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  • Sca-1+ progenitors derived from embryonic stem cells differentiate into endothelial cells capable of vascular repair after arterial injury. 16902164

    BACKGROUND: Embryonic stem cells possess the ability to differentiate into endothelium. The ability to produce large volumes of endothelium from embryonic stem cells could provide a potential therapeutic modality for vascular injury. We describe an approach that selects endothelial cells using magnetic beads that may be used therapeutically to treat arterial injury. METHODS AND RESULTS: Large numbers of endothelial cells (ECs) with high purity were produced using Sca-1+ cells isolated with magnetic beads from predifferentiated embryonic stem cells (ESCs) cultured in alpha-MEM containing 10 ng/mL VEGF165 for a minimum of 21 days (esEC). The transcription regulator histone deacetylase (HDAC3) was essential for VEGF-induced EC differentiation. Immunofluorescence or fluorescence-activated cell sorter (FACS) analysis revealed that esECs expressed a full range of EC lineage-specific markers including CD31, CD106, CD144, Flk-1, Flt-1, and von Willebrand factor (vWF). FACS analysis confirmed that 99% of esECs were CD31-positive and 75% vWF-positive. Furthermore, almost all cells were positive for DiI-acLDL uptake. When matrigel containing esECs was subcutaneously implanted into mice, various vessel-like structures were observed indicating their endothelial cell like phenotype. In keeping with this, when esECs infected with adenovirus-LacZ were injected into denuded femoral arteries of mice, they were found to form a neo-endothelium that covered the injured areas (86%+/-13.6%), which resulted in a 73% decrease in neointimal area 2 weeks after injury. CONCLUSIONS: We conclude that Sca-1+ cells can differentiate into functional ECs via activation of HDAC3, accelerating re-endothelialization of injured arteries and reducing neointima formation.
    Document Type:
    Reference
    Product Catalog Number:
    64-101
    Product Catalog Name:
    siIMPORTER™

  • Sphere formation reverses the metastatic and cancer stem cell phenotype of the murine mammary tumour 4T1, independently of the putative cancer stem cell marker Sca-1. 22459350

    Breast cancer stem cells (BCSCs) initiate and sustain breast cancers, and several putative markers have been proposed to prospectively isolate BCSC from the non-cancer stem cell population. The candidate BCSC marker Sca-1 is a GPI-linked membrane protein expressed on activated lymphocytes, hematopoietic stem cells and mammary stem cells. Sca-1+ cells were purified from the murine mammary tumour cell line 4T1. However, this did not enrich for a stem-like, tumour initiating or metastatic cell population in vitro or in vivo. Sphere formation, which induced high levels of Sca-1, reduced BCSC gene expression with near complete loss of spontaneous metastasis from sphere-derived tumours. This was associated with decreased expression of TGFB2 and reduced activation of the TGFβ signalling pathway in spheres. Both TGFB2 expression in vitro and spontaneous metastasis in vivo could be restored upon re-differentiation of sphere cells by exposure to serum, and this occurred with retention of the majority of Sca-1 expression. We conclude that while putative BCSC, including spheres, can have high Sca-1 expression, Sca-1 itself is not a marker of BCSC in established 4T1 tumours or the cell line.
    Document Type:
    Reference
    Product Catalog Number:
    07-408
    Product Catalog Name:
    Anti-Smad2/3 Antibody

  • Systemically transferred hematopoietic stem cells home to the subretinal space and express RPE-65 in a mouse model of retinal pigment epithelium damage. 16949576

    Stem cell regeneration of damaged tissue has recently been reported in many different organs. Since the loss of retinal pigment epithelium (RPE) in the eye is associated with a major cause of visual loss - specifically, age-related macular degeneration - we investigated whether hematopoietic stem cells (HSC) given systemically can home to the damaged subretinal space and express markers of RPE lineage. Green fluorescent protein (GFP) cells of bone marrow origin were used in a sodium iodate (NaIO(3)) model of RPE damage in the mouse. The optimal time for adoptive transfer of bone marrow-derived stem cells relative to the time of injury and the optimal cell type [whole bone marrow, mobilized peripheral blood, HSC, facilitating cells (FC)] were determined by counting the number of GFP(+) cells in whole eye flat mounts. Immunocytochemistry was performed to identify the bone marrow origin of the cells in the RPE using antibodies for CD45, Sca-1, and c-kit, as well as the expression of the RPE-specific marker, RPE-65. The time at which bone marrow-derived cells were adoptively transferred relative to the time of NaIO(3) injection did not significantly influence the number of cells that homed to the subretinal space. At both one and two weeks after intravenous (i.v.) injection, GFP(+) cells of bone marrow origin were observed in the damaged subretinal space, at sites of RPE loss, but not in the normal subretinal space. The combined transplantation of HSC+FC cells appeared to favor the survival of the homed stem cells at two weeks, and RPE-65 was expressed by adoptively transferred HSC by four weeks. We have shown that systemically injected HSC homed to the subretinal space in the presence of RPE damage and that FC promoted survival of these cells. Furthermore, the RPE-specific marker RPE-65 was expressed on adoptively transferred HSC in the denuded areas.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5428
    Product Catalog Name:
    Anti-Retinal Pigment Epithelium 65 Antibody

  • mAb C19 targets a novel surface marker for the isolation of human cardiac progenitor cells from human heart tissue and differentiated hESCs. 25820071

    Cardiac progenitor cells (CPCs) have been isolated from adult and developing hearts using an anti-mouse Sca-1 antibody. However, the absence of a human Sca-1 homologue has hampered the clinical application of the CPCs. Therefore, we generated novel monoclonal antibodies (mAbs) specifically raised against surface markers expressed by resident human CPCs. Here, we explored the suitability of one of these mAbs, mAb C19, for the identification, isolation and characterization of CPCs from fetal heart tissue and differentiating cultures of human embryonic stem cells (hESCs).Using whole-cell immunization, mAbs were raised against Sca-1+ CPCs and screened for reactivity to various CPC lines by flow cytometry. mAb C19 was found to be specific for Sca-1+ CPCs, with high cell surface binding capabilities. mAb C19 stained small stem-like cells in cardiac tissue sections. Moreover, during differentiation of hESCs towards cardiomyocytes, a transient population of cells with mAb C19 reactivity was identified and isolated using magnetic-activated cell sorting. Their cell fate was tracked and found to improve cardiomyocyte purity from hESC-derived cultures. mAb C19+ CPCs, from both hESC differentiation and fetal heart tissues, were maintained and expanded in culture, while retaining their CPC-like characteristics and their ability to further differentiate into cardiomyocytes by stimulation with TGFβ1. Finally, gene expression profiling of these mAb C19+ CPCs suggested a highly angiogenic nature, which was further validated by cell-based angiogenesis assays.mAb C19 is a new surface marker for the isolation of multipotent CPCs from both human heart tissues and differentiating hESCs.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Screening for proteins with polyglutamine expansions in autosomal dominant cerebellar ataxias. 8968739

    Expansion of trinucleotide CAG repeats coding for polyglutamine has been implicated in five neurodegenerative disorders, including spinocerebellar ataxia (SCA) 1 and SCA3 or Machado-Joseph disease (SCA3/MJD), two forms of type I autosomal dominant cerebellar ataxias (ADCA). Using the 1C2 antibody which specifically recognizes large polyglutamine tracts, particularly those that are expanded, we recently reported the detection of proteins with pathological glutamine expansions in lymphoblasts from another form of ADCA type I, SCA2, as well as from patients presenting with the distinct phenotype of ADCA type II. We now have screened a large series of patients with ADCA or isolated cases with cerebellar ataxia, for the presence of proteins with polyglutamine expansions. A 150 kDa SCA2 protein was detected in 16 out of 40 families with ADCA type I. This corresponds to 24% of all ADCA type I families, which is much more frequent than SCA1 in this series of patients (13%). The signal intensity of the SCA2 protein was negatively correlated to age at onset, as expected for an expanded and unstable trinucleotide repeat mutation. The disease segregated with markers closely linked to the SCA2 locus in all identified SCA2 families. In addition, a specific 130 kDa protein, which segregated with the disease, was detected in lymphoblasts of patients from nine families with ADCA type II. It was also visualized in the cerebral cortex of one of the patients, demonstrating its translation in the nervous system. Finally, no new disease-related proteins containing expanded polyglutamine tracts could be detected in lymphoblasts from the remaining patients with ADCA or isolated cases with cerebellar ataxia.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1574
    Product Catalog Name:
    Anti-Polyglutamine-Expansion Diseases Marker Antibody, clone 5TF1-1C2

  • Mesenchymal stem cells secrete multiple cytokines that promote angiogenesis and have contrasting effects on chemotaxis and apoptosis. 22558198

    We have previously shown that mesenchymal stem cells (MSC) improve function upon integration in ischemic myocardium. We examined whether specific cytokines and growth factors produced by MSCs are able to affect angiogenesis, cellular migration and apoptosis. Conditioned media (CM) was prepared by culturing MSC for 48 hours. CM displayed significantly elevated levels of VEGF, Monocyte Chemoattractant Protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), MIP-1β and monokine induced by IFN-γ (MIG) compared to control media. MSC contained RNA for these factors as detected by RT-PCR. CM was able to induce angiogenesis in canine vascular endothelial cells. MCP-1 and MIP-1α increased cell migration of MSC while VEGF reduced it. H9c2 cells treated with CM under hypoxic conditions for 24 hours displayed a 16% reduction in caspase-3 activity compared to controls. PI 3-kinase γ inhibitor had no effect on controls but reversed the effect of CM on caspase-3 activity. MCP-1 alone mimicked the protective effect of CM while the PI 3-Kγ inhibitor did not reverse the effect of MCP-1. CM reduced phospho-BAD (Ser112) and phospho-Akt (Ser473) while increasing phospho-Akt (Thr308). MCP-1 reduced the level of phospho-Akt (Ser473) while having no effect on the other two; the PI 3-Kγ inhibitor did not alter the MCP-1 effect. ERK 1/2 phosphorylation was reduced in CM treated H9c2 cells, and inhibition of ERK 1/2 reduced the phosphorylation of Akt (Ser473), Akt (Thr308) and Bad (Ser112). In conclusion, MSC synthesize and secrete multiple paracrine factors that are able to affect MSC migration, promote angiogenesis and reduce apoptosis. While both MCP-1 and PI3-kinase are involved in the protective effect, they are independent of each other. It is likely that multiple pro-survival factors in addition to MCP-1 are secreted by MSC which act on divergent intracellular signaling pathways.
    Document Type:
    Reference
    Product Catalog Number:
    ECM630
    Product Catalog Name:
    Fibrin In Vitro Angiogenesis Assay

  • Evidence that the population of quiescent bone marrow-residing very small embryonic/epiblast-like stem cells (VSELs) expands in response to neurotoxic treatment. 24895014

    The concept that bone marrow (BM)-derived cells may participate in neural regeneration remains controversial, and the identity of the specific cell type(s) involved remains unknown. We recently reported that the adult murine BM contains a highly mobile population of Sca-1(+) Lin(-) CD45(-) cells known as very small embryonic/epiblast-like stem cells (VSELs) that express several markers of pluripotency such as Oct-4. In the BM microenvironment, these cells are kept quiescent because of epigenetic modification of certain paternally imprinted genes. However, as reported, these cells can be mobilized in mice in an experimental model of stroke and express several genes involved in neurogenesis while circulating in peripheral blood (PB). In the current work, we employed a model of toxic brain damage, which is induced by administration of kainic acid, to see not only whether VSELs can be mobilized into PB in response to this neurotoxin, but, more importantly, whether they proliferate and expand in BM tissue. We report here for the first time that brain damage leads to activation and expansion of the BM pool of quiescent VSELs, which precedes their subsequent egress into PB. Harnessing these cells in neural tissue regeneration is currently one of the challenges in regenerative medicine.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4419
    Product Catalog Name:
    Anti-OCT-4 [POU5F1] Antibody, clone 7F9.2

  • Identification of pulmonary Oct-4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus (SARS-CoV) infection in vitro. 16772384

    In this study, we report a serum-free culture system for primary neonatal pulmonary cells that can support the growth of octamer-binding transcription factor 4+ (Oct-4+) epithelial colonies with a surrounding mesenchymal stroma. In addition to Oct-4, these cells also express other stem cell markers such as stage-specific embryonic antigen 1 (SSEA-1), stem cell antigen 1 (Sca-1), and Clara cell secretion protein (CCSP) but not c-Kit, CD34, and p63, indicating that they represent a subpopulation of Clara cells that have been implicated as lung stem/progenitor cells in lung injury models. These colony cells can be kept for weeks in primary cultures and undergo terminal differentiation to alveolar type-2- and type-1-like pneumocytes sequentially when removed from the stroma. In addition, we have demonstrated the presence of Oct-4+ long-term BrdU label-retaining cells at the bronchoalveolar junction of neonatal lung, providing a link between the Oct-4+ cells in vivo and in vitro and strengthening their identity as putative neonatal lung stem/progenitor cells. Lastly, these Oct-4+ epithelial colony cells, which also express angiotensin-converting enzyme 2, are the target cells for severe acute respiratory syndrome coronavirus infection in primary cultures and support active virus replication leading to their own destruction. These observations imply the possible involvement of lung stem/progenitor cells, in addition to pneumocytes, in severe acute respiratory syndrome coronavirus infection, accounting for the continued deterioration of lung tissues and apparent loss of capacity for lung repair.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple