, YS; Yung, KK
PloS one 7 e51294 2012
Here we report a previously unknown self repair mechanism during extremely early stages of rat Parkinsonism. Two important cell survival signaling cascades, Phosphatidylinositol-3 kinases (PI3K)/Akt pathway and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, could be responsible for this potential endogenous rescue system. In the 6-hydroxydopamine-lesioned rat, the phosphorylated p44/42 MAPK and its downstream target, the phosphorylated Bad at Ser 112, were up-regulated at post-lesion day 3 and lasted for a couple of weeks. Although the change in the phosphorylated Akt kinase was negligible throughout the studied period, its downstream target, the phosphorylated Bad at 136, was increased from post-lesion day 3 to post-lesion day 14. In the mean time, nestin-positive reactive astrocytes with low levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) appeared at post-lesion day 3 in 6-hydroxydopamine-lesioned rat. BDNF was expressed in both striatum and substantia nigra whereas GDNF was displayed in striatum only. At post-lesion day 14, nestin, BDNF and GDNF expressions were diminished. These neurotrophic factors were believed to initiate the above anti-apoptotic signal transduction cascades as we could see that their expression patterns were similar. The data strongly suggest that there is an endogenous repair effort by evoking the cell survival signaling and possibly via the releases of BDNF and GDNF from nestin-immunoreactive reactive astrocytes. ERK/MAPK pathway was proposed to be the key endogenous neuroprotective mechanisms, particularly in early stages of rat Parkinsonism. However, the self repair effort is only functional within an extremely short time window immediately after onset.
|Cellular host responses to gliomas. |
Najbauer, J; Huszthy, PC; Barish, ME; Garcia, E; Metz, MZ; Myers, SM; Gutova, M; Frank, RT; Miletic, H; Kendall, SE; Glackin, CA; Bjerkvig, R; Aboody, KS
PloS one 7 e35150 2012
Glioblastoma multiforme (GBM) is the most aggressive type of malignant primary brain tumors in adults. Molecular and genetic analysis has advanced our understanding of glioma biology, however mapping the cellular composition of the tumor microenvironment is crucial for understanding the pathology of this dreaded brain cancer. In this study we identified major cell populations attracted by glioma using orthotopic rodent models of human glioma xenografts. Marker-specific, anatomical and morphological analyses revealed a robust influx of host cells into the main tumor bed and tumor satellites.Human glioma cell lines and glioma spheroid orthotopic implants were used in rodents. In both models, the xenografts recruited large numbers of host nestin-expressing cells, which formed a 'network' with glioma. The host nestin-expressing cells appeared to originate in the subventricular zone ipsilateral to the tumor, and were clearly distinguishable from pericytes that expressed smooth muscle actin. These distinct cell populations established close physical contact in a 'pair-wise' manner and migrated together to the deeper layers of tumor satellites and gave rise to tumor vasculature. The GBM biopsy xenografts displayed two different phenotypes: (a) low-generation tumors (first in vivo passage in rats) were highly invasive and non-angiogenic, and host nestin-positive cells that infiltrated into these tumors displayed astrocytic or elongated bipolar morphology; (b) high-generation xenografts (fifth passage) had pronounced cellularity, were angiogenic with 'glomerulus-like' microvascular proliferations that contained host nestin-positive cells. Stromal cell-derived factor-1 and its receptor CXCR4 were highly expressed in and around glioma xenografts, suggesting their role in glioma progression and invasion.Our data demonstrate a robust migration of nestin-expressing host cells to glioma, which together with pericytes give rise to tumor vasculature. Mapping the cellular composition of glioma microenvironment and deciphering the complex 'crosstalk' between tumor and host may ultimately aid the development of novel anti-glioma therapies.
|Identification of a novel set of genes reflecting different in vivo invasive patterns of human GBM cells. |
Monticone, M; Daga, A; Candiani, S; Romeo, F; Mirisola, V; Viaggi, S; Melloni, I; Pedemonte, S; Zona, G; Giaretti, W; Pfeffer, U; Castagnola, P
BMC cancer 12 358 2012
Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting.We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type).We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis.Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion.This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting.Massimiliano Monticone and Antonio Daga contributed equally to this work.
|ALKBH1 is a histone H2A dioxygenase involved in neural differentiation. |
Ougland, R; Lando, D; Jonson, I; Dahl, JA; Moen, MN; Nordstrand, LM; Rognes, T; Lee, JT; Klungland, A; Kouzarides, T; Larsen, E
Stem cells (Dayton, Ohio) 30 2672-82 2012
AlkB homolog 1 (ALKBH1) is one of nine members of the family of mammalian AlkB homologs. Most Alkbh1(-/-) mice die during embryonic development, and survivors are characterized by defects in tissues originating from the ectodermal lineage. In this study, we show that deletion of Alkbh1 prolonged the expression of pluripotency markers in embryonic stem cells and delayed the induction of genes involved in early differentiation. In vitro differentiation to neural progenitor cells (NPCs) displayed an increased rate of apoptosis in the Alkbh1(-/-) NPCs when compared with wild-type cells. Whole-genome expression analysis and chromatin immunoprecipitation revealed that ALKBH1 regulates both directly and indirectly, a subset of genes required for neural development. Furthermore, our in vitro enzyme activity assays demonstrate that ALKBH1 is a histone dioxygenase that acts specifically on histone H2A. Mass spectrometric analysis demonstrated that histone H2A from Alkbh1(-/-) mice are improperly methylated. Our results suggest that ALKBH1 is involved in neural development by modifying the methylation status of histone H2A.
|Querkopf is a key marker of self-renewal and multipotency of adult neural stem cells. |
Sheikh, BN; Dixon, MP; Thomas, T; Voss, AK
Journal of cell science 125 295-309 2012
Adult neural stem cells (NSCs) reside in the subventricular zone (SVZ) and produce neurons throughout life. Although their regenerative potential has kindled much interest, few factors regulating NSCs in vivo are known. Among these is the histone acetyltransferase querkopf (QKF, also known as MYST4, MORF, KAT6B), which is strongly expressed in a small subset of cells in the neurogenic subventricular zone. However, the relationship between Qkf gene expression and the hierarchical levels within the neurogenic lineage is currently unknown. We show here that the 10% of SVZ cells with the highest Qkf expression possess the defining NSC characteristics of multipotency and self-renewal and express markers previously shown to enrich for NSCs. A fraction of cells expressing Qkf at medium to high levels is enriched for multipotent progenitor cells with limited self-renewal, followed by a population containing migrating neuroblasts. Cells low in Qkf promoter activity are predominantly ependymal cells. In addition, we show that mice deficient for Bmi1, a central regulator of NSC self-renewal, show an age-dependent decrease in the strongest Qkf-expressing cell population in the SVZ. Our results show a strong relationship between Qkf promoter activity and stem cell characteristics, and a progressive decrease in Qkf gene activity as lineage commitment and differentiation proceed in vivo.
|MiR-125b orchestrates cell proliferation, differentiation and migration in neural stem/progenitor cells by targeting Nestin. |
Cui, Y; Xiao, Z; Han, J; Sun, J; Ding, W; Zhao, Y; Chen, B; Li, X; Dai, J
BMC neuroscience 13 116 2012
The emerging concept is that microRNAs (miRNAs) play a central role in controlling stem cell self-renewal and fate determination by regulating the expression of stem cell regulators. miR-125b, one of neuronal miRNAs, recently was found to be necessary for neural differentiation of neural stem/progenitor cells (NS/PCs). However, the other specific biological role of miR-125b in NS/PCs is little known. We used rat NS/PCs as a model system to study the role of miR-125b in governing the behavior of NS/PCs.We report here the transfection of exogenous miR-125b inhibited proliferation of NS/PCs but promoted differentiation and migration. Whereas anti-miR-125b had the opposite effect. Similar results were observed when Nestin was knocked down by siRNA. Subsequently, we demonstrated that Nestin was a direct functional target of miR-125b. MiR-125b downregulates the expression of luciferase through Nestin 3'untranslated region (3'-UTR), and the regulation was abolished by mutations in the miR-125b binding site. MiR-125b targeted the 3'-UTR of Nestin and reduced the abundance of Nestin at both mRNA and protein levels.The results provided new insight into the function by which miR-125b modulates NS/PCs proliferation, differentiation and migration. The data also indicated the regulatory role of miR-125b in NS/PCs might through the suppression of Nestin expression.
|RP58/ZNF238 directly modulates proneurogenic gene levels and is required for neuronal differentiation and brain expansion. |
Xiang, C; Baubet, V; Pal, S; Holderbaum, L; Tatard, V; Jiang, P; Davuluri, RV; Dahmane, N
Cell death and differentiation 19 692-702 2012
Although neurogenic pathways have been described in the developing neocortex, less is known about mechanisms ensuring correct neuronal differentiation thus also preventing tumor growth. We have shown that RP58 (aka zfp238 or znf238) is highly expressed in differentiating neurons, that its expression is lost or diminished in brain tumors, and that its reintroduction blocks their proliferation. Mice with loss of RP58 die at birth with neocortical defects. Using a novel conditional RP58 allele here we show that its CNS-specific loss yields a novel postnatal phenotype: microencephaly, agenesis of the corpus callosum and cerebellar hypoplasia that resembles the chr1qter deletion microcephaly syndrome in human. RP58 mutant brains maintain precursor pools but have reduced neuronal and increased glial differentiation. Well-timed downregulation of pax6, ngn2 and neuroD1 depends on RP58 mediated transcriptional repression, ngn2 and neuroD1 being direct targets. Thus, RP58 may act to favor neuronal differentiation and brain growth by coherently repressing multiple proneurogenic genes in a timely manner.
|Targeted disruption in mice of a neural stem cell-maintaining, KRAB-Zn finger-encoding gene that has rapidly evolved in the human lineage. |
Chien, HC; Wang, HY; Su, YN; Lai, KY; Lu, LC; Chen, PC; Tsai, SF; Wu, CI; Hsieh, WS; Shen, CK
PloS one 7 e47481 2012
Understanding the genetic basis of the physical and behavioral traits that separate humans from other primates is a challenging but intriguing topic. The adaptive functions of the expansion and/or reduction in human brain size have long been explored. From a brain transcriptome project we have identified a KRAB-Zn finger protein-encoding gene (M003-A06) that has rapidly evolved since the human-chimpanzee separation. Quantitative RT-PCR analysis of different human tissues indicates that M003-A06 expression is enriched in the human fetal brain in addition to the fetal heart. Furthermore, analysis with use of immunofluorescence staining, neurosphere culturing and Western blotting indicates that the mouse ortholog of M003-A06, Zfp568, is expressed mainly in the embryonic stem (ES) cells and fetal as well as adult neural stem cells (NSCs). Conditional gene knockout experiments in mice demonstrates that Zfp568 is both an NSC maintaining- and a brain size-regulating gene. Significantly, molecular genetic analyses show that human M003-A06 consists of 2 equilibrated allelic types, H and C, one of which (H) is human-specific. Combined contemporary genotyping and database mining have revealed interesting genetic associations between the different genotypes of M003-A06 and the human head sizes. We propose that M003-A06 is likely one of the genes contributing to the uniqueness of the human brain in comparison to other higher primates.
|Prenatal cocaine exposure alters progenitor cell markers in the subventricular zone of the adult rat brain. |
Patel, DA; Booze, RM; Mactutus, CF
International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience 30 1-9 2012
Long-term consequences of early developmental exposure to drugs of abuse may have deleterious effects on the proliferative plasticity of the brain. The purpose of this study was to examine the long-term effects of prenatal exposure to cocaine, using the IV route of administration and doses that mimic the peak arterial levels of cocaine use in humans, on the proliferative cell types of the subventricular zones (SVZ) in the adult (180 days-old) rat brain. Employing immunocytochemistry, the expression of GFAP(+) (type B cells) and nestin(+)(GFAP(-)) (type C and A cells) staining was quantified in the subcallosal area of the SVZ. GFAP(+) expression was significantly different between the prenatal cocaine treated group and the vehicle (saline) control group. The prenatal cocaine treated group possessed significantly lower GFAP(+) expression relative to the vehicle control group, suggesting that prenatal cocaine exposure significantly reduced the expression of type B neural stem cells of the SVZ. In addition, there was a significant sex difference in nestin(+) expression with females showing approximately 8-13% higher nestin(+) expression compared to the males. More importantly, a significant prenatal treatment condition (prenatal cocaine, control) by sex interaction in nestin(+) expression was confirmed, indicating different effects of cocaine based on sex of the animal. Specifically, prenatal cocaine exposure eliminated the basal difference between the sexes. Collectively, the present findings suggest that prenatal exposure to cocaine, when delivered via a protocol designed to capture prominent features of recreational usage, can selectively alter the major proliferative cell types in the subcallosal area of the SVZ in an adult rat brain, and does so differently for males and females.
|Proximal tubular injury and rapid formation of atubular glomeruli in mice with unilateral ureteral obstruction: a new look at an old model. |
Forbes, MS; Thornhill, BA; Chevalier, RL
American journal of physiology. Renal physiology 301 F110-7 2011
Unilateral ureteral obstruction (UUO), employed extensively as a model of progressive renal interstitial fibrosis, results in rapid parenchymal deterioration. Atubular glomeruli are formed in many renal disorders, but their identification has been limited by labor-intensive available techniques. The formation of atubular glomeruli was therefore investigated in adult male mice subjected to complete UUO under general anesthesia. In this species, the urinary pole of Bowman's capsule is normally lined by tall parietal epithelial cells similar to those of the proximal tubule, and both avidly bind Lotus tetragonolobus lectin. Following UUO, these cells became flattened, lost their affinity for Lotus lectin, and no longer generated superoxide (revealed by nitroblue tetrazolium infusion). Based on Lotus lectin staining, stereological measurements, and serial section analysis, over 80% of glomeruli underwent marked transformation after 14 days of UUO. The glomerulotubular junction became stenotic and atrophic due to cell death by apoptosis and autophagy, with concomitant remodeling of Bowman's capsule to form atubular glomeruli. In this degenerative process, transformed epithelial cells sealing the urinary pole expressed α-smooth muscle actin, vimentin, and nestin. Although atubular glomeruli remained perfused, renin immunostaining was markedly increased along afferent arterioles, and associated maculae densae disappeared. Numerous progressive kidney disorders, including diabetic nephropathy, are characterized by the formation of atubular glomeruli. The rapidity with which glomerulotubular junctions degenerate, coupled with Lotus lectin as a marker of glomerular integrity, points to new investigative uses for the model of murine UUO focusing on mechanisms of epithelial cell injury and remodeling in addition to fibrogenesis.
|Differentiation of induced pluripotent stem cells into functional oligodendrocytes. |
Czepiel M, Balasubramaniyan V, Schaafsma W, Stancic M, Mikkers H, Huisman C, Boddeke E, Copray S
Glia 59 882-92 2011
The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells. © 2011 Wiley-Liss, Inc. Copyright © 2011 Wiley-Liss, Inc.
|Pattern of invasion of the embryonic mouse spinal cord by microglial cells at the time of the onset of functional neuronal networks. |
C Rigato,R Buckinx,H Le-Corronc,J M Rigo,P Legendre
Glia 59 2011
Microglial cells invade the central nervous system during embryonic development, but their developmental functional roles in vivo remain largely unknown. Accordingly, their invasion pattern during early embryonic development is still poorly understood. To address this issue, we analyzed the initial developmental pattern of microglial cell invasion in the spinal cord of CX3CR1-eGFP mouse embryos using immunohistochemistry. Microglial cells began to invade the mouse embryonic spinal cord at a developmental period corresponding to the onset of spontaneous electrical activity and of synaptogenesis. Microglial cells reached the spinal cord through the peripheral vasculature and began to invade the parenchyma at 11.5 days of embryonic age (E11.5). Remarkably, at E12.5, activated microglial cells aggregated in the dorsolateral region close to terminals of dying dorsal root ganglia neurons. At E13.5, microglial cells in the ventral marginal zone interacted with radial glial cells, whereas ramified microglial cells within the parenchyma interacted with growing capillaries. At this age, activated microglial cells (Mac-2 staining) also accumulated within the lateral motor columns at the onset of the developmental cell death of motoneurons. This cell aggregation was still observed at E14.5, but microglial cells no longer expressed Mac-2. At E15.5, microglial cells were randomly distributed within the parenchyma. Our results provide the essential basis for further studies on the role of microglial cells in the early development of spinal cord neuronal networks in vivo.
|The temporal and spatial expression pattern of the LGI1 epilepsy predisposition gene during mouse embryonic cranial development. |
Silva, J; Wang, G; Cowell, JK
BMC neuroscience 12 43 2011
Mutations in the LGI1 gene predispose to a rare, hereditary form of temporal epilepsy. Currently, little is known about the temporal and spatial expression pattern of Lgi1 during normal embryogenesis and so to define this more clearly we used a transgenic mouse line that expresses GFP under the control of Lgi1 cis-regulatory elements.During embryonic brain growth, high levels of Lgi1 expression were found in the surface ectoderm, the neuroepithelium, mesenchymal connective tissue, hippocampus, and sensory organs, such as eye, tongue, and the olfactory bulb. Lgi1 was also found in the cranial nerve nuclei and ganglia, such as vestibular, trigeminal, and dorsal ganglia. Expression of Lgi1 followed an orchestrated pattern during mouse development becoming more subdued in areas of the neocortex of the mid- and hind-brain in early postnatal animals, although high expression levels were retained in the choroid plexus and hippocampus. In late postnatal stages, Lgi1 expression continued to be detected in many areas in the brain including, hippocampus, paraventricular thalamic nuclei, inferior colliculus, and the cerebral aqueduct. We also showed that Lgi1-expressing cells co-express nestin, DCX, and beta-III tubulin suggesting that Lgi1-expressing cells are migratory neuroblasts.These observations imply that Lgi1 may have a role in establishing normal brain architecture and neuronal functions during brain development suggesting that it may be involved in neurogenesis and neuronal plasticity, which become more specifically defined in the adult animal.Artículo Texto completo
|Functional neural stem cell isolation from brains of adult mutant SOD1 (SOD1(G93A)) transgenic amyotrophic lateral sclerosis (ALS) mice. |
Lee JC, Jin Y, Jin J, Kang BG, Nam DH, Joo KM, Cha CI
Neurol Res 33 33-7. Epub 2010 Aug 31. 2011
OBJECTIVES: The aim of present study is to investigate more functional neural stem cells (NSCs) could be isolated from brains with amyotrophic lateral sclerosis (ALS) and expanded in vitro, based on previous reports demonstrating de novo neurogenesis is enhanced to replace degenerating neural tissue.METHODS: Thirteen- or eighteen-week-old mutant human Cu/Zn superoxide dismutase (SOD1(G93A)) transgenic ALS and wild-type SOD1 transgenic control mice were utilized. Changes in numbers of NSCs in the dentate gyrus were analyzed by immunohistochemistry against nestin and CD133. NSCs were primarily cultured from hippocampus of ALS or control mice. Expression of NSC markers, in vitro expansion capacity, and differentiating potential were compared.RESULTS: Hippocampus of 13-week-old pre-symptomatic ALS mice harbor more cells that can be propagated for more than 12 passages in vitro, compared with same age control mice. Primarily-cultured cells formed neurospheres in the NSC culture medium, expressed NSC markers, and differentiated into cells with differentiated neural cell characteristics in the differentiation condition confirming that they are NSCs. In contrast, long-term expansible NSCs could not be derived from brains of 18-week-old symptomatic ALS mice with the same experimental techniques, although they had comparable nestin-immunoreactive cells in the dentate gyrus.DISCUSSION: These results would suggest that increased neuroregeneration in early phase of ALS could be translated to regenerative approaches; however, long-term exposure to ALS microenvironments could abolish functional capacities of NSCs.
|Differentiation of nestin-positive cells derived from bone marrow into pancreatic endocrine and ductal cells in vitro. |
Milanesi, A; Lee, JW; Xu, Q; Perin, L; Yu, JS
The Journal of endocrinology 209 193-201 2011
Promising results of pancreatic islet transplantation to treat type 1 diabetes mellitus, combined with severe shortage of donor pancreata, have spurred efforts to generate pancreatic islet-like cells and insulin-producing β-cells from various progenitor populations. In this study, we show for the first time that multipotent nestin-positive stem cells selected from rat bone marrow can be differentiated into pancreatic ductal and insulin-producing β-cells in vitro. We report an effective multistep protocol in a serum-free system, which could efficiently induce β-cell differentiation from multipotent nestin-positive bone marrow stem cells. To enhance the induction and differentiation toward pancreatic lineage we used trichostatin A, an important regulator of chromatin remodeling, and 5-aza 2' deoxycytidine, an inhibitor of DNA methylase. All-trans retinoic acid was then utilized to promote pancreatic differentiation. We sequentially induced important transcription factor genes, such as Pdx1, Ngn3, and Pax6, following the in vivo development timeline of the pancreas in rats. Furthermore, in the final stage with the presence of nicotinamide, the induced cells expressed islet and ductal specific markers. The differentiated cells not only expressed insulin and glucose transporter 2, but also displayed a glucose-responsive secretion of the hormone. Our results delineate a new model system to study islet neogenesis and possible pharmaceutical targets. Nestin-positive bone marrow stem cells may be therapeutically relevant for β-cell replacement in type 1 diabetes.
|Induced differentiation of neural stem cells of astrocytic origin to motor neurons in the rat. |
Zhicheng Shao,Qian Luo,Dandan Liu,Yajing Mi,Ping Zhang,Gong Ju
Stem cells and development 20 2011
Destruction of the motor neurons will lead to loss of innervation of the somatic muscle, which has long been considered an illness with no remedy. The only possible treatment is to substitute the injured motor neurons by neurons differentiated from stem cells. It has been recently reported that embryonic stems cells can be induced to differentiate to motor neurons. However, the use of embryonic stem cells has innate problems. The ideal source of motor neurons should be the cells from the patients themselves, which have the potential to be induced to motor neurons. Our previous study demonstrated that mature astrocyte has the potential of being dedifferentiated to neural stem cell. The present study was aimed to investigate if the neural stem cells of astrocytic origin can be induced to motor neurons. The results demonstrated that neural stem cells of astrocytic origin could be induced to differentiate into motor neurons and their progenitor cells with rich harvest. Further, it has been reported that astrocytes can be readily obtained via biopsy from the cerebral cortex of the patient, rendering autologous transplantation possible. In conclusion, matured astrocytes can be induced to motor neurons and be autologously transplanted to patients suffering from motor neuron destruction.
|Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing. |
Zimmer, B; Kuegler, PB; Baudis, B; Genewsky, A; Tanavde, V; Koh, W; Tan, B; Waldmann, T; Kadereit, S; Leist, M
Cell death and differentiation 18 383-95 2011
As neuronal differentiation of embryonic stem cells (ESCs) recapitulates embryonic neurogenesis, disturbances of this process may model developmental neurotoxicity (DNT). To identify the relevant steps of in vitro neurodevelopment, we implemented a differentiation protocol yielding neurons with desired electrophysiological properties. Results from focussed transcriptional profiling suggested that detection of non-cytotoxic developmental disturbances triggered by toxicants such as retinoic acid (RA) or cyclopamine was possible. Therefore, a broad transcriptional profile of the 20-day differentiation process was obtained. Cluster analysis of expression kinetics, and bioinformatic identification of overrepresented gene ontologies revealed waves of regulation relevant for DNT testing. We further explored the concept of superimposed waves as descriptor of ordered, but overlapping biological processes. The initial wave of transcripts indicated reorganization of chromatin and epigenetic changes. Then, a transient upregulation of genes involved in the formation and patterning of neuronal precursors followed. Simultaneously, a long wave of ongoing neuronal differentiation started. This was again superseded towards the end of the process by shorter waves of neuronal maturation that yielded information on specification, extracellular matrix formation, disease-associated genes and the generation of glia. Short exposure to lead during the final differentiation phase, disturbed neuronal maturation. Thus, the wave kinetics and the patterns of neuronal specification define the time windows and end points for examination of DNT.
|Jarid1b targets genes regulating development and is involved in neural differentiation. |
Sandra U Schmitz,Mareike Albert,Martina Malatesta,Lluis Morey,Jens V Johansen,Mads Bak,Niels Tommerup,Iratxe Abarrategui,Kristian Helin
The EMBO journal 30 2011
H3K4 methylation is associated with active transcription and in combination with H3K27me3 thought to keep genes regulating development in a poised state. The contribution of enzymes regulating trimethylation of lysine 4 at histone 3 (H3K4me3) levels to embryonic stem cell (ESC) self-renewal and differentiation is just starting to emerge. Here, we show that the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) is dispensable for ESC self-renewal, but essential for ESC differentiation along the neural lineage. By genome-wide location analysis, we demonstrate that Jarid1b localizes predominantly to transcription start sites of genes encoding developmental regulators, of which more than half are also bound by Polycomb group proteins. Virtually all Jarid1b target genes are associated with H3K4me3 and depletion of Jarid1b in ESCs leads to a global increase of H3K4me3 levels. During neural differentiation, Jarid1b-depleted ESCs fail to efficiently silence lineage-inappropriate genes, specifically stem and germ cell genes. Our results delineate an essential role for Jarid1b-mediated transcriptional control during ESC differentiation.
|Control of neural cell composition in poly(ethylene glycol) hydrogel culture with soluble factors. |
Mooney, R; Haeger, S; Lawal, R; Mason, M; Shrestha, N; Laperle, A; Bjugstad, K; Mahoney, M
Tissue engineering. Part A 17 2805-15 2011
Poly(ethylene glycol) (PEG) hydrogels are being developed as cell delivery vehicles that have great potential to improve neuronal replacement therapies. Current research priorities include (1) characterizing neural cell growth within PEG hydrogels relative to standard culture systems and (2) generating neuronal-enriched populations within the PEG hydrogel environment. This study compares the percentage of neural precursor cells (NPCs), neurons, and glia present when dissociated neural cells are seeded within PEG hydrogels relative to standard monolayer culture. Results demonstrate that PEG hydrogels enriched the initial cell population for NPCs, which subsequently gave rise to neurons, then to glia. Relative to monolayer culture, PEG hydrogels maintained an increased percentage of NPCs and a decreased percentage of glia. This neurogenic advantage of PEG hydrogels is accentuated in the presence of basic fibroblast growth factor and epidermal growth factor, which more potently increase NPC and neuronal expression markers when applied to cells cultured within PEG hydrogels. Finally, this work demonstrates that glial differentiation can be selectively eliminated upon supplementation with a γ-secretase inhibitor. Together, this study furthers our understanding of how the PEG hydrogel environment influences neural cell composition and also describes select soluble factors that are useful in generating neuronal-enriched populations within the PEG hydrogel environment.
|Primary cilia regulate proliferation of amplifying progenitors in adult hippocampus: implications for learning and memory. |
Amador-Arjona, A; Elliott, J; Miller, A; Ginbey, A; Pazour, GJ; Enikolopov, G; Roberts, AJ; Terskikh, AV
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 9933-44 2011
Integration of new neurons into the adult hippocampus has been linked to specific types of learning. Primary cilia were found to be required for the formation of adult neural stem cells (NSCs) in the hippocampal dentate gyrus during development. However, the requirement of cilia in maintenance of adult NSCs is unknown. We developed a genetic mouse model in which fetal/perinatal brain development is unaffected, but adult hippocampal neurogenesis is constantly reduced by conditional ablation of primary cilia in adult GFAP(+) neural stem/progenitor cells. We found that this approach specifically reduces the number of hippocampal amplifying progenitors (also called type 2a cells) without affecting the number of radial NSCs (or type 1 cells). Constant reduction of adult hippocampal neurogenesis produced a delay rather than a permanent deficiency in spatial learning without affecting the retention of long-term memories. Decreased neurogenesis also altered spatial novelty recognition and hippocampus-independent cue conditioning. Here, we propose that adult hippocampal newborn neurons increase the efficiency of generating the new representations of spatial memories and that reduction of adult hippocampal neurogenesis may be biased toward cue-based strategies. This novel mouse model provides evidences that cognitive deficits associated with ciliary defects (ciliopathies) might be, in part, mediated by the deficiency of primary cilia in adult hippocampal stem/progenitor cells.
|Acute pancreatitis accelerates initiation and progression to pancreatic cancer in mice expressing oncogenic Kras in the nestin cell lineage. |
Carrière, C; Young, AL; Gunn, JR; Longnecker, DS; Korc, M
PloS one 6 e27725 2011
Targeting of oncogenic Kras to the pancreatic Nestin-expressing embryonic progenitor cells and subsequently to the adult acinar compartment and Nestin-expressing cells is sufficient for the development of low grade pancreatic intraepithelial neoplasia (PanIN) between 2 and 4 months. The mice die around 6 month-old of unrelated causes, and it is therefore not possible to assess whether the lesions will progress to carcinoma. We now report that two brief episodes of caerulein-induced acute pancreatitis in 2 month-old mice causes rapid PanIN progression and pancreatic ductal adenocarcinoma (PDAC) development by 4 months of age. These events occur with similar frequency as observed in animals where the oncogene is targeted during embryogenesis to all pancreatic cell types. Thus, these data show that oncogenic Kras-driven PanIN originating in a non-ductal compartment can rapidly progress to PDAC when subjected to a brief inflammatory insult.
|Increased re-entry into cell cycle mitigates age-related neurogenic decline in the murine subventricular zone. |
Stoll, EA; Habibi, BA; Mikheev, AM; Lasiene, J; Massey, SC; Swanson, KR; Rostomily, RC; Horner, PJ
Stem cells (Dayton, Ohio) 29 2005-17 2011
Although new neurons are produced in the subventricular zone (SVZ) of the adult mammalian brain, fewer functional neurons are produced with increasing age. The age-related decline in neurogenesis has been attributed to a decreased pool of neural progenitor cells (NPCs), an increased rate of cell death, and an inability to undergo neuronal differentiation and develop functional synapses. The time between mitotic events has also been hypothesized to increase with age, but this has not been directly investigated. Studying primary-cultured NPCs from the young adult and aged mouse forebrain, we observe that fewer aged cells are dividing at a given time; however, the mitotic cells in aged cultures divide more frequently than mitotic cells in young cultures during a 48-hour period of live-cell time-lapse imaging. Double-thymidine-analog labeling also demonstrates that fewer aged cells are dividing at a given time, but those that do divide are significantly more likely to re-enter the cell cycle within a day, both in vitro and in vivo. Meanwhile, we observed that cellular survival is impaired in aged cultures. Using our live-cell imaging data, we developed a mathematical model describing cell cycle kinetics to predict the growth curves of cells over time in vitro and the labeling index over time in vivo. Together, these data surprisingly suggest that progenitor cells remaining in the aged SVZ are highly proliferative.
|Deficiency of mDia, an actin nucleator, disrupts integrity of neuroepithelium and causes periventricular dysplasia. |
Thumkeo, D; Shinohara, R; Watanabe, K; Takebayashi, H; Toyoda, Y; Tohyama, K; Ishizaki, T; Furuyashiki, T; Narumiya, S
PloS one 6 e25465 2011
During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that mDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both mDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.
|While p73 is essential, p63 is completely dispensable for the development of the central nervous system. |
Holembowski, L; Schulz, R; Talos, F; Scheel, A; Wolff, S; Dobbelstein, M; Moll, U
Cell cycle (Georgetown, Tex.) 10 680-9 2011
The ancient p53 paralogs p63 and p73 regulate specific tissue formation, cell survival and cell death via their TA and ΔN isoforms. Targeted disruption of the p73 locus leads to severe defects in the development of the central nervous system (CNS), and p73 has recently been shown to be an essential regulator of neural stem cell maintenance and differentiation in both embryonal and adult neurogenesis. In contrast, global p63-/- mice lack skin and limbs. Moreover, p63 is detectable in embryonic cortex. It has previously been proposed to also play critical pro-death and pro-survival roles in neural precursors of the developing sympathetic and central nervous system, respectively, based on experimental overexpression and siRNA-mediated knockdown of p63. Here we perform an extensive analysis of the developing central nervous system in global p63-/- mice and their wildtype littermates. Brain and spinal cord of embryos and newborn mice were assessed in vivo for neuroanatomy, histology, apoptosis, proliferation, stemness and differentiation, and in vitro for self-renewal and maturation in neurosphere assays. None of these analyses revealed a detectable phenotype in p63-/- mice. Hence, despite the profound impact of p63 on the development of stratified epithelia and limbs, p63 is completely dispensable for proper development of the central nervous system. Thus, despite their strong homology, the non-overlapping tissue specificity of p63 and p73 functions appears more pronounced than previously anticipated.
|miR-132 mediates the integration of newborn neurons into the adult dentate gyrus. |
Luikart, BW; Bensen, AL; Washburn, EK; Perederiy, JV; Su, KG; Li, Y; Kernie, SG; Parada, LF; Westbrook, GL
PloS one 6 e19077 2011
Neuronal activity enhances the elaboration of newborn neurons as they integrate into the synaptic circuitry of the adult brain. The role microRNAs play in the transduction of neuronal activity into growth and synapse formation is largely unknown. MicroRNAs can influence the expression of hundreds of genes and thus could regulate gene assemblies during processes like activity-dependent integration. Here, we developed viral-based methods for the in vivo detection and manipulation of the activity-dependent microRNA, miR-132, in the mouse hippocampus. We find, using lentiviral and retroviral reporters of miR-132 activity, that miR-132 is expressed at the right place and right time to influence the integration of newborn neurons. Retroviral knockdown of miR-132 using a specific 'sponge' containing multiple target sequences impaired the integration of newborn neurons into the excitatory synaptic circuitry of the adult brain. To assess potential miR-132 targets, we used a whole-genome microarray in PC12 cells, which have been used as a model of neuronal differentiation. miR-132 knockdown in PC12 cells resulted in the increased expression of hundreds of genes. Functional grouping indicated that genes involved in inflammatory/immune signaling were the most enriched class of genes induced by miR-132 knockdown. The correlation of miR-132 knockdown to increased proinflammatory molecular expression may indicate a mechanistic link whereby miR-132 functions as an endogenous mediator of activity-dependent integration in vivo.
|PPARbeta activation inhibits melanoma cell proliferation involving repression of the Wilms' tumour suppressor WT1. |
Michiels, JF; Perrin, C; Leccia, N; Massi, D; Grimaldi, P; Wagner, N
Pflügers Archiv : European journal of physiology 459 689-703 2010
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that strongly influence molecular signalling in normal and cancer cells. Although increasing evidence suggests a role of PPARs in skin carcinogenesis, only expression of PPARgamma has been investigated in human melanoma tissues. Activation of PPARalpha has been shown to inhibit the metastatic potential, whereas stimulation of PPARgamma decreased melanoma cell proliferation. We show here that the third member of the PPAR family, PPARbeta/delta is expressed in human melanoma samples. Specific pharmacological activation of PPARbeta using GW0742 or GW501516 in low concentrations inhibits proliferation of human and murine melanoma cells. Inhibition of proliferation is accompanied by decreased expression of the Wilms' tumour suppressor 1 (WT1), which is implicated in melanoma proliferation. We demonstrate that PPARbeta directly represses WT1 as (1) PPARbeta activation represses WT1 promoter activity; (2) in chromatin immunoprecipitation and electrophoretic mobility shift assays, we identified a binding element for PPARbeta in the WT1 promoter; (3) deletion of this binding element abolishes repression by PPARbeta and (4) the WT1 downstream molecules nestin and zyxin are down-regulated upon PPARbeta activation. Our findings elucidate a novel mechanism of signalling by ligands of PPARbeta, which leads to suppression of melanoma cell growth through direct repression of WT1.Artículo Texto completo
|Toll-like receptor 3 inhibits memory retention and constrains adult hippocampal neurogenesis. |
Okun E, Griffioen K, Barak B, Roberts NJ, Castro K, Pita MA, Cheng A, Mughal MR, Wan R, Ashery U, Mattson MP
Proc Natl Acad Sci U S A 2010
Toll-like receptors (TLRs) are innate immune receptors that have recently emerged as regulators of neuronal survival and developmental neuroplasticity. Adult TLR3-deficient mice exhibited enhanced hippocampus-dependent working memory in the Morris water maze, novel object recognition, and contextual fear-conditioning tasks. In contrast, TLR3-deficient mice demonstrated impaired amygdala-related behavior and anxiety in the cued fear-conditioning, open field, and elevated plus maze tasks. Further, TLR3-deficient mice exhibited increased hippocampal CA1 and dentate gyrus volumes, increased hippocampal neurogenesis, and elevated levels of the AMPA receptor subunit GluR1 in the CA1 region of the hippocampus. In addition, levels of activated forms of the kinase ERK and the transcription factor CREB were elevated in the hippocampus of TLR3-deficient mice, suggesting that constitutive TLR3 signaling negatively regulates pathways known to play important roles in hippocampal plasticity. Direct activation of TLR3 by intracerebroventricular infusion of a TLR3 ligand impaired working memory, but not reference memory. Our findings reveal previously undescribed roles for TLR3 as a suppressor of hippocampal cellular plasticity and memory retention.
|Analysis of the expression and function of BRINP family genes during neuronal differentiation in mouse embryonic stem cell-derived neural stem cells. |
Terashima M, Kobayashi M, Motomiya M, Inoue N, Yoshida T, Okano H, Iwasaki N, Minami A, Matsuoka I
J Neurosci Res 88 1387-93. 2010
We previously identified a novel family of genes, BRINP1, 2, and 3, that are predominantly and widely expressed in both the central nervous system (CNS) and peripheral nervous system (PNS). In the present study, we analyzed the expression pattern of three BRINP genes during differentiation of mouse embryonic stem (ES) cell-derived neural stem cells (NSCs) and their effects on the cell-cycle regulation of NSCs. While there was no significant expression of any BRINP-mRNA expressed in mouse ES cells, BRINP 1 and 2-mRNAs was expressed at high levels in the ES cell-derived neural stem cells. Upon differentiation into neuronal cells in the presence of retinoic acid and BDNF, all three types of BRINP-mRNA were induced with a similar time course peaking at day three of treatment. Upon differentiation into astroglial cells in the presence of serum, BRINP1-mRNA was slightly up-regulated, while BRINP2- and BRINP3-mRNAs were almost abolished in the astrocytes. While 69.2, 26.1, and 7.7% of cells in a population of NSCs in the exponentially growing phase were in the G1, S and G2 phases, respectively, over-expression of any one of the three BRINP genes completely abolished cells in the G2 phase and significantly reduced the cells in S phase to 11.8-13.8%. Based on these results, the physiological roles of induced BRINP genes in the cell-cycle suppression of terminally differentiated post-mitotic neurons are discussed. (c) 2009 Wiley-Liss, Inc.
|Folic acid remodels chromatin on Hes1 and Neurog2 promoters during caudal neural tube development. |
Ichi, S; Costa, FF; Bischof, JM; Nakazaki, H; Shen, YW; Boshnjaku, V; Sharma, S; Mania-Farnell, B; McLone, DG; Tomita, T; Soares, MB; Mayanil, CS
The Journal of biological chemistry 285 36922-32 2010
The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.
|Prdm16 promotes stem cell maintenance in multiple tissues, partly by regulating oxidative stress. |
Chuikov, S; Levi, BP; Smith, ML; Morrison, SJ
Nature cell biology 12 999-1006 2010
To better understand the mechanisms that regulate stem cell identity and function, we sought to identify genes that are preferentially expressed by stem cells and critical for their function in multiple tissues. Prdm16 is a transcription factor that regulates leukaemogenesis, palatogenesis and brown-fat development, but which was not known to be required for stem cell function. We demonstrate that Prdm16 is preferentially expressed by stem cells throughout the nervous and haematopoietic systems and is required for their maintenance. In the haematopoietic and nervous systems, Prdm16 deficiency led to changes in the levels of reactive oxygen species (ROS), depletion of stem cells, increased cell death and altered cell-cycle distribution. In neural stem/progenitor cells, Prdm16 binds to the Hgf promoter, and Hgf expression declined in the absence of Prdm16. Addition of recombinant HGF to Prdm16-deficient neural stem cells in cell culture reduced the depletion of these cells and partially rescued the increase in ROS levels. Administration of the anti-oxidant, N-acetyl-cysteine, to Prdm16-deficient mice partially rescued defects in neural stem/progenitor cell function and neural development. Prdm16 therefore promotes stem cell maintenance in multiple tissues, partly by modulating oxidative stress.Artículo Texto completo
|G1 arrest and differentiation can occur independently of Rb family function. |
Wirt, SE; Adler, AS; Gebala, V; Weimann, JM; Schaffer, BE; Saddic, LA; Viatour, P; Vogel, H; Chang, HY; Meissner, A; Sage, J
The Journal of cell biology 191 809-25 2010
The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.
|Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system. |
Nishino, J; Saunders, TL; Sagane, K; Morrison, SJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 30 15228-40 2010
The mechanisms that regulate peripheral nervous system (PNS) gliogenesis are incompletely understood. For example, gut neural crest stem cells (NCSCs) do not respond to known gliogenic factors, suggesting that yet-unidentified factors regulate gut gliogenesis. To identify new mechanisms, we performed gene expression profiling to identify factors secreted by gut NCSCs during the gliogenic phase of development. These cells highly expressed leucine-rich glioma inactivated 4 (Lgi4) despite the fact that Lgi4 has never been implicated in stem cell function or enteric nervous system development. Lgi4 is known to regulate peripheral nerve myelination (having been identified as the mutated gene in spontaneously arising claw paw mutant mice), but Lgi4 is not known to play any role in PNS development outside of peripheral nerves. To systematically analyze Lgi4 function, we generated gene-targeted mice. Lgi4-deficient mice exhibited a more severe phenotype than claw paw mice and had gliogenic defects in sensory, sympathetic, and enteric ganglia. We found that Lgi4 is required for the proliferation and differentiation of glial-restricted progenitors throughout the PNS. Analysis of compound-mutant mice revealed that the mechanism by which Lgi4 promotes enteric gliogenesis involves binding the ADAM22 receptor. Our results identify a new mechanism regulating enteric gliogenesis as well as novel functions for Lgi4 regulating the proliferation and maturation of glial lineage cells throughout the PNS.Artículo Texto completo
|The effect of variation in expression of the candidate dyslexia susceptibility gene homolog Kiaa0319 on neuronal migration and dendritic morphology in the rat. |
Peschansky, VJ; Burbridge, TJ; Volz, AJ; Fiondella, C; Wissner-Gross, Z; Galaburda, AM; Lo Turco, JJ; Rosen, GD
Cerebral cortex (New York, N.Y. : 1991) 20 884-97 2010
We investigated the postnatal effects of embryonic knockdown and overexpression of the candidate dyslexia gene homolog Kiaa0319. We used in utero electroporation to transfect cells in E15/16 rat neocortical ventricular zone with either 1) small hairpin RNA (shRNA) vectors targeting Kiaa0319, 2) a KIAA0319 expression construct, 3) Kiaa0319 shRNA along with KIAA0319 expression construct ("rescue"), or 4) a scrambled version of Kiaa0319 shRNA. Knockdown, but not overexpression, of Kiaa0319 resulted in periventricular heterotopias that contained large numbers of both transfected and non-transfected neurons. This suggested that Kiaa0319 shRNA disrupts neuronal migration by cell autonomous as well as non-cell autonomous mechanisms. Of the Kiaa0319 shRNA-transfected neurons that migrated into the cortical plate, most migrated to their appropriate lamina. In contrast, neurons transfected with the KIAA0319 expression vector attained laminar positions subjacent to their expected positions. Neurons transfected with Kiaa0319 shRNA exhibited apical, but not basal, dendrite hypertrophy, which was rescued by overexpression of KIAA0319. The results provide additional supportive evidence linking candidate dyslexia susceptibility genes to migrational disturbances during brain development, and extends the role of Kiaa0319 to include growth and differentiation of dendrites.Artículo Texto completo
|A COMPARISON OF DIFFERENTIATION PROTOCOLS FOR RGC-5 CELLS. |
Wood JP, Chidlow G, Tran T, Crowston J, Casson RJ
Invest Ophthalmol Vis Sci 2010
Purpose: Although the RGC-5 cell line is widely employed in retinal ganglion cell (RGC) research, recent data have raised questions about the nature of these cells. We therefore performed a systematic analysis of RGC-5 cells in order to determine which RGC or neuronal markers are expressed after treatment with known differentiating agents. This provided further insights into the nature of these cells and assisted in defining their future use. Methods: RGC-5 cells were treated for 5 days with either staurosporine (STSN; 316nM), trichostatin A (TSA; 500nM) or succinyl-concanavalin A (sConA; 50microg/ml), whereafter they were assayed for specific marker antigen/mRNA expression. Treated cells were also assayed for excitotoxic responsiveness. Results: Neither treated nor untreated RGC-5 cells expressed any specific RGC marker mRNAs or proteins (Brn-3, neurofilaments, Thy-1), or any of calbindin, calretinin, synaptophysin, PKCalpha or glial fibrillary acidic protein (GFAP). However, control RGC-5 cells did express the neuronal markers, tau, betaIII-tubulin, MAP-1b, MAP2 and PGP9.5. Although treatment with sConA had no effect on expression of these markers, STSN and TSA (the latter, dose-dependently) increased their expression and induced excitotoxic responsiveness. All cells, whether treated or not, expressed high levels of nestin, but no other progenitor cell markers. All cells also expressed cone-specific but not rod-specific opsin indicative of being of cone photoreceptor lineage. Conclusions: RGC-5 cells expressed neuronal but not RGC-specific markers which were dose-dependently upregulated by TSA. Hence, TSA provided the best tested means to terminally differentiate the cells to a neuronal phenotype from a precursor-like lineage.
|Drosophila CAF-1 regulates HP1-mediated epigenetic silencing and pericentric heterochromatin stability. |
Huang H, Yu Z, Zhang S, Liang X, Chen J, Li C, Ma J, Jiao R
J Cell Sci 123 2853-61. Epub 2010 Jul 27. 2010
Chromatin assembly factor 1 (CAF-1) was initially characterized as a histone deliver in the process of DNA-replication-coupled chromatin assembly in eukaryotic cells. Here, we report that CAF-1 p180, the largest subunit of Drosophila CAF-1, participates in the process of heterochromatin formation and functions to maintain pericentric heterochromatin stability. We provide evidence that Drosophila CAF-1 p180 plays a role in both classes of position effect variegation (PEV) and in the expression of heterochromatic genes. A decrease in the expression of Drosophila CAF-1 p180 leads to a decrease in both H3K9 methylation at pericentric heterochromatin regions and the recruitment of heterochromatin protein 1 (HP1) to the chromocenter of the polytene chromosomes. The artificial targeting of HP1 to a euchromatin location leads to the enrichment of Drosophila CAF-1 p180 at this ectopic heterochromatin, suggesting the mutual recruitment of HP1 and CAF-1 p180. We also show that the spreading of heterochromatin is compromised in flies that have reduced CAF-1 p180. Furthermore, reduced CAF-1 p180 causes a defect in the dynamics of heterochromatic markers in early Drosophila embryos. Together, these findings suggest that Drosophila CAF-1 p180 is an essential factor in the epigenetic control of heterochromatin formation and/or maintenance.
|Angiogenic factors stimulate growth of adult neural stem cells. |
Androutsellis-Theotokis, A; Rueger, MA; Park, DM; Boyd, JD; Padmanabhan, R; Campanati, L; Stewart, CV; LeFranc, Y; Plenz, D; Walbridge, S; Lonser, RR; McKay, RD
PloS one 5 e9414 2010
The ability to grow a uniform cell type from the adult central nervous system (CNS) is valuable for developing cell therapies and new strategies for drug discovery. The adult mammalian brain is a source of neural stem cells (NSC) found in both neurogenic and non-neurogenic zones but difficulties in culturing these hinders their use as research tools.Here we show that NSCs can be efficiently grown in adherent cell cultures when angiogenic signals are included in the medium. These signals include both anti-angiogenic factors (the soluble form of the Notch receptor ligand, Dll4) and pro-angiogenic factors (the Tie-2 receptor ligand, Angiopoietin 2). These treatments support the self renewal state of cultured NSCs and expression of the transcription factor Hes3, which also identifies the cancer stem cell population in human tumors. In an organotypic slice model, angiogenic factors maintain vascular structure and increase the density of dopamine neuron processes.We demonstrate new properties of adult NSCs and a method to generate efficient adult NSC cultures from various central nervous system areas. These findings will help establish cellular models relevant to cancer and regeneration.Artículo Texto completo
|Administration of the bisphosphonate zoledronic acid during tooth development inhibits tooth eruption and formation and induces dental abnormalities in rats. |
Hiraga T, Ninomiya T, Hosoya A, Nakamura H
Calcif Tissue Int 86 502-10. Epub 2010 Apr 22. 2010
Bisphosphonates (BPs) are potent inhibitors of osteoclastic bone resorption and widely used for the treatment of osteoporosis and metastatic bone diseases. Recently, BPs have also been shown to benefit children with primary and secondary osteoporosis, including osteogenesis imperfecta; however, their long-term safety has not been established yet. Clinical and experimental studies have demonstrated that BPs delay or inhibit tooth eruption. The failure of tooth eruption causes several dental abnormalities. In this study, to determine the effects of BPs on tooth formation, the BP zoledronic acid (ZOL) was injected into 7- and 14-day-old rats, and the development of the mandibular teeth was examined. X-ray analysis demonstrated that ZOL inhibited the eruption of both incisors and molars and their formation, especially in the molar roots. Histological examination showed that, in ZOL-treated animals, alveolar bone remained unresorbed around tooth crowns, which injured ameloblasts and enamel matrix, leading to defects of the enamel. Furthermore, haphazard proliferation of odontogenic epithelium and mesenchyme associated with primitive tooth structures, which resembles human odontomas, was induced at the basal end of incisors but not around the molars. Tooth ankylosis to alveolar bone was occasionally observed in molars. These results suggest that administration of BPs during tooth development has the potential to inhibit tooth eruption and formation and to induce several types of dental abnormalities, which may be attributed to the altered osteoclastic activities.
|The effect of hyperglycemia on hypoperfusion-induced injury. |
Holman, MC; Chidlow, G; Wood, JP; Casson, RJ
Investigative ophthalmology & visual science 51 2197-207 2010
Purpose. Because of differences in energy metabolism between the brain and retina, the hypothesis for the study was that, in a model of ocular and cerebral hypoperfusion, the retina would be protected by short-term hyperglycemia, whereas brain injury would be exacerbated. Methods. Hyperglycemia was induced by intraperitoneal streptozotocin. An initial experiment determined the effect of hyperglycemia alone in sham-surgery rats. Simultaneous retinal and cerebral hypoperfusion was achieved by two-vessel occlusion (2VO; permanent ligation of both common carotid arteries). Hyperglycemia was induced 3 days before 2VO by streptozotocin injection. The rats were killed 7 days after 2VO or sham surgery. The retina of one eye was collected for histology/immunohistochemistry and the fellow retina was collected for real-time RT-PCR. The retinas were analyzed for neuronal and glial markers and heat shock protein-27. The brains were processed for histology and immunohistochemistry. Results. Short-term (approximately 10 days) hyperglycemia alone caused no discernible injury to the retina. The retinas of the normoglycemic 2VO animals showed a marked loss of retinal ganglion cells and horizontal cells, thinning of the inner retina, glial cell activation, and infiltration of macrophages. The hyperglycemic 2VO rats displayed remarkable protection of the retinal structure and reduced glial cell activation compared with the normoglycemic 2VO animals. There was a significantly greater number of heat shock protein-27-positive retinal ganglion cells in the normoglycemic animals than in the hyperglycemic ones. Brains of both the normoglycemic and hyperglycemic 2VO animals displayed scattered ischemic infarcts and mild white matter injury. Conclusions. Short-term hyperglycemia affords robust protection against retinal hypoperfusion injury, but in the same animals, brain injury is not ameliorated. The mechanism of this retinal hyperglycemia-induced neuroprotection requires further study.
|Involvement of Notch1 inhibition in serum-stimulated glia and oligodendrocyte differentiation from human mesenchymal stem cells. |
Lee, YJ; Hung, SC; Chu, MS
Stem cells and cloning : advances and applications 3 165-73 2010
The use of in vitro oligodendrocyte differentiation for transplantation of stem cells to treat demyelinating diseases is an important consideration. In this study, we investigated the effects of serum on glia and oligodendrocyte differentiation from human mesenchymal stem cells (KP-hMSCs). We found that serum deprivation resulted in a reversible downregulation of glial- and oligodendrocyte-specific markers. Serum stimulated expression of oligodendrocyte markers, such as galactocerebroside, as well as Notch1 and JAK1 transcripts. Inhibition of Notch1 activation by the Notch inhibitor, MG132, led to enhanced expression of a serum-stimulated oligodendrocyte marker. This marker was undetectable in serum-deprived KP-hMSCs treated with MG132, suggesting that inhibition of Notch1 function is additive to serum-stimulated oligodendrocyte differentiation. Furthermore, a dominant-negative mutant RBP-J protein also inhibited Notch1 function and led to upregulation of oligodendrocyte-specific markers. Our results demonstrate that serum-stimulated oligodendrocyte differentiation is enhanced by the inhibition of Notch1-associated functions.
|Origins of gliogenic stem cell populations within adult skin and bone marrow. |
Hunt, DP; Sajic, M; Phillips, H; Henderson, D; Compston, A; Smith, K; Chandran, S
Stem cells and development 19 1055-65 2010
The generation of Schwann cells from precursors within adult skin and bone marrow is of significant clinical interest because of the opportunities for disease modelling and strategies for remyelination. Recent evidence has suggested that glial cells can be generated from (i) mesenchymal stem cells (MSCs) within adult bone marrow and (ii) skin-derived precursor cells (SKPs) within adult skin. However, there is a need to clarify the developmental mechanism whereby such multipotent adult stem cell populations generate glia. We used Wnt1-Cre/Rosa26R(LacZ) and Wnt1-Cre/Rosa26R(YFP) neural crest reporter mice to test the hypothesis that (i) MSCs and (ii) SKPs represent adult gliogenic precursor cells of neural crest origin. We demonstrate that, although labeled cells can be identified within long bone preparation, such cells are rarely found in marrow plugs. Moreover, we did not find evidence of a neural crest origin of bone marrow-derived MSCs and were not able to provide a developmental rationale for the derivation of glial cells from MSCs using this approach. In contrast, we provide robust evidence for the neural crest origin of SKPs derived from adult skin. These precursor cells reliably generate cells with a Schwann cell phenotype, expressing appropriate transcription factors and Schwann cell markers. We demonstrate multiple anatomical origins of gliogenic SKPs within adult skin. We conclude that SKPs, rather than bone marrow-derived MSCs, represent a more defined and developmentally rational source for the study and generation of Schwann cells from readily accessible adult tissues.
|Neuronatin promotes neural lineage in ESCs via Ca(2+) signaling. |
Lin, HH; Bell, E; Uwanogho, D; Perfect, LW; Noristani, H; Bates, TJ; Snetkov, V; Price, J; Sun, YM
Stem cells (Dayton, Ohio) 28 1950-60 2010
Neural induction is the first step in the formation of the vertebrate central nervous system. The emerging consensus of the mechanisms underlying neural induction is the combined influences from inhibiting bone morphogenetic protein (BMP) signaling and activating fibroblast growth factor (FGF)/Erk signaling, which act extrinsically via either autocrine or paracrine fashions. However, do intrinsic forces (cues) exist and do they play decisive roles in neural induction? These questions remain to be answered. Here, we have identified a novel neural initiator, neuronatin (Nnat), which acts as an intrinsic factor to promote neural fate in mammals and Xenopus. ESCs lacking this intrinsic factor fail to undergo neural induction despite the inhibition of the BMP pathway. We show that Nnat initiates neural induction in ESCs through increasing intracellular Ca(2+) ([Ca(2+) ](i)) by antagonizing Ca(2+) -ATPase isoform 2 (sarco/endoplasmic reticulum Ca(2+) -ATPase isoform 2) in the endoplasmic reticulum, which in turn increases the phosphorylation of Erk1/2 and inhibits the BMP4 pathway and leads to neural induction in conjunction with FGF/Erk pathway.
|Astroglial cells in the external granular layer are precursors of cerebellar granule neurons in neonates. |
Silbereis, J; Heintz, T; Taylor, MM; Ganat, Y; Ment, LR; Bordey, A; Vaccarino, F
Molecular and cellular neurosciences 44 362-73 2010
It is well established that cerebellar granule cell precursors (GCPs) initially derive from progenitors in the rhombic lip of the embryonic cerebellar primordium. GCPs proliferate and migrate tangentially across the cerebellum to form the external granule cell layer (EGL) in late embryogenesis and early postnatal development. It is unclear whether GCPs are specified exclusively in the embryonic rhombic lip or whether their precursor persists in the neonate. Using transgenic mice expressing DsRed under the human glial fibrillary acidic protein (hGFAP) promoter, we found 2 populations of DsRed(+) cells in the EGL in the first postnatal week defined by bright and faint DsRed-fluorescent signal. Bright DsRed(+) cells have a protein expression profile and electrophysiological characteristics typical of astrocytes, but faint DsRed(+) cells in the EGL and internal granule cell layer (IGL) express markers and physiological properties of immature neurons. To determine if these astroglial cells gave rise to GCPs, we genetically tagged them with EGFP or betagal reporter genes at postnatal day (P)3-P5 using a hGFAP promoter driven inducible Cre recombinase. We found that GFAP promoter(+) cells in the EGL are proliferative and express glial and neural stem cell markers. In addition, immature granule cells (GCs) en route to the IGL at P12 as well as GCs in the mature cerebellum, 30days after recombination, express the reporter protein, suggesting that GFAP promoter(+) cells in the EGL generate a subset of granule cells. The identification of glial cells which function as neuronal progenitor cells profoundly impacts our understanding of cellular plasticity in the developing cerebellum.
|Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains. |
Androutsellis-Theotokis, A; Walbridge, S; Park, DM; Lonser, RR; McKay, RD
PloS one 5 e10841 2010
New mechanisms that regulate neural stem cell (NSC) expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers.Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen.Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer.
|Loss of Bright/ARID3a function promotes developmental plasticity. |
An, G; Miner, CA; Nixon, JC; Kincade, PW; Bryant, J; Tucker, PW; Webb, CF
Stem cells (Dayton, Ohio) 28 1560-7 2010
B-cell regulator of immunoglobulin heavy chain transcription (Bright)/ARID3a, an A+T-rich interaction domain protein, was originally discovered in B lymphocyte lineage cells. However, expression patterns and high lethality levels in knockout mice suggested that it had additional functions. Three independent lines of evidence show that functional inhibition of Bright results in increased developmental plasticity. Bright-deficient cells from two mouse models expressed a number of pluripotency-associated gene products, expanded indefinitely, and spontaneously differentiated into cells of multiple lineages. Furthermore, direct knockdown of human Bright resulted in colonies capable of expressing multiple lineage markers. These data suggest that repression of this single molecule confers adult somatic cells with new developmental options.
|Gene expression and differentiation characteristics in mice E13.5 and E17.5 neural retinal progenitors. |
Sun X, Jiang R, Zhang Y, Chen M, Xiang P, Qi Y, Gao Q, Huang B, Ge J
Molecular vision 15 2503-14 2009
PURPOSE: Retinal progenitor cells (RPCs) are the most valuable seed cells in replacement therapy for neural retinal diseases. The competence of RPCs changes with retinal development. Gene expression plays a fundamental role in determining the competence. To improve the selection of the right-timing RPCs for replacement therapy, we compared the gene expression between embryonic day (E) 13.5 and E17.5 RPCs and further explored their gene expression and differentiation capacity in vitro. METHODS: Timed-pregnant E13.5 and E17.5 RPCs were freshly harvested and cultured in proliferation conditions for 4 days and then in differentiation conditions for 8 days. At different time points, the expression of key genes involved in retinal development was investigated by quantitative reverse transcription-PCR or immunofluorescence. RESULTS: The expression of 14 key genes involved in retinal development was investigated in freshly harvested E13.5 and E17.5 RPCs. The freshly harvested E13.5 RPCs showed a high expression of retinal ganglion cell (RGC)-related genes, including Math5, Brn3b, Islet1, and Nfl, while the freshly harvested E17.5 RPCs displayed a high expression for Nrl, GFAP, and Thy1, the key genes involved in rod photoreceptor development, glial cell development, and synaptogenesis, respectively. During proliferation culture in vitro, the gene expression changed dramatically in both RPCs. After the 4 days of proliferation culture, the expression levels of most genes (11 of the 14 genes) in E13.5 RPCs came close to those in the freshly harvested E17.5 RPCs. Differentiation of RPCs in vitro was verified by the significant decrease in Nestin expression and BruU incorporation efficiency. After the 8 days of differentiation in vitro, the expression level of RGC-related genes (Math5, Brn3b, and Islet1) was still significantly higher in E13.5 RPCs than in E17.5 RPCs. In contrast, the expression level of Nrl and GFAP was significantly higher in E17.5 RPCs than in E13.5 RPCs. In morphology, the differentiated E13.5 RPCs displayed more robust process outgrowth than did the differentiated E17.5 RPCs. Immunofluorescence showed that, after the 8 days of differentiation, E13.5 RPCs contained more Brn3b- and Map2-positive cells, while E17.5 RPCs contained more GFAP-, GS-, and Rhodopsin-positive cells. CONCLUSIONS: The results implied that E13.5 RPCs might be a better choice for RGC replacement therapy, while E17.5 RPCs might be better for photoreceptor replacement therapy. The duration of in vitro culture should be timed, since the expression of key genes kept changing in the proliferating RPCs.Artículo Texto completo
|c-kit expression identifies cardiovascular precursors in the neonatal heart. |
Yvonne N Tallini, Kai Su Greene, Michael Craven, Alyson Spealman, Martin Breitbach, James Smith, Patricia J Fisher, Michele Steffey, Michael Hesse, Robert M Doran, Ashley Woods, Babu Singh, Andrew Yen, Bernd K Fleischmann, Michael I Kotlikoff, Yvonne N Tallini, Kai Su Greene, Michael Craven, Alyson Spealman, Martin Breitbach, James Smith, Patricia J Fisher, Michele Steffey, Michael Hesse, Robert M Doran, Ashley Woods, Babu Singh, Andrew Yen, Bernd K Fleischmann, Michael I Kotlikoff, Yvonne N Tallini, Kai Su Greene, Michael Craven, Alyson Spealman, Martin Breitbach, James Smith, Patricia J Fisher, Michele Steffey, Michael Hesse, Robert M Doran, Ashley Woods, Babu Singh, Andrew Yen, Bernd K Fleischmann, Michael I Kotlikoff, Yvonne N Tallini, Kai Su Greene, Michael Craven, Alyson Spealman, Martin Breitbach, James Smith, Patricia J Fisher, Michele Steffey, Michael Hesse, Robert M Doran, Ashley Woods, Babu Singh, Andrew Yen, Bernd K Fleischmann, Michael I Kotlikoff, Yvonne N Tallini, Kai Su
Proceedings of the National Academy of Sciences of the United States of America 106 1808-13 2009
Directed differentiation of embryonic stem cells indicates that mesodermal lineages in the mammalian heart (cardiac, endothelial, and smooth muscle cells) develop from a common, multipotent cardiovascular precursor. To isolate and characterize the lineage potential of a resident pool of cardiovascular progenitor cells (CPcs), we developed BAC transgenic mice in which enhanced green fluorescent protein (EGFP) is placed under control of the c-kit locus (c-kit(BAC)-EGFP mice). Discrete c-kit-EGFP(+) cells were observed at different stages of differentiation in embryonic hearts, increasing in number to a maximum at about postnatal day (PN) 2; thereafter, EGFP(+) cells declined and were rarely observed in the adult heart. EGFP(+) cells purified from PN 0-5 hearts were nestin(+) and expanded in culture; 67% of cells were fluorescent after 9 days. Purified cells differentiated into endothelial, cardiac, and smooth muscle cells, and differentiation could be directed by specific growth factors. CPc-derived cardiac myocytes displayed rhythmic beating and action potentials characteristic of multiple cardiac cell types, similar to ES cell-derived cardiomyocytes. Single-cell dilution studies confirmed the potential of individual CPcs to form all 3 cardiovascular lineages. In adult hearts, cryoablation resulted in c-kit-EGFP(+) expression, peaking 7 days postcryolesion. Expression occurred in endothelial and smooth muscle cells in the revascularizing infarct, and in terminally differentiated cardiomyocytes in the border zone surrounding the infarct. Thus, c-kit expression marks CPc in the neonatal heart that are capable of directed differentiation in vitro; however, c-kit expression in cardiomyocytes in the adult heart after injury does not identify cardiac myogenesis.Artículo Texto completo
|In utero exposure to di-(2-ethylhexyl) phthalate decreases mineralocorticoid receptor expression in the adult testis. |
Martinez-Arguelles DB, Culty M, Zirkin BR, Papadopoulos V
Endocrinology 150 5575-85 2009
In utero exposure to di-(2-ethylhexyl) phthalate (DEHP) has been shown to result in decreased androgen formation by fetal and adult rat testes. In the fetus, decreased androgen is accompanied by the reduced expression of steroidogenic enzymes. The mechanism by which in utero exposure results in reduced androgen formation in the adult, however, is unknown. We hypothesized that deregulation of the nuclear steroid receptors might explain the effects of in utero DEHP exposure on adult testosterone production. To test this hypothesis, pregnant Sprague Dawley dams were gavaged with 100-950 mg DEHP per kilogram per day from gestational d 14-19, and testes were collected at gestational d 20 and postnatal days (PND) 3, 21, and 60. Among the nuclear receptors studied, the mineralocorticoid receptor (MR) mRNA and protein levels were reduced in PND60 interstitial Leydig cells, accompanied by reduced mRNA expression of MR-regulated genes. Methylation-sensitive PCR showed effects on the nuclear receptor subfamilies NR3A and -3C, but only MR was affected at PND60. Pyrosequencing of two CpG islands within the MR gene promoter revealed a loss of methylation in DEHP-treated animals that was correlated with reduced MR. Because MR activation is known to stimulate Leydig cell testosterone formation, and MR inhibition to be repressive, our results are consistent with the hypothesis that in utero exposure to DEHP leads to MR dysfunction and thus to depressed testosterone production in the adult. We suggest that decreased MR, possibly epigenetically mediated, is a novel mechanism by which phthalates may affect diverse functions later in life.Artículo Texto completo
|Detection of calcium transients in embryonic stem cells and their differentiated progeny. |
Jason S Meyer,Gregory Tullis,Christopher Pierret,Kathleen M Spears,Jason A Morrison,Mark D Kirk
Cellular and molecular neurobiology 29 2009
A central issue in stem cell biology is the determination of function and activity of differentiated stem cells, features that define the true phenotype of mature cell types. Commonly, physiological mechanisms are used to determine the functionality of mature cell types, including those of the nervous system. Calcium imaging provides an indirect method of determining the physiological activities of a mature cell. Camgaroos are variants of yellow fluorescent protein that act as intracellular calcium sensors in transfected cells. We expressed one version of the camgaroos, Camgaroo-2, in mouse embryonic stem (ES) cells under the control of the CAG promoter system. Under the control of this promoter, Camgaroo-2 fluorescence was ubiquitously expressed in all cell types derived from the ES cells that were tested. In response to pharmacological stimulation, the fluorescence levels in transfected cells correlated with cellular depolarization and hyperpolarization. These changes were observed in both undifferentiated ES cells as well as ES cells that had been neurally induced, including putative neurons that were differentiated from transfected ES cells. The results presented here indicate that Camgaroo-2 may be used like traditional fluorescent proteins to track cells as well as to study the functionality of stem cells and their progeny.
|Impaired terminal differentiation of hippocampal granule neurons and defective contextual memory in PC3/Tis21 knockout mice. |
Farioli-Vecchioli, S; Saraulli, D; Costanzi, M; Leonardi, L; Cinà, I; Micheli, L; Nutini, M; Longone, P; Oh, SP; Cestari, V; Tirone, F
PloS one 4 e8339 2009
Neurogenesis in the dentate gyrus of the adult hippocampus has been implicated in neural plasticity and memory, but the molecular mechanisms controlling the proliferation and differentiation of newborn neurons and their integration into the synaptic circuitry are still largely unknown. To investigate this issue, we have analyzed the adult hippocampal neurogenesis in a PC3/Tis21-null mouse model. PC3/Tis21 is a transcriptional co-factor endowed with antiproliferative and prodifferentiative properties; indeed, its upregulation in neural progenitors has been shown to induce exit from cell cycle and differentiation. We demonstrate here that the deletion of PC3/Tis21 causes an increased proliferation of progenitor cells in the adult dentate gyrus and an arrest of their terminal differentiation. In fact, in the PC3/Tis21-null hippocampus postmitotic undifferentiated neurons accumulated, while the number of terminally differentiated neurons decreased of 40%. As a result, PC3/Tis21-null mice displayed a deficit of contextual memory. Notably, we observed that PC3/Tis21 can associate to the promoter of Id3, an inhibitor of proneural gene activity, and negatively regulates its expression, indicating that PC3/Tis21 acts upstream of Id3. Our results identify PC3/Tis21 as a gene required in the control of proliferation and terminal differentiation of newborn neurons during adult hippocampal neurogenesis and suggest its involvement in the formation of contextual memories.Artículo Texto completo
|Effective cryopreservation of neural stem or progenitor cells without serum or proteins by vitrification. |
L L Kuleshova, F C K Tan, R Magalhães, S S Gouk, K H Lee, G S Dawe, L L Kuleshova, F C K Tan, R Magalhães, S S Gouk, K H Lee, G S Dawe
Cell transplantation 18 135-44 2009
Development of effective cryopreservation protocols will be essential to realizing the potential for clinical application of neural stem and progenitor cells. Current cryopreservation protocols have been largely employed in research, which does not require as stringent consideration of viability and sterility. Therefore, these protocols involve the use of serum and protein additives, which can potentially introduce contaminants, and slow cooling with DMSO/glycerol-based cryopreservation solutions, which impairs cell survival. We investigated whether serum- and protein-free vitrification is effective for functional cryopreservation of neurosphere cultures of neural stem or progenitor cells. To protect the samples from introduction of other contaminants during handling and cryostorage, an original "straw-in-straw" method (250 microl sterile straw placed in 500 microl straw) for direct immersion into liquid nitrogen and storing the samples was also introduced. The protocol employed brief step-wise exposure to vitrification solution composed of ethylene glycol (EG) and sucrose (40% v/v EG, 0.6 M sucrose) and removal of vitrification solution at room temperature. Evaluation of the effects of vitrification revealed that there were no differences between control and vitrified neural stem or progenitor cells in expression of the neural stem or progenitor cell markers, proliferation, or multipotent differentiation. This sterile method for the xeno-free cryopreservation of murine neurospheres without animal or human proteins may have the potential to serve as a starting point for the development of cryopreservation protocols for human neural stem and progenitor cells for clinical use.
|Isoflurane inhibits growth but does not cause cell death in hippocampal neural precursor cells grown in culture. |
Sall, JW; Stratmann, G; Leong, J; McKleroy, W; Mason, D; Shenoy, S; Pleasure, SJ; Bickler, PE
Anesthesiology 110 826-33 2009
Isoflurane causes long-term hippocampal-dependent learning deficits in rats despite limited isoflurane-induced hippocampal cell death, raising questions about the causality between isoflurane-induced cell death and isoflurane-induced cognitive function. Neurogenesis in the dentate gyrus is required for hippocampal-dependent learning and thus constitutes a potential alternative mechanism by which cognition can be altered after neonatal anesthesia. The authors tested the hypothesis that isoflurane alters proliferation and differentiation of hippocampal neural progenitor cells.Multipotent neural progenitor cells were isolated from pooled rat hippocampi (postnatal day 2) and grown in culture. These cells were exposed to isoflurane and evaluated for cell death using lactate dehydrogenase release, caspase activity, and immunocytochemistry for nuclear localization of cleaved caspase 3. Growth was assessed by cell counting and BrdU incorporation. Expression of markers of stemness (Sox2) and cell division (Ki67) were determined by quantitative polymerase chain reaction. Cell fate selection was assessed using immunocytochemistry to stain for neuronal and glial markers.Isoflurane did not change lactate dehydrogenase release, activity of caspase 3/7, or the amount of nuclear cleaved caspase 3. Isoflurane decreased caspase 9 activity, inhibited proliferation, and decreased the proportion of cells in s-phase. messenger ribonucleic acid expression of Sox2 (stem cells) and Ki67 (proliferation) were decreased. Differentiating neural progenitor cells more often select a neuronal fate after isoflurane exposure.The authors conclude that isoflurane does not cause cell death, but it does act directly on neural progenitor cells independently of effects on the surrounding brain to decrease proliferation and increase neuronal fate selection. These changes could adversely affect cognition after isoflurane anesthesia.
|Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. |
Zhu, L; Gibson, P; Currle, DS; Tong, Y; Richardson, RJ; Bayazitov, IT; Poppleton, H; Zakharenko, S; Ellison, DW; Gilbertson, RJ
Nature 457 603-7 2009
Cancer stem cells are remarkably similar to normal stem cells: both self-renew, are multipotent and express common surface markers, for example, prominin 1 (PROM1, also called CD133). What remains unclear is whether cancer stem cells are the direct progeny of mutated stem cells or more mature cells that reacquire stem cell properties during tumour formation. Answering this question will require knowledge of whether normal stem cells are susceptible to cancer-causing mutations; however, this has proved difficult to test because the identity of most adult tissue stem cells is not known. Here, using an inducible Cre, nuclear LacZ reporter allele knocked into the Prom1 locus (Prom1(C-L)), we show that Prom1 is expressed in a variety of developing and adult tissues. Lineage-tracing studies of adult Prom1(+/C-L) mice containing the Rosa26-YFP reporter allele showed that Prom1(+) cells are located at the base of crypts in the small intestine, co-express Lgr5 (ref. 2), generate the entire intestinal epithelium, and are therefore the small intestinal stem cell. Prom1 was reported recently to mark cancer stem cells of human intestinal tumours that arise frequently as a consequence of aberrant wingless (Wnt) signalling. Activation of endogenous Wnt signalling in Prom1(+/C-L) mice containing a Cre-dependent mutant allele of beta-catenin (Ctnnb1(lox(ex3))) resulted in a gross disruption of crypt architecture and a disproportionate expansion of Prom1(+) cells at the crypt base. Lineage tracing demonstrated that the progeny of these cells replaced the mucosa of the entire small intestine with neoplastic tissue that was characterized by focal high-grade intraepithelial neoplasia and crypt adenoma formation. Although all neoplastic cells arose from Prom1(+) cells in these mice, only 7% of tumour cells retained Prom1 expression. Our data indicate that Prom1 marks stem cells in the adult small intestine that are susceptible to transformation into tumours retaining a fraction of mutant Prom1(+) tumour cells.
|Circumventricular organs: a novel site of neural stem cells in the adult brain. |
Bennett, L; Yang, M; Enikolopov, G; Iacovitti, L
Molecular and cellular neurosciences 41 337-47 2009
Neurogenesis in the adult mammalian nervous system is now well established in the subventricular zone of the anterolateral ventricle and subgranular zone of the hippocampus. In these regions, neurons are thought to arise from neural stem cells, identified by their expression of specific intermediate filament proteins (nestin, vimentin, GFAP) and transcription factors (Sox2). In the present study, we show that in adult rat and mouse, the circumventricular organs (CVOs) are rich in nestin+, GFAP+, vimentin+ cells which express Sox2 and the cell cycle-regulating protein Ki67. In culture, these cells proliferate as neurospheres and express neuronal (doublecortin+, beta-tubulin III+) and glial (S100beta+, GFAP+, RIP+) phenotypic traits. Further, our in vivo studies using bromodeoxyuridine show that CVO cells proliferate and undergo constitutive neurogenesis and gliogenesis. These findings suggest that CVOs may constitute a heretofore unknown source of stem/progenitor cells, capable of giving rise to new neurons and/or glia in the adult brain.
|Chd1 regulates open chromatin and pluripotency of embryonic stem cells. |
Gaspar-Maia, A; Alajem, A; Polesso, F; Sridharan, R; Mason, MJ; Heidersbach, A; Ramalho-Santos, J; McManus, MT; Plath, K; Meshorer, E; Ramalho-Santos, M
Nature 460 863-8 2009
An open chromatin largely devoid of heterochromatin is a hallmark of stem cells. It remains unknown whether an open chromatin is necessary for the differentiation potential of stem cells, and which molecules are needed to maintain open chromatin. Here we show that the chromatin remodelling factor Chd1 is required to maintain the open chromatin of pluripotent mouse embryonic stem cells. Chd1 is a euchromatin protein that associates with the promoters of active genes, and downregulation of Chd1 leads to accumulation of heterochromatin. Chd1-deficient embryonic stem cells are no longer pluripotent, because they are incapable of giving rise to primitive endoderm and have a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. Our results indicate that Chd1 is essential for open chromatin and pluripotency of embryonic stem cells, and for somatic cell reprogramming to the pluripotent state.
|Convergence of cells from the progenitor fraction of adult olfactory bulb tissue to remyelinating glia in demyelinating spinal cord lesions. |
Markakis, EA; Sasaki, M; Lankford, KL; Kocsis, JD
PloS one 4 e7260 2009
Progenitor cells isolated from adult brain tissue are important tools for experimental studies of remyelination. Cells harvested from neurogenic regions in the adult brain such as the subependymal zone have demonstrated remyelination potential. Multipotent cells from the progenitor fraction have been isolated from the adult olfactory bulb (OB) but their potential to remyelinate has not been studied.We used the buoyant density gradient centrifugation method to isolate the progenitor fraction and harvest self-renewing multipotent neural cells grown in monolayers from the adult green-fluorescent protein (GFP) transgenic rat OB. OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient. The progenitor fraction was isolated and these cells were plated in growth media with serum for 24 hrs. Cells were then propagated in N2 supplemented serum-free media containing b-FGF. Cells at passage 4 (P4) were introduced into a demyelinated spinal cord lesion. The GFP(+) cells survived and integrated into the lesion, and extensive remyelination was observed in plastic sections. Immunohistochemistry revealed GFP(+) cells in the spinal cord to be glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and neurofilament negative. The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present. Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings.We report that neural cells from the progenitor fraction of the adult rat OB grown in monolayers can be expanded for several passages in culture and that upon transplantation into a demyelinated spinal cord lesion provide extensive remyelination without ectopic neuronal differentiation.
|Up-regulation of nestin in the infarcted myocardium potentially indicates differentiation of resident cardiac stem cells into various lineages including cardiomyocytes. |
Scobioala, S; Klocke, R; Kuhlmann, M; Tian, W; Hasib, L; Milting, H; Koenig, S; Stelljes, M; El-Banayosy, A; Tenderich, G; Michel, G; Breithardt, G; Nikol, S
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 22 1021-31 2008
To identify proteins involved in cardiac regeneration, a proteomics approach was applied. A total of 26 proteins, which displayed aberrant expression in mouse hearts infarcted through ligation of the left anterior descending coronary artery, were identified. These included the intermediate filament protein nestin, which was up-regulated in the infarct border zone. Corresponding changes were observed for its mRNA. Nestin mRNA was also up-regulated in hearts from 17 of 19 patients with end-stage heart failure, including 4 with acute myocardial infarction in comparison with 8 donor hearts. Immunofluorescence confocal laser scanning microscopy revealed that nestin is expressed, on the one hand, in small proportions of cardiomyocytes, endothelial cells, smooth muscle cells, neuronal cells, and fibroblasts. On the other hand, it was found to be coexpressed with the stem cell markers c-kit, Sca-1, Mdr-1, and Abcg2 in small interstitial cells. In infarcted hearts from chimeric mice transplanted with bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice, less than 1% of nestin-positive cells coexpressed EGFP, although EGFP-positive cells were abundant in these. Consequently, enhanced expression of nestin in the injured myocardium might reflect spontaneous regenerative processes supposedly based on the differentiation of resident cardiac stem cells into diverse cardiac cell types.
|Generating neurons from stem cells. |
Andreas Androutsellis-Theotokis, Sachiko Murase, Justin D Boyd, Deric M Park, Daniel J Hoeppner, Rea Ravin, Ronald D G McKay
Methods in molecular biology (Clifton, N.J.) 438 31-8 2008
Recent work shows that major developmental and clinical processes such as central nervous system regeneration and carcinogenesis involve stem cells (SCs) in the brain. In spite of this importance, the requirements of these SCs and their differentiated offspring (neurons, astrocytes, and oligodendrocytes) for survival and proper function are little understood. In vivo, the SCs themselves interact with their environment. This SC niche may be complex because it likely includes cells of the vascular and immune systems. The ability to maintain (1) and differentiate (1 -4) central nervous system (CNS) SCs in tissue culture where they can be pharmacologically or genetically (5) manipulated provides a powerful starting point for understanding their behavior. We present detailed information on the methods that permit CNS SCs to differentiate into functional neurons in tissue culture. Important aspects of the culture systems include (1) homogeneity, so that the input and output of a manipulation is known to involve the SC itself; (2) growth in monolayer to visualize and study individual SCs and their offspring; and (3) the use of fully defined culture components to exclude unknown factors from the culture. These conditions support the differentiation of functional, electrically active neurons. These methods allow cell growth and differentiation from normal adult and diseased tissue derived from both animal models and clinical samples. Ultimate validation of such a system comes from accurate prediction of in vivo effects, and the methods we present for CNS SC culture have also successfully predicted regenerative responses in the injured adult nervous system.
|Markers of adult neural stem cells. |
Methods in molecular biology (Clifton, N.J.) 438 2008
I provide detailed protocols for conduction and troubleshooting the key steps in our three most used experimental designs: (1) prospectively counting and sorting of human neural stem cells (NSCs)/committed progenitors before placing them in culture; (2) high-throughput methods of quantifying changes in NSC/progenitor proliferation, in vitro; and (3) retrovirally tagging NSCs before differentiation to assess cell fates in individual clones. Detailed troubleshooting of immunohistochemical and fluorescence-activated cell sorting staining is described. Some of these techniques overlap with other chapters in this volume. Ultimately, this provision of complementary technical information should help ensure the reader's experimental success.
|Endothelial cell-derived bone morphogenetic proteins regulate glial differentiation of cortical progenitors. |
Tetsuya Imura, Kotoha Tane, Nana Toyoda, Shinji Fushiki
The European journal of neuroscience 27 1596-606 2008
Gliogenesis is an important component of cortical development during the postnatal period. Two macroglial cells are generated in a particular order, i.e. astrocytes first and oligodendrocytes later. The mechanisms underlying this sequence of glial differentiation are unknown but interactions with blood vessels are postulated to play a role. We show, using a mouse in-vitro coculture system, that endothelial cells promote astrocyte differentiation but inhibit oligodendrocyte differentiation of postnatal cortical progenitors. Endothelial cells produce bone morphogenetic proteins (BMPs) to activate Sma- and Mad-related protein (Smad) signalling in progenitors and the effects of endothelial cells on glial differentiation are blocked by the BMP antagonist Noggin. Differentiation of progenitors into astrocytes results in the inhibition of endothelial cell growth, accompanied by changes in gene expression of angiogenic factors, indicating bidirectional interactions between progenitors and endothelial cells. In vivo, Smad signalling is activated in various types of cortical cells including progenitors in association with astrogenesis but is inactivated before the peak of oligodendrogenesis. Capillary vessels isolated from the developing cortex express high levels of BMPs. Together, these results demonstrate that endothelial cells regulate glial differentiation by secreting BMPs in vitro and suggest a similar role in cortical gliogenesis in vivo.
|Ramified microglial cells promote astrogliogenesis and maintenance of neural stem cells through activation of Stat3 function. |
Zhu, P; Hata, R; Cao, F; Gu, F; Hanakawa, Y; Hashimoto, K; Sakanaka, M
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 22 3866-77 2008
The differentiation and proliferation of neural stem cells (NSCs) are regulated by a combination of their intrinsic properties (e.g., transcription factors, epigenetic factors, and microRNA regulation) and cell-extrinsic properties from the microenvironment around NSC (e.g., cytokines, growth factors, and cell-cell contact). Recently, there has been a great interest in clarifying the mechanism of the influence of the microenvironment on NSCs, especially cell-cell contact between NSCs and other types of cells nearby. In this study, we investigated whether microglial (Mi) cells influence the fate of NSCs. Coculture study showed that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs through their paracrine effects. This microglia-induced astrogliogenesis was inhibited by AG490 and by overexpression of the dominant-negative form of Stat3 and SOCS3. Promoter assay revealed transactivation of Stat3 function in NSCs by Mi cells. Gene expression study revealed that mRNA of Notch family members (notch1-3) and sox9 in NSCs was significantly upregulated by Mi cells, and this up-regulation was inhibited by AG490. These results demonstrated that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs by activating Stat3 function and via notch and sox9 signaling pathways.
|Delayed rectifier and A-type potassium channels associated with Kv 2.1 and Kv 4.3 expression in embryonic rat neural progenitor cells. |
Smith, DO; Rosenheimer, JL; Kalil, RE
PloS one 3 e1604 2008
Because of the importance of voltage-activated K(+) channels during embryonic development and in cell proliferation, we present here the first description of these channels in E15 rat embryonic neural progenitor cells derived from the subventricular zone (SVZ). Activation, inactivation, and single-channel conductance properties of recorded progenitor cells were compared with those obtained by others when these Kv gene products were expressed in oocytes.Neural progenitor cells derived from the subventricular zone of E15 embryonic rats were cultured under conditions that did not promote differentiation. Immunocytochemical and Western blot assays for nestin expression indicated that almost all of the cells available for recording expressed this intermediate filament protein, which is generally accepted as a marker for uncommitted embryonic neural progenitor cells. However, a very small numbers of the cells expressed GFAP, a marker for astrocytes, O4, a marker for immature oligodendrocytes, and betaIII-tubulin, a marker for neurons. Using immunocytochemistry and Western blots, we detected consistently the expression of Kv2.1, and 4.3. In whole-cell mode, we recorded two outward currents, a delayed rectifier and an A-type current.We conclude that Kv2.1, and 4.3 are expressed in E15 SVZ neural progenitor cells, and we propose that they may be associated with the delayed-rectifier and the A-type currents, respectively, that we recorded. These results demonstrate the early expression of delayed rectifier and A-type K(+) currents and channels in embryonic neural progenitor cells prior to the differentiation of these cells.Artículo Texto completo
|Extensive contribution of embryonic stem cells to the development of an evolutionarily divergent host. |
Xiang, AP; Mao, FF; Li, WQ; Park, D; Ma, BF; Wang, T; Vallender, TW; Vallender, EJ; Zhang, L; Lee, J; Waters, JA; Zhang, XM; Yu, XB; Li, SN; Lahn, BT
Human molecular genetics 17 27-37 2008
The full potential of embryonic stem (ES) cells to generate precise cell lineages and complex tissues can be best realized when they are differentiated in vivo-i.e. in developing blastocysts. Owing to various practical and ethical constraints, however, it is impossible to introduce ES cells of certain species into blastocysts of the same species. One solution is to introduce ES cells into blastocysts of a different species. However, it is not known whether ES cells can contribute extensively to chimerism when placed into blastocysts of a distantly related species. Here, we address this question using two divergent species, Apodemus sylvaticus and Mus musculus, whose genome sequence differs by approximately 18% from each other. Despite this considerable evolutionary distance, injection of Apodemus ES cells into Mus blastocysts led to viable chimeras bearing extensive Apodemus contributions to all major organs, including the germline, with Apodemus contribution reaching approximately 40% in some tissues. Immunostaining showed that Apodemus ES cells have differentiated into a wide range of cell types in the chimeras. Our results thus provide a proof of principle for the feasibility of differentiating ES cells into a wide range of cell types and perhaps even complex tissues by allowing them to develop in vivo in an evolutionarily divergent host-a strategy that may have important applications in research and therapy. Furthermore, our study demonstrates that mammalian evolution can proceed at two starkly contrasting levels: significant divergence in genome and proteome sequence, yet striking conservation in developmental programs.
|Rest-mediated regulation of extracellular matrix is crucial for neural development. |
Sun, YM; Cooper, M; Finch, S; Lin, HH; Chen, ZF; Williams, BP; Buckley, NJ
PloS one 3 e3656 2008
Neural development from blastocysts is strictly controlled by intricate transcriptional programmes that initiate the down-regulation of pluripotent genes, Oct4, Nanog and Rex1 in blastocysts followed by up-regulation of lineage-specific genes as neural development proceeds. Here, we demonstrate that the expression pattern of the transcription factor Rest mirrors those of pluripotent genes during neural development from embryonic stem (ES) cells and an early abrogation of Rest in ES cells using a combination of gene targeting and RNAi approaches causes defects in this process. Specifically, Rest ablation does not alter ES cell pluripotency, but impedes the production of Nestin(+) neural stem cells, neural progenitor cells and neurons, and results in defective adhesion, decrease in cell proliferation, increase in cell death and neuronal phenotypic defects typified by a reduction in migration and neurite elaboration. We also show that these Rest-null phenotypes are due to the dysregulation of its direct or indirect target genes, Lama1, Lamb1, Lamc1 and Lama2 and that these aberrant phenotypes can be rescued by laminins.Artículo Texto completo
|Ethanol exposure during neurogenesis induces persistent effects on neural maturation: evidence from an ex vivo model of fetal cerebral cortical neuroepithelial progenitor maturation. |
Camarillo, C; Miranda, RC
Gene expression 14 159-71 2008
Ethanol is a significant neuroteratogen. We previously used fetal cortical-derived neurosphere cultures as an ex vivo model of the second trimester ventricular neuroepithelium, and showed that ethanol directly induced fetal stem and progenitor cell proliferation and maturation without inducing death. However, ethanol is defined as a teratogen because of its capacity to persistently disrupt neural maturation beyond a specific exposure period. We therefore utilized a simplified neuronal maturation paradigm to examine the immediate and persistent changes in neuronal migration following ethanol exposure during the phase of neuroepithelial proliferation. Our data indicate that mRNA transcripts for migration-associated genes RhoA, Paxillin (Pxn), and CDC42 were immediately induced following ethanol exposure, whereas dynein light chain, LC8-type 1 (DYNLL1), and growth-associated protein (Gap)-43 were suppressed. With the exception of Gap43, ethanol did not induce persistent changes in the other mRNAs, suggesting that ethanol had an activational, rather than organizational, impact on migration-associated mRNAs. However, despite this lack of persistent effects on these mRNAs, ethanol exposure during the proliferation period significantly increased subsequent neuronal migration. Moreover, differentiating neurons, pretreated with ethanol during the proliferation phase, exhibited reduced neurite branching and an increased length of primary neurites, indicating a persistent destabilization of neuronal maturation. Collectively, our data indicate that ethanol-exposed proliferating neuroepithelial precursors exhibit subsequent differentiation-associated increases in migratory behavior, independent of mRNA transcript levels. These data help explain the increased incidence of cerebral cortical neuronal heterotopias associated with the fetal alcohol syndrome.
|Legume lectin FRIL preserves neural progenitor cells in suspension culture in vitro. |
Yao, H; Xie, X; Li, Y; Wang, D; Han, S; Shi, S; Nan, X; Bai, C; Wang, Y; Pei, X
Clinical & developmental immunology 2008 531317 2008
In vitro maintenance of stem cells is crucial for many clinical applications. Stem cell preservation factor FRIL (Flt3 receptor-interacting lectin) is a plant lectin extracted from Dolichos Lablab and has been found preserve hematopoietic stem cells in vitro for a month in our previous studies. To investigate whether FRIL can preserve neural progenitor cells (NPCs), it was supplemented into serum-free suspension culture media. FRIL made NPC grow slowly, induced cell adhesion, and delayed neurospheres formation. However, FRIL did not initiate NPC differentiation according to immunofluorescence and semiquantitive RT-PCR results. In conclusion, FRIL could also preserve neural progenitor cells in vitro by inhibiting both cell proliferation and differentiation.
|Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. |
Mirzadeh, Z; Merkle, FT; Soriano-Navarro, M; Garcia-Verdugo, JM; Alvarez-Buylla, A
Cell stem cell 3 265-78 2008
Neural stem cells (NSCs, B1 cells) are retained in the walls of the adult lateral ventricles but, unlike embryonic NSCs, are displaced from the ventricular zone (VZ) into the subventricular zone (SVZ) by ependymal cells. Apical and basal compartments, which in embryonic NSCs play essential roles in self-renewal and differentiation, are not evident in adult NSCs. Here we show that SVZ B1 cells in adult mice extend a minute apical ending to directly contact the ventricle and a long basal process ending on blood vessels. A closer look at the ventricular surface reveals a striking pinwheel organization specific to regions of adult neurogenesis. The pinwheel's core contains the apical endings of B1 cells and in its periphery two types of ependymal cells: multiciliated (E1) and a type (E2) characterized by only two cilia and extraordinarily complex basal bodies. These results reveal that adult NSCs retain fundamental epithelial properties, including apical and basal compartmentalization, significantly reshaping our understanding of this adult neurogenic niche.
|The timing of differentiation of adult hippocampal neurons is crucial for spatial memory. |
Farioli-Vecchioli, S; Saraulli, D; Costanzi, M; Pacioni, S; Cinà, I; Aceti, M; Micheli, L; Bacci, A; Cestari, V; Tirone, F
PLoS biology 6 e246 2008
Adult neurogenesis in the dentate gyrus plays a critical role in hippocampus-dependent spatial learning. It remains unknown, however, how new neurons become functionally integrated into spatial circuits and contribute to hippocampus-mediated forms of learning and memory. To investigate these issues, we used a mouse model in which the differentiation of adult-generated dentate gyrus neurons can be anticipated by conditionally expressing the pro-differentiative gene PC3 (Tis21/BTG2) in nestin-positive progenitor cells. In contrast to previous studies that affected the number of newly generated neurons, this strategy selectively changes their timing of differentiation. New, adult-generated dentate gyrus progenitors, in which the PC3 transgene was expressed, showed accelerated differentiation and significantly reduced dendritic arborization and spine density. Functionally, this genetic manipulation specifically affected different hippocampus-dependent learning and memory tasks, including contextual fear conditioning, and selectively reduced synaptic plasticity in the dentate gyrus. Morphological and functional analyses of hippocampal neurons at different stages of differentiation, following transgene activation within defined time-windows, revealed that the new, adult-generated neurons up to 3-4 weeks of age are required not only to acquire new spatial information but also to use previously consolidated memories. Thus, the correct unwinding of these key memory functions, which can be an expression of the ability of adult-generated neurons to link subsequent events in memory circuits, is critically dependent on the correct timing of the initial stages of neuron maturation and connection to existing circuits.
|Cooperation between the Hic1 and Ptch1 tumor suppressors in medulloblastoma. |
Briggs, KJ; Corcoran-Schwartz, IM; Zhang, W; Harcke, T; Devereux, WL; Baylin, SB; Eberhart, CG; Watkins, DN
Genes & development 22 770-85 2008
Medulloblastoma is an embryonal tumor thought to arise from the granule cell precursors (GCPs) of the cerebellum. PATCHED (PTCH), an inhibitor of Hedgehog signaling, is the best-characterized tumor suppressor in medulloblastoma. However, less than 20% of medulloblastomas have mutations in PTCH. In the search for other tumor suppressors, interest has focused on the deletion events at the 17p13.3 locus, the most common genetic defect in medulloblastoma. This chromosomal region contains HYPERMETHYLATED IN CANCER 1 (HIC1), a transcriptional repressor that is a frequent target of epigenetic gene silencing in medulloblastoma. Here we use a mouse model of Ptch1 heterozygosity to reveal a critical tumor suppressor function for Hic1 in medulloblastoma. When compared with Ptch1 heterozygous mutants, compound Ptch1/Hic1 heterozygotes display a fourfold increased incidence of medulloblastoma. We show that Hic1 is a direct transcriptional repressor of Atonal Homolog 1 (Atoh1), a proneural transcription factor essential for cerebellar development, and show that ATOH1 expression is required for human medulloblastoma cell growth in vitro. Given that Atoh1 is also a putative target of Hh signaling, we conclude that the Hic1 and Ptch1 tumor suppressors cooperate to silence Atoh1 expression during a critical phase in GCP differentiation in which malignant transformation may lead to medulloblastoma.
|The role of thioredoxin reductases in brain development. |
Soerensen, J; Jakupoglu, C; Beck, H; Förster, H; Schmidt, J; Schmahl, W; Schweizer, U; Conrad, M; Brielmeier, M
PloS one 3 e1813 2008
The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells.
|The receptor for granulocyte-colony stimulating factor (G-CSF) is expressed in radial glia during development of the nervous system. |
Kirsch, F; Krüger, C; Schneider, A
BMC developmental biology 8 32 2008
Granulocyte colony-stimulating (G-CSF) factor is a well-known hematopoietic growth factor stimulating the proliferation and differentiation of myeloid progenitors. Recently, we uncovered that G-CSF acts also as a neuronal growth factor in the brain, which promotes adult neural precursor differentiation and enhances regeneration of the brain after insults. In adults, the receptor for G-CSF is predominantly expressed in neurons in many brain areas. We also described expression in neurogenic regions of the adult brain, such as the subventricular zone and the subgranular layer of the dentate gyrus. In addition, we found close co-localization of the G-CSF receptor and its ligand G-CSF. Here we have conducted a systematic expression analysis of G-CSF receptor and its ligand in the developing embryo.Outside the central nervous system (CNS) we found G-CSF receptor expression in blood vessels, muscles and their respective precursors and neurons. The expression of the G-CSF receptor in the developing CNS was most prominent in radial glia cells.Our data imply that in addition to the function of G-CSF and its receptor in adult neurogenesis, this system also has a role in embryonic neurogenesis and nervous system development.
|p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation. |
Zheng, H; Ying, H; Yan, H; Kimmelman, AC; Hiller, DJ; Chen, AJ; Perry, SR; Tonon, G; Chu, GC; Ding, Z; Stommel, JM; Dunn, KL; Wiedemeyer, R; You, MJ; Brennan, C; Wang, YA; Ligon, KL; Wong, WH; Chin, L; DePinho, RA
Nature 455 1129-33 2008
Glioblastoma (GBM) is a highly lethal brain tumour presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as a high-grade disease that typically harbours mutations in EGFR, PTEN and INK4A/ARF (also known as CDKN2A), and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF and TP53 events. Here we show that concomitant central nervous system (CNS)-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with notable clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted TP53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of TP53 as well as the expected PTEN mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives increased Myc protein levels and its associated signature. Functional studies validated increased Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of NSCs doubly null for p53 and Pten (p53(-/-) Pten(-/-)) as well as tumour neurospheres (TNSs) derived from this model. Myc also serves to maintain robust tumorigenic potential of p53(-/-) Pten(-/-) TNSs. These murine modelling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumour suppressor mutation profile in human primary GBM and establish Myc as an important target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential.
|Tonic activation of CXC chemokine receptor 4 in immature granule cells supports neurogenesis in the adult dentate gyrus. |
Kolodziej, A; Schulz, S; Guyon, A; Wu, DF; Pfeiffer, M; Odemis, V; Höllt, V; Stumm, R
The Journal of neuroscience : the official journal of the Society for Neuroscience 28 4488-500 2008
Stromal-cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) play a well-established role during embryonic development of dentate gyrus granule cells. However, little is known about the regulation and function of CXCR4 in the postnatal dentate gyrus. Here, we identify a striking mismatch between intense CXCR4 mRNA and limited CXCR4 protein expression in adult rat subgranular layer (SGL) neurons. We demonstrate that CXCR4 protein expression in SGL neurons is progressively lost during postnatal day 15 (P15) to P21. This loss of CXCR4 protein expression was paralleled by a reduction in the number of SDF-1-responsive SGL neurons and a massive upregulation of SDF-1 mRNA in granule cells. Intraventricular infusion of the CXCR4-antagonist AMD3100 dramatically increased CXCR4 protein expression in SGL neurons, suggesting that CXCR4 is tonically activated and downregulated by endogenous SDF-1. Infusion of AMD3100 also facilitated detection of CXCR4 protein in bromodeoxyuridine-, nestin-, and doublecortin-labeled cells and showed that the vast majority of adult-born granule cells transiently expressed CXCR4. Chronic AMD3100 administration impaired formation of new granule cells as well as neurogenesis-dependent long-term recognition of novel objects. Therefore, our findings suggest that tonic activation of CXCR4 in newly formed granule cells by endogenous SDF-1 is essential for neurogenesis-dependent long-term memory in the adult hippocampus.
|A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival. |
Meikle, L; Talos, DM; Onda, H; Pollizzi, K; Rotenberg, A; Sahin, M; Jensen, FE; Kwiatkowski, DJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 27 5546-58 2007
Tuberous sclerosis (TSC) is a hamartoma syndrome caused by mutations in TSC1 or TSC2 in which cerebral cortical tubers and seizures are major clinical issues. We have engineered mice in which most cortical neurons lose Tsc1 expression during embryonic development. These Tsc1 mutant mice display several neurological abnormalities beginning at postnatal day 5 with subsequent failure to thrive and median survival of 35 d. The mice also display clinical and electrographic seizures both spontaneously and with physical stimulation, and some seizures end in a fatal tonic phase. Many cortical and hippocampal neurons are enlarged and/or dysplastic in the Tsc1 mutant mice, strongly express phospho-S6, and are ectopic in multiple sites in the cortex and hippocampus. There is a striking delay in myelination in the mutant mice, which appears to be caused by an inductive neuronal defect. This new TSC brain model replicates several features of human TSC brain lesions and implicates an important function of Tsc1/Tsc2 in neuronal development.
|Developmental changes and injury induced disruption of the radial organization of the cortex in the immature rat brain revealed by in vivo diffusion tensor MRI. |
Sizonenko, SV; Camm, EJ; Garbow, JR; Maier, SE; Inder, TE; Williams, CE; Neil, JJ; Huppi, PS
Cerebral cortex (New York, N.Y. : 1991) 17 2609-17 2007
During brain development, morphological changes modify the cortex from its immature radial organization to its mature laminar appearance. Applying in vivo diffusion tensor imaging (DTI), the microstructural organization of the cortex in the immature rat was analyzed and correlated to neurohistopathology. Significant differences in apparent diffusion coefficient (ADC) and fractional anisotropy (FA) were detected between the external (I-III) and deep (IV-VI) cortical layers in postnatal day 3 (P3) and P6 pups. With cortical maturation, ADC was reduced in both cortical regions, whereas a decrease in FA was only seen in the deep layers. A distinct radial organization of the external cortical layers with the eigenvectors perpendicular to the pial surface was observed at both ages. Histology revealed maturational differences in the cortical architecture with increased neurodendritic density and reduction in the radial glia scaffolding. Early DTI after hypoxia-ischemia at P3 shows reduced ADC and FA in the ipsilateral cortex that persisted at P6. Cortical DTI eigenvector maps reveal microstructural disruption of the radial organization corresponding to regions of neuronal death, radial glial disruption, and astrocytosis. Thus, the combined use of in vivo DTI and histopathology can assist in delineating normal developmental changes and postinjury modifications in the immature rodent brain.
|Expression change of stem cell-derived neural stem/progenitor cell supporting factor gene in injured spinal cord of rats. |
Yi Feng, Yi-Lu Gao, Fei Ding, Yan Liu
Neuroscience bulletin 23 165-9 2007
OBJECTIVE: To explore the expression change of stem cell-derived neural stem/progenitor cell supporting factor (SDNSF) gene in the injuried spinal cord tissues of rats, and the relation between the expressions of SDNSF and nestin. METHODS: The spinal cord contusion model of rat was established according to Allen's falling strike method. The expression of SDNSF was studied by RT-PCR and in situ hybridization (ISH), and the expression of nestin was detected by immunochemistry. RESULTS: RT-PCR revealed that SDNSF mRNA was upregulated on day 4 after injury, peaked on day 8-12, and decreased to the sham operation level on day 16. ISH revealed that SDNSF mRNA was mainly expressed in the gray matter cells, probably neurons, of spinal cord. The immunohistochemistry showed that accompanied with SDNSF mRNA upregulation, the nestin-positive cells showed erupted roots, migrated peripherad and proliferation on the 8-day slice. However, the distribution pattern of these new cells was different from that of SDNSF-positive cells. CONCLUSION: (1) SDNSF is expressed in the gray matter of spinal cord. The expression of SDNSF mRNA in the spinal cord varies with injured time. (2) The nestin-positive cells proliferate accompanied with spinal cord injury repair, but do not secrete SDNSF.
|The diffuse stellate cell system. |
Liena Zhao,Alastair D Burt
Journal of molecular histology 38 2007
Hepatic stellate cells (HSCs) play an important role in liver fibrogenesis. Morphologically similar cells have been found at extrahepatic sites such as pancreas, kidney and colon. The true phenotypic relationship between these cells has not been fully established. We carried out immunohistochemical staining in normal tissues from liver, kidney, colon, pancreas, lung and heart, obtained from a range of species. Immunoreactivity to antibodies directed to synemin, glial fibrillary acidic protein (GFAP), nestin, neurofilament-L, beta-tubulin, protein gene product 9.5 (PGP9.5), S100, desmin, alpha-smooth muscle actin (alpha-SMA) and vimentin was examined. Synemin was identified in HSCs, pancreatic stellate cells, mesangial cells and in peribronchiolar stellate-shaped fibroblasts. GFAP positivity was detected in HSCs and peribronchiolar stellate-shaped fibroblasts. Desmin immunoreactivity was detected in HSCs, pancreatic stellate cells, mesangial cells, periglomerular and peritubular fibroblasts, subepithelial fibroblasts, as well as in peribronchiolar stellate-shaped fibroblasts. Vimentin expression was evident in HSCs, periductal fibroblasts, pancreatic stellate cells, fibroblasts within the fibroconnective tissue capsule, mesangial cells, subepithelial fibroblasts and the interstitial cells of Cajal, as well as in peribronchiolar fibroblasts. Mesangial cells and peritubular fibroblasts showed nestin immunoreactivity. Our data indicates that mesenchymal cells at extrahepatic sites express many of the neural and muscle-associated proteins seen in HSCs; there are however species differences in the expression pattern of these proteins. The findings support the concept of a diffuse stellate cell system in mammals.
|Stress and electroconvulsive seizure differentially alter GPR56 expression in the adult rat brain. |
Go Suzuki, Yasunari Kanda, Masashi Nibuya, Takeshi Hiramoto, Teppei Tanaka, Kunio Shimizu, Yasuhiro Watanabe, Soichiro Nomura
Brain research 1183 21-31 2007
GPR56, a member of the G-protein-coupled receptor family, plays a role in the formation of the frontal and parietal brain lobes and cortical lamination in the embryonic stage. A recent report indicated the existence of GPR56 transcripts in the subventricular zone (SVZ) and hippocampal subgranular zone (SGZ) of the adult mouse brain. Both these regions are known to continually produce neural progenitor cells in the adult brain. Here, we demonstrate abundant GPR56 protein expression in the ependymal cell layer and SVZ as well as its reciprocal translational regulation by a 12-day behavioral stress paradigm and 10-day electroconvulsive seizure (ECS) treatment. Our study revealed that GPR56 transcript expression in the hippocampus was regulated by stress and seizure in a manner identical to that in the SVZ. GPR56 expression was downregulated by stress and upregulated by the ECS treatment in both regions, whereas nestin expression showed no changes. Western blot analysis revealed a robust ECS-induced increase in brain-derived neurotrophic factor expression in the wall of the lateral ventricle including the ependymal cell layer and the SVZ, which may provide a possible regulatory mechanism for GPR56 expression. We consider that GPR56 is expressed in the ependymal cell layer and in immature progenitor cells and that its expression is regulated by functional stimulation.
|Citron kinase is required for postnatal neurogenesis in the hippocampus. |
James B Ackman,Raddy L Ramos,Matthew R Sarkisian,Joseph J Loturco
Developmental neuroscience 29 2007
The dentate gyrus is a site of continual neurogenesis in the postnatal mammalian brain. Here we investigated postnatal neurogenesis in the citron kinase (citron-K) null-mutant rat (flathead). The flathead rat has substantial deficits in embryonic neurogenesis that are due to failed cytokinesis and cell death. We report here the loss of citron-K function has an even severer effect on postnatal neurogenesis in the dentate gyrus. Analysis of phosphorylated histone H3 expression in postnatal neurogenic regions of the flathead mutant revealed a complete lack of mitotic cells in the dentate gyrus and a large reduction in the number of dividing cells in the flathead subventricular zone. Examination of 5-bromodeoxyuridine incorporation in the flathead rat revealed that the flathead rat had a 99% reduction in the number of newly generated cells in the dentate gyrus at postnatal day 10. In addition, doublecortin-positive cells were essentially absent from the postnatal flathead dentate gyrus which also lacked the vimentin- and nestin-positive radial glia scaffold that defines the neurogenic niche in the postnatal subgranular zone. Together these results indicate that postnatal neurogenesis in the dentate gyrus is eliminated by loss of citron-K function, and suggests that a citron-K-dependent progenitor lineage forms the postnatal neuronal progenitor population in the dentate gyrus.Artículo Texto completo
|Smad-interacting protein-1 (Zfhx1b) acts upstream of Wnt signaling in the mouse hippocampus and controls its formation. |
Miquelajauregui, A; Van de Putte, T; Polyakov, A; Nityanandam, A; Boppana, S; Seuntjens, E; Karabinos, A; Higashi, Y; Huylebroeck, D; Tarabykin, V
Proceedings of the National Academy of Sciences of the United States of America 104 12919-24 2007
Smad-interacting protein-1 (Sip1) [Zinc finger homeobox (Zfhx1b)] is a transcription factor implicated in the genesis of Mowat-Wilson syndrome in humans. Sip1 expression in the dorsal telencephalon of mouse embryos was documented from E12.5. We inactivated the gene specifically in cortical precursors. This resulted in the lack of the entire hippocampal formation. Sip1 mutant mice exhibited death of differentiating cells and decreased proliferation in the region of the prospective hippocampus and dentate gyrus. The expression of the Wnt antagonist Sfrp1 was ectopically activated, whereas the activity of the noncanonical Wnt effector, JNK, was down-regulated in the embryonic hippocampus of mutant mice. In cortical cells, Sip1 protein was detected on the promoter of Sfrp1 gene and both genes showed a mutually exclusive pattern of expression suggesting that Sfrp1 expression is negatively regulated by Sip1. Sip1 is therefore essential to the development of the hippocampus and dentate gyrus, and is able to modulate Wnt signaling in these regions.
|Conditional ablation of Stat3 or Socs3 discloses a dual role for reactive astrocytes after spinal cord injury |
Okada, Seiji, et al
Nat Med, 12:829-34 (2006) 2006
|Non-psychoactive CB2 cannabinoid agonists stimulate neural progenitor proliferation. |
Palazuelos, Javier, et al.
FASEB J., 20: 2405-7 (2006) 2006
|Designer self-assembling Peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures |
Gelain, Fabrizio, et al.
PLoS ONE, 1:e119 (2006) 2006
|Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells. |
Hongxiu Ning, Guiting Lin, Tom F Lue, Ching-Shwun Lin
Differentiation; research in biological diversity 74 510-8 2006
Adipose tissue-derived stromal cells (ADSC) have previously been shown to possess stem cell properties such as transdifferentiation and self-renewal. Because future clinical applications are likely to use these adult stem cells in an autologous fashion, we wished to establish and characterize rat ADSC for pre-clinical tests. In the present study, we showed that rat ADSC expressed stem cell markers CD34 and STRO-1 at passage 1 but only STRO-1 at passage 3. These cells could also be induced to differentiate into adipocytes, smooth muscle cells, and neuron-like cells, the latter of which expressed neuronal markers S100, nestin, and NF70. Isobutylmethylxanthine (IBMX), indomethacin (INDO), and insulin were the active ingredients in a previously established neural induction medium (NIM); however, here we showed that IBMX alone was as effective as NIM in the induction of morphological changes as well as neuronal marker expression. Finally, we showed that vascular smooth muscle cells could also be induced by either NIM or IBMX to differentiate into neuron-like cells that expressed NF70.
|The endocannabinoid system promotes astroglial differentiation by acting on neural progenitor cells. |
Aguado, Tania, et al.
J. Neurosci., 26: 1551-61 (2006) 2006
Endocannabinoids exert an important neuromodulatory role via presynaptic cannabinoid CB1 receptors and may also participate in the control of neural cell death and survival. The function of the endocannabinoid system has been extensively studied in differentiated neurons, but its potential role in neural progenitor cells remains to be elucidated. Here we show that the CB1 receptor and the endocannabinoid-inactivating enzyme fatty acid amide hydrolase are expressed, both in vitro and in vivo, in postnatal radial glia (RC2+ cells) and in adult nestin type I (nestin(+)GFAP+) neural progenitor cells. Cell culture experiments show that CB1 receptor activation increases progenitor proliferation and differentiation into astroglial cells in vitro. In vivo analysis evidences that, in postnatal CB1(-/-) mouse brain, progenitor proliferation and astrogliogenesis are impaired. Likewise, in adult CB1-deficient mice, neural progenitor proliferation is decreased but is increased in fatty acid amide hydrolase-deficient mice. In addition, endocannabinoid signaling controls neural progenitor differentiation in the adult brain by promoting astroglial differentiation of newly born cells. These results show a novel physiological role of endocannabinoids, which constitute a new family of signaling cues involved in the regulation of neural progenitor cell function.
|Sustained angiopoietin-2 expression disrupts vessel formation and inhibits glioma growth. |
Lee, OH; Fueyo, J; Xu, J; Yung, WK; Lemoine, MG; Lang, FF; Bekele, BN; Zhou, X; Alonso, MA; Aldape, KD; Fuller, GN; Gomez-Manzano, C
Neoplasia (New York, N.Y.) 8 419-28 2006
Systematic analyses of the expression of angiogenic regulators in cancer models should yield useful information for the development of novel therapies for malignant gliomas. In this study, we analyzed tumor growth, vascularization, and angiopoietin-2 (Ang2) expression during the development of U-87 MG xenografts. We found that tumoral angiogenesis in this model follows a multistage process characterized by avascular, prolific peripheral angiogenesis, and late vascular phases. On day 4, we observed an area of central necrosis, a peripheral ring of Ang2-positive glioma cells, and reactive Ang2-positive vascular structures in the tumor/brain interface. When the tumor had developed a vascular network, Ang2 was expressed only in peripheral vascular structures. Because Ang2 expression was downmodulated in the late stages of development, probably to maintain a stable tumoral vasculature, we next studied whether sustained Ang2 expression might impair vascular development and, ultimately, tumor growth. Ang2 prevented the formation of capillary-like structures by and impaired angiogenesis in a chorioallantoic membrane chicken model. Finally, we tested the effect of sustained Ang2 expression on U-87 MG xenograft development. Ang2 significantly prolonged the survival of intracranial U-87 MG tumor-bearing animals. Examination of Ang2-treated xenografts revealed areas of tumor necrosis and vascular damage. We therefore conclude that deregulated Ang2 expression during gliomagenesis hindered successful angiogenesis and that therapies that sustain Ang2 expression might be effective against malignant gliomas.Artículo Texto completo
|Overexpression of SOCS3 inhibits astrogliogenesis and promotes maintenance of neural stem cells. |
Fang Cao, Ryuji Hata, Pengxiang Zhu, Yong-Jie Ma, Junya Tanaka, Yasushi Hanakawa, Koji Hashimoto, Michio Niinobe, Kazuaki Yoshikawa, Masahiro Sakanaka
Journal of neurochemistry 98 459-70 2006
To investigate the effects of suppressors of cytokine signaling 3 (SOCS3) on neural stem cell fate, stem cells were infected with an adenoviral vector expressing SOCS3. Three days later, western blot analysis and immunocytochemical analysis revealed that the protein level of MAP2 and the number of MAP2-positive cells were significantly increased in SOCS3-transfected cells, whereas the protein level of GFAP and the number of GFAP-positive cells were significantly decreased. Furthermore, promoter assay revealed a significant reduction in the transcriptional level of signal transducer and activator of transcription 3 (Stat3) in the transfected cells. In addition, the mRNA levels of Notch family member (notch1) and inhibitory basic helix-loop-helix (bHLH) factors (hes5 and id3) were significantly up-regulated 1 day after overexpression of SOCS3. Three days after transfection, the mRNA level of hes5 was significantly decreased, whereas that of notch1 was still up-regulated. Moreover, all of SOCS3-positive cells expressed Nestin protein but did not express MAP2 or GFAP proteins. These data indicate that overexpression of SOCS3 induced neurogenesis and inhibited astrogliogenesis in neural stem cells. Our data also show that SOCS3 promoted maintenance of neural stem cells.
|Use of differentiating embryonic stem cells in the Parkinsonian mouse model. |
Fumihiko Nishimura, Hayato Toriumi, Shigeaki Ishizaka, Toshisuke Sakaki, Masahide Yoshikawa, Fumihiko Nishimura, Hayato Toriumi, Shigeaki Ishizaka, Toshisuke Sakaki, Masahide Yoshikawa
Methods in molecular biology (Clifton, N.J.) 329 485-93 2006
Progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the following reduction in striatal dopamine cause Parkinson's disease (PD). Transplantation of dopamine-producing cells into the striatum is a proposed treatment modality. In this report, we describe a model experiment assessing the effectiveness of mouse embryonic stem (ES) cell-derived dopaminergic neurons using a mouse model of PD. ES cells were shown to be an attractive and promising source for the generation of dopaminergic neurons, and the mouse PD model was useful to assess the efficacy of transplantation therapy with dopamine-producing cells, including ES cell-derived dopaminergic neurons.
|The transcriptional coactivator Querkopf controls adult neurogenesis. |
Merson, TD; Dixon, MP; Collin, C; Rietze, RL; Bartlett, PF; Thomas, T; Voss, AK
The Journal of neuroscience : the official journal of the Society for Neuroscience 26 11359-70 2006
The adult mammalian brain maintains populations of neural stem cells within discrete proliferative zones. Understanding of the molecular mechanisms regulating adult neural stem cell function is limited. Here, we show that MYST family histone acetyltransferase Querkopf (Qkf, Myst4, Morf)-deficient mice have cumulative defects in adult neurogenesis in vivo, resulting in declining numbers of olfactory bulb interneurons, a population of neurons produced in large numbers during adulthood. Qkf-deficient mice have fewer neural stem cells and fewer migrating neuroblasts in the rostral migratory stream. Qkf gene expression is strong in the neurogenic subventricular zone. A population enriched in multipotent cells can be isolated from this region on the basis of Qkf gene expression. Neural stem cells/progenitor cells isolated from Qkf mutant mice exhibited a reduced self-renewal capacity and a reduced ability to produce differentiated neurons. Together, our data show that Qkf is essential for normal adult neurogenesis.
|Intermediate filament protein nestin is expressed in developing kidney and heart and might be regulated by the Wilms' tumor suppressor Wt1. |
Wagner, N; Wagner, KD; Scholz, H; Kirschner, KM; Schedl, A
American journal of physiology. Regulatory, integrative and comparative physiology 291 R779-87 2006
Nestin is an intermediate filament protein originally described in neural stem cells and a variety of progenitor cells. More recently, nestin was detected in rat kidney podocytes. We show here that nestin is expressed in a developmentally regulated pattern in the kidney. Nestin was detected by immunohistochemistry in the condensing mesenchyme surrounding the ureter, in developing glomeruli, in podocytes of the adult kidney, and in a podocyte cell line. Nestin shared a striking overlap in expression with the Wilms' tumor suppressor Wt1. Nestin was significantly upregulated in a cell line with inducible Wt1 expression upon induction of Wt1. Cotransfection experiments in human embryonic kidney cells (HEK293) revealed stimulation of a nestin intron 2 enhancer element up to six-fold by the Wt1(-KTS) splice variant. Nestin expression was significantly reduced in an inducible mouse model of glomerular disease. This model is based on podocyte-specific overexpression of Pax2 and associated with a loss of Wt1 expression. Furthermore, also in the developing heart, nestin was found in an overlapping pattern with Wt1 in the epicardium and the forming coronary vessels. Strikingly, in the hearts of Wt1 knockout mice, nestin was barely detectable compared with the hearts of wild-type embryos. Our results show that nestin is expressed at different stages of kidney and cardiac development and suggest that its expression in these organs might be regulated by the Wilms' tumor suppressor Wt1.
|Human embryonic stem cells express an immunogenic nonhuman sialic acid. |
Martin, Maria J, et al.
Nat. Med., 11: 228-32 (2005) 2005
|Neuro-glial differentiation of human bone marrow stem cells in vitro. |
Bossolasco, P, et al.
Exp. Neurol., 193: 312-25 (2005) 2005
Bone marrow (BM) is a rich source of stem cells and may represent a valid alternative to neural or embryonic cells in replacing autologous damaged tissues for neurodegenerative diseases. The purpose of the present study is to identify human adult BM progenitor cells capable of neuro-glial differentiation and to develop effective protocols of trans-differentiation to surmount the hematopoietic commitment in vitro. Heterogeneous cell populations such as whole BM, low-density mononuclear and mesenchymal stem (MSCs), and several immunomagnetically separated cell populations were investigated. Among them, MSCs and CD90+ cells were demonstrated to express neuro-glial transcripts before any treatment. Several culture conditions with the addition of stem cell or astroblast conditioned media, different concentrations of serum, growth factors, and supplements, used alone or in combinations, were demonstrated to alter the cellular morphology in some cell subpopulations. In particular, MSCs and CD90+ cells acquired astrocytic and neuron-like morphologies in specific culture conditions. They expressed several neuro-glial specific markers by RT-PCR and glial fibrillary acid protein by immunocytochemistry after co-culture with astroblasts, both in the absence or presence of cell contact. In addition, floating neurosphere-like clones have been observed when CD90+ cells were grown in neural specific media. In conclusion, among the large variety of human adult BM cell populations analyzed, we demonstrated the in vitro neuro-glial potential of both the MSC and CD90+ subset of cells. Moreover, unidentified soluble factors provided by the conditioned media and cellular contacts in co-culture systems were effective in inducing the neuro-glial phenotype, further supporting the adult BM neural differentiative capability.
|Suppression of Stat3 promotes neurogenesis in cultured neural stem cells. |
Feng Gu, Ryuji Hata, Yong-Jie Ma, Junya Tanaka, Noriaki Mitsuda, Yoshiaki Kumon, Yasushi Hanakawa, Koji Hashimoto, Koichi Nakajima, Masahiro Sakanaka
Journal of neuroscience research 81 163-71 2005
To investigate the effects of signal transducer and activator of transcription 3 (Stat3) on neural stem cell fate, stem cells were inoculated with an adenovirus vector expressing dominant negative form of Stat3 (Stat3F). One day later, a promoter assay revealed significant reduction of the transcriptional level in the transfected cells. Three days later, Western blot analysis and immunocytochemical analysis revealed that the protein level of microtubule-associated protein (MAP)2 and the number of MAP2-positive cells were increased significantly in the transfected cells whereas the protein level of glial fibrillary acidic protein (GFAP) and the number of GFAP-positive cells were decreased significantly. In addition, mRNA levels of Notch family members (Notch1, 2, and 3) and of inhibitory basic helix-loop-helix (bHLH) factors (Hes5, Id2, and Id3) were significantly downregulated at 3 days after viral inoculation with Stat3F; however, mRNA levels of bHLH determination factors (Math1 and Neurogenin3) and bHLH differentiation factors (NeuroD1 and NeuroD2) were significantly upregulated. These data indicated that suppression of Stat3 directly induced neurogenesis and inhibited astrogliogenesis in neural stem cells.
|Differentiation of mouse embryonic stem cells into neurons using conditioned medium of dorsal root ganglia. |
Ayako Kitazawa, Norio Shimizu
Journal of bioscience and bioengineering 100 94-9 2005
Mouse embryonic stem (ES) cells, which are continuously growing cell lines, have a pluripotent ability to differentiate into various cell lineages in vitro including neurons. We investigated the effects of chick dorsal root ganglion (DRG) conditioned medium (CM) and nerve growth factor (NGF) on the directed differentiation of ES cells into neurons. Because DRGs from 8-day-old chick embryos are often used in bioassays of neurotrophic factors, DRGs may release soluble factors that can induce ES cell differentiation into neurons in a culture broth. When cultivated in a Dulbecco's modified Eagle's medium (DMEM)/F-12K medium containing DRG-CM or NGF, the ES cell colonies clearly showed neurite outgrowths. Of particular significance, the immunofluorescence analysis of ES cell colonies using an anti-betaIII-tubulin antibody indicated that the addition of DRG-CM effectively promoted the differentiation of ES cells into neurons. We confirmed the effect of DRG-CM addition on ES cell differentiation into neurons via neuronal stem cells by the immunofluorescence analysis of ES cell colonies. Thus, DRG-CM appeared to effectively promote ES cell differentiation into neurons.
|Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation. |
Jung, CG; Kim, HJ; Kawaguchi, M; Khanna, KK; Hida, H; Asai, K; Nishino, H; Miura, Y
Development (Cambridge, England) 132 5137-45 2005
The present study aimed to elucidate the function of AT motif-binding factor 1 (ATBF1) during neurogenesis in the developing brain and in primary cultures of neuroepithelial cells and cell lines (Neuro 2A and P19 cells). Here, we show that ATBF1 is expressed in the differentiating field in association with the neuronal differentiation markers beta-tubulin and MAP2 in the day E14.5 embryo rat brain, suggesting that it promotes neuronal differentiation. In support of this, we show that ATBF1 suppresses nestin expression, a neural stem cell marker, and activates the promoter of Neurod1 gene, a marker for neuronal differentiation. Furthermore, we show that in Neuro 2A cells, overexpressed ATBF1 localizes predominantly in the nucleus and causes cell cycle arrest. In P19 cells, which formed embryonic bodies in the floating condition, ATBF1 is mainly cytoplasmic and has no effect on the cell cycle. However, the cell cycle was arrested when ATBF1 became nuclear after transfer of P19 cells onto adhesive surfaces or in isolated single cells. The nuclear localization of ATBF1 was suppressed by treatment with caffeine, an inhibitor of PI(3)K-related kinase activity of ataxa-telangiectasia mutated (ATM) gene product. The cytoplasmic localization of ATBF1 in floating/nonadherent cells is due to CRM1-dependent nuclear export of ATBF1. Moreover, in the embryonic brain ATBF1 was expressed in the cytoplasm of proliferating stem cells on the ventricular zone, where cells are present at high density and interact through cell-to-cell contact. Conversely, in the differentiating field, where cell density is low and extracellular matrix is dense, the cell-to-matrix interaction triggered nuclear localization of ATBF1, resulting in the cell cycle arrest. We propose that ATBF1 plays an important role in the nucleus by organizing the neuronal differentiation associated with the cell cycle arrest.
|Dedifferentiation of adult human myoblasts induced by ciliary neurotrophic factor in vitro. |
Chen, X; Mao, Z; Liu, S; Liu, H; Wang, X; Wu, H; Wu, Y; Zhao, T; Fan, W; Li, Y; Yew, DT; Kindler, PM; Li, L; He, Q; Qian, L; Wang, X; Fan, M
Molecular biology of the cell 16 3140-51 2005
Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells--they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These "progenitor cells" retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.
|Trimethyltin-induced neurogenesis in the murine hippocampus. |
Harry, G Jean, et al.
Neurotoxicity research, 5: 623-7 (2004) 2004
Neurogenesis continues to occur in the mature rodent brain with one of the most prominent sources for new neurons being the subgranular layer (SGL) of the dentate gyrus (DG) in the hippocampus. A number of factors can stimulate this process including synaptic activity and injury. To determine if this process would occur upon a direct injury to the dentate region, we exposed young, 21 day old male CD-1 mice to the hippocampal toxicant, trimethyltin (TMT). An acute i.p. injection of TMT (2 mg/kg) produced extensive damage and loss of dentate granule neurons within 72 h. This active period of degeneration was accompanied by an increase in the generation of progenitor cells within the SGL as identified by BrdU uptake and Ki-67 immunostaining. As additional markers for neurogenesis, both nestin and doublecortin showed increased staining patterns within the blades of the dentate. In these young weanling mice, the level of proliferation was sufficient to significantly repopulate the dentate region by 4 weeks post-TMT, suggesting a high level of regenerative potential. Our data indicate a significant level of neurogenesis occurring during the active process of degeneration and in an environment of microglia activation. The TMT-induced injury offers a model system for further examination of the process of neurogenesis, neural adaptation, and the influence of inflammatory factors and glia interactions.
|Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases |
Noctor, Stephen C, et al
Nat Neurosci, 7:136-44 (2004) 2004
|Reelin promotes hippocampal dendrite development through the VLDLR/ApoER2-Dab1 pathway. |
Niu, Sanyong, et al.
Neuron, 41: 71-84 (2004) 2004
Reelin is a secreted glycoprotein that regulates neuronal positioning in cortical brain structures through the VLDLR and ApoER2 receptors and the adaptor protein Dab1. In addition to cellular disorganization, dendrite abnormalities are present in the brain of reeler mice lacking Reelin. It is unclear whether these defects are due primarily to cellular ectopia or the absence of Reelin. Here we examined dendrite development in the hippocampus of normal and mutant mice and in dissociated cultures. We found that dendrite complexity is severely reduced in homozygous mice deficient in Reelin signaling both in vivo and in vitro, and it is also reduced in heterozygous mice in the absence of cellular ectopia. Addition of Reelin interfering antibodies, receptor antagonists, and Dab1 phosphorylation inhibitors prevented dendrite outgrowth from normal neurons, whereas addition of recombinant Reelin rescued the deficit in reeler cultures. Thus, the same signaling pathway controls both neuronal migration and dendrite maturation.
|VIP and PACAP induce selective neuronal differentiation of mouse embryonic stem cells. |
Michèle Cazillis, Bruno J Gonzalez, Claude Billardon, Alain Lombet, Alexandre Fraichard, Jacques Samarut, Pierre Gressens, Hubert Vaudry, William Rostène
The European journal of neuroscience 19 798-808 2004
The capacity of embryonic stem cells (ES cells) to differentiate into neuronal cells represents a potential source for neuronal replacement and a model for studying factors controlling early stages of neuronal differentiation. Various molecules have been used to induce such differentiation but so far neuropeptides acting via functional G-protein-coupled receptors (GPCRs) have not been investigated. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides expressed in early development which affect neuronal precursor proliferation and neuronal differentiation. VIP and PACAP share two common receptors (VPAC1 and VPAC2 receptors) while only PACAP binds with high affinity to PAC1 receptors. The aim of the study was to determine whether VIP and PACAP could produce functional neuronal differentiation of ES cells. Mouse ES cells were allowed to aggregate in embryoid bodies (EBs) in the presence or not of VIP and PACAP for 1 week. VIP and PACAP potently increased the proportion of EB-derived cells expressing specifically a neuronal phenotype shown by immunocytochemistry and neurite outgrowth without altering glial cell number. Binding and RT-PCR analyses demonstrated the presence of VPAC2 and PAC1 receptors on ES cells. Accordingly, both peptides increased cyclic AMP and intracellular calcium. In contrast, EB-derived cells only expressed a functional PAC1 receptor, suggesting a switch in GPCR phenotype during ES cell differentiation. These original data demonstrate that functional GPCRs for VIP and PACAP are present on ES cells and that these neuropeptides may induce their differentiation into a neuronal phenotype. It opens an exciting new field for neuropeptide regulation of tissue ontogenesis.
|Enrichment and identification of human 'fetal' epidermal stem cells. |
Zhou, JX; Chen, SY; Liu, WM; Cao, YJ; Duan, EK
Human reproduction (Oxford, England) 19 968-74 2004
Human epidermis, a continuously renewing tissue, is maintained throughout life by stem cells that proliferate and replenish worn-out or damaged cells in the tissue. Cultured human epidermal stem cells have great potential in clinical application. However, isolating and culturing a pure population of epidermal stem cells has proven to be challenging.We show that p63, a new marker for epidermal stem cells, is expressed in the basal layer of human fetal epidermis using immunohistochemistry, and that keratinocytes with the characteristics of stem cells can be isolated from the epidermis of aborted human fetuses aged greater than /=20 weeks based on high expression of beta(1) integrins by fluorescence-activated cell sorting. Furthermore, the enriched population showed the expression of molecular markers of putative human epidermal stem cells under a confocal microscope and a high colony formation efficiency when it was cultured at a clonal density. Under an electron microscope the sorted stem cells exhibited a high nuclear:cytoplasmic ratio and fewer organelles than the transit amplifying cells. The cultured epidermal stem cells can also be amplified and induced to terminal differentiation by suspension in vitro.Human 'fetal' epidermal stem cells have been successfully isolated and cultured in vitro. The cultured human epidermal stem cells could be used as a tool for studying stem cell biology and testing stem cell therapy.
|The remyelinating potential and in vitro differentiation of MOG-expressing oligodendrocyte precursors isolated from the adult rat CNS. |
A J Crang, J M Gilson, W-W Li, W F Blakemore
The European journal of neuroscience 20 1445-60 2004
There is a long-standing controversy as to whether oligodendrocytes may be capable of cell division and thus contribute to remyelination. We recently published evidence that a subpopulation of myelin oligodendrocyte glycoprotein (MOG)-expressing cells in the adult rat spinal cord co-expressed molecules previously considered to be restricted to oligodendrocyte progenitors [G. Li et al. (2002) Brain Pathol., 12, 463-471]. To further investigate the properties of MOG-expressing cells, anti-MOG-immunosorted cells were grown in culture and transplanted into acute demyelinating lesions. The immunosorting protocol yielded a cell preparation in which over 98% of the viable cells showed anti-MOG- and O1-immunoreactivity; 12-15% of the anti-MOG-immunosorted cells co-expressed platelet-derived growth factor alpha receptor (PDGFRalpha) or the A2B5-epitope. When cultured in serum-free medium containing EGF and FGF-2, 15-18% of the anti-MOG-immunosorted cells lost anti-MOG- and O1-immunoreactivity and underwent cell division. On removal of these growth factors, cells differentiated into oligodendrocytes, or astrocytes and Schwann cells when the differentiation medium contained BMPs. Transplantation of anti-MOG-immunosorted cells into areas of acute demyelination immediately after isolation resulted in the generation of remyelinating oligodendrocytes and Schwann cells. Our studies indicate that the adult rat CNS contains a significant number of oligodendrocyte precursors that express MOG and galactocerebroside, molecules previously considered restricted to mature oligodendrocytes. This may explain why myelin-bearing oligodendrocytes were considered capable of generating remyelinating cells. Our study also provides evidence that the adult oligodendrocyte progenitor can be considered as a source of the Schwann cells that remyelinate demyelinated CNS axons following concurrent destruction of oligodendrocytes and astrocytes.
|The critical role of cyclin D2 in adult neurogenesis. |
Kowalczyk, A; Filipkowski, RK; Rylski, M; Wilczynski, GM; Konopacki, FA; Jaworski, J; Ciemerych, MA; Sicinski, P; Kaczmarek, L
The Journal of cell biology 167 209-13 2004
Adult neurogenesis (i.e., proliferation and differentiation of neuronal precursors in the adult brain) is responsible for adding new neurons in the dentate gyrus of the hippocampus and in the olfactory bulb. We describe herein that adult mice mutated in the cell cycle regulatory gene Ccnd2, encoding cyclin D2, lack newly born neurons in both of these brain structures. In contrast, genetic ablation of cyclin D1 does not affect adult neurogenesis. Furthermore, we show that cyclin D2 is the only D-type cyclin (out of D1, D2, and D3) expressed in dividing cells derived from neuronal precursors present in the adult hippocampus. In contrast, all three cyclin D mRNAs are present in the cultures derived from 5-day-old hippocampi, when developmental neurogenesis in the dentate gyrus takes place. Thus, our results reveal the existence of molecular mechanisms discriminating adult versus developmental neurogeneses.Artículo Texto completo
|Ethanol induces heterotopias in organotypic cultures of rat cerebral cortex. |
Mooney, SM; Siegenthaler, JA; Miller, MW
Cerebral cortex (New York, N.Y. : 1991) 14 1071-80 2004
Abnormalities in the migration of cortical neurons to ectopic sites can be caused by prenatal exposure to ethanol. In extreme cases, cells migrate past the pial surface and form suprapial heterotopias or 'warts'. We used organotypic slice cultures from 17-day-old rat fetuses to examine structural and molecular changes that accompany wart formation. Cultures were exposed to ethanol (0, 200, 400 or 800 mg/dl) and maintained for 2-32 h. Fixed slices were sectioned and immunolabeled with antibodies directed against calretinin, reelin, nestin, GFAP, doublecortin, MAP-2 and NeuN. Ethanol promoted the widespread infiltration of the marginal zone (MZ) with neurons and the focal formation of warts. The appearance of warts is time- and concentration-dependent. Heterotopias comprised migrating neurons and were not detected in control slices. Warts were associated with breaches in the array of Cajal-Retzius cells and with translocation of reelin-immunoexpression from the MZ to the outer limit of the wart. Ethanol also altered the morphology of the radial glia. Thus, damage to the integrity of superficial cortex allows neurons to infiltrate the MZ, and if the pial-subpial glial barrier is also compromised these ectopic neurons can move beyond the normal cerebral limit to form a wart.
|Enrichment and characterization of mouse putative epidermal stem cells. |
Jia-Xi Zhou, Li-Wei Jia, Yong-Jun Yang, Sha Peng, Yu-Jing Cao, En-Kui Duan
Cell biology international 28 523-9 2004
Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life. Cultured epidermal stem cells have great potential in scientific research and clinical application. However, isolating a pure and viable population of epidermal stem cells and culturing them has been challenging. In this study, putative epidermal stem cells of mouse were isolated by combining Hoechst 33342 and propidium iodide staining with fluorescence-activated cell sorting. Molecular markers expression pattern analysis showed that cytokeratin 14, integrin beta1 and p63 are expressed in the sorted putative stem cells, but not active beta-catenin, nestin and involucrin. Our results provide further supporting data that mouse putative epidermal stem cells could be successfully isolated by combining Hoechst dye staining with fluorescence-activated cell sorting and cultured in vitro. The cultured mouse putative epidermal stem cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy.
|Reduced expression of P2Y1 receptors in connexin43-null mice alters calcium signaling and migration of neural progenitor cells |
Scemes, E. et al.
J Neurosci, 23:11444-11452 (2003) 2003
|Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons. |
Sumrejkanchanakij, P; Tamamori-Adachi, M; Matsunaga, Y; Eto, K; Ikeda, MA
Oncogene 22 8723-30 2003
Cyclin D-dependent kinases phosphorylate the retinoblastoma (Rb) protein and play a critical role in neuronal cell cycle control and apoptosis. Here we show that cyclin D1 became predominantly cytoplasmic as primary cortical progenitor cells underwent cell cycle withdrawal and terminal differentiation. Furthermore, ectopically expressed cyclin D1 sequestered in the cytoplasm of postmitotic neurons, whereas it efficiently entered the nucleus of proliferating progenitor cells. Cytoplasmic cyclin D1 were complexed with cyclin-dependent kinase 4 (CDK4), and also with CDK inhibitors, p27(Kip)(I) or p21(Cip)(I), which positively regulate assembly and nuclear accumulation of the cyclin D1-CDK4 complex. Although overexpression of p21(Cip)(I) promoted cyclin D1 nuclear localization, inhibition of either glycogen synthase kinase 3beta- or CRM1-mediated cyclin D1 nuclear export did not, suggesting that the inhibition of its nuclear import, rather than the acceleration of nuclear export, contributes to cytoplasmic sequestration of cyclin D1 in postmitotic neurons. In differentiated progenitor cells, nuclear localization of ectopic cyclin D1 induced apoptosis, and the DNA-damaging compound camptothecin caused nuclear accumulation of endogenous cyclin D1, accompanied by Rb phosphorylation. These results indicate that nuclear accumulation of cyclin D1 is inhibited in postmitotic neurons and suggest a role of its subcellular localization in neuronal death and survival.
|The Use of Interleukin 12-secreting Neural Stem Cells for the Treatment of Intracranial Glioma |
Ehtesham, M., et al.
Cancer Research , 62:5657-5663 (2002) 2002
|Distribution, differentiation, and survival of intravenously administered neural stem cells in a rat model of amyotrophic lateral sclerosis. |
Mitrecić D, Nicaise C, Gajović S, Pochet R
Cell Transplant 19 537-48. Epub 2010 Mar 26. 2001
The transplantation of neural stem cells (NSCs) is a challenging therapeutic strategy for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). To provide insight into the potential of the intravenous delivery of NSCs, we evaluated the delivery of NSCs marked with green fluorescent protein to the central nervous system (CNS) via intravenous tail vein injections in an ALS model. The injected cell fates were followed 1, 3, and 7 days after transplantation. The highest efficiency of cell delivery to the CNS was found in symptomatic ALS (up to 13%), moderate in presymptomatic ALS (up to 6%), and the lowest in wild-type animals (up to 0.3%). NSCs injected into ALS animals preferentially colonized the motor cortex, hippocampus, and spinal cord, and their differentiation was characterized by a decrease of nestin expression and the appearance of MAP2-, GFAP-, O4-, and CD68-positive cells. Tumor necrosis factor (TNF) administration increased the CNS delivery of transplanted cells in wild-type and presymptomatic, but not ALS symptomatic animals. Moreover, a TNF-related increase in NSC differentiation and survival was detected. Apoptosis was detected as the main cause of the loss of transplanted cells and it was influenced by TNF. Although 3 days after TNF treatment cell death was accelerated, TNF slowed down apoptosis after 7 days. This study provides elementary facts about the process occurring after NSCs leave the blood stream and enter the nervous tissue affected by inflammation/degeneration, which should help facilitate the planning of future bench-to-bedside translational projects.
|A novel p75NTR signaling pathway promotes survival, not death, of immunopurified neocortical subplate neurons. |
M F DeFreitas, P S McQuillen, C J Shatz
The Journal of neuroscience : the official journal of the Society for Neuroscience 21 5121-9 2001
Subplate neurons of mammalian neocortex undergo pronounced cell death postnatally, long after they have matured and become incorporated into functional cortical circuits. They express the p75 neurotrophin receptor (p75NTR), which is known to signal cell death in some types of neurons via the activation of sphingomyelinase and the concomitant increase in the sphingolipid ceramide. To evaluate the role of p75NTR in subplate neurons, they were immunopurified and cultured in vitro. Contrary to its known function as a death receptor, ligand binding to p75NTR promotes subplate neuron survival. Moreover, p75NTR-dependent survival is blocked by inhibition of ceramide synthesis and rescued by addition of its precursor sphingomyelin. Inhibition of Trk signaling does not block survival, nor is Trk signaling alone sufficient to promote survival. Thus, ligand-dependent p75NTR regulation of the ceramide pathway mediates survival in certain neurons and may represent an important target for neuroprotective drugs in degenerative diseases involving p75NTR-expressing neurons, such as Alzheimer's disease.
|Immature human NT2 cells grafted into mouse brain differentiate into neuronal and glial cell types. |
A Ferrari, E Ehler, R M Nitsch, J Götz
FEBS letters 486 121-5 2000
NT2 cells are a transfectable human embryonal carcinoma cell line, that can be differentiated into postmitotic neuron-like cells (NT2N cells), and transplanted into rodent brains. Differentiation requires a 5-week-long treatment with retinoic acid prior to transplantation. Here, we show that this step can be omitted, and that undifferentiated NT2 cells migrate over long distances and differentiate into both neuron- and oligodendrocyte-like cell types upon grafting into brains of immunocompetent newborn mice. Grafted cells can be traced by fluorogold, with no evidence for tumor formation. Our approach provides an experimental model system which allows the immunohistological and biochemical study of neuronal and glial differentiation of human cells in vivo, and which may be suitable as an in vivo model for pharmacological studies.
|Angiogenesis of extra- and intraembryonic blood vessels is associated with expression of nestin in endothelial cells |
Mokry J and Nemecek S.
Folia Biologica, 44:155-161 (1998) 1998
|Neurons produce a neuronal cell surface-associated chondroitin sulfate proteoglycan |
Lander, C. et al.
J. Neuroscience, 18:174-183 (1998) 1998
|Intermediate filaments in cardiac myogenesis: nestin in the developing mouse heart |
Kachinsky, A. et al.
J Histochem Cytochem. , 43:843-847 (1995) 1995
|Radial glia in the neocortex of adult rats: effects of neonatal brain injury. |
Rosen, G D, et al.
Brain Res. Dev. Brain Res., 82: 127-35 (1994) 1994
Microgyria can be induced in otherwise normal rat neocortex by a freezing injury to the cortical plate before the completion of neuronal migration. We had previously reported radial glial like-immunoreactive fibers in the area of the microgyria in 32-day-old rats. Here we demonstrate that these glial fibers, which are immunoreactive to Rat-401, vimentin, and glial fibrillary acidic protein (GFAP) antibodies, are seen in adult rats. The appearance of these fibers is hypothesized to result from the release of a trophic factor during the recovery from neonatal injury which acts to either (1) halt the transformation of radial glia to astrocytes and/or dedifferentiate already committed astrocytes, (2) create a hybrid cell, or (3) induce increased proliferation of glia.
|Anti-Nestin, clone rat-401 - Data Sheet|