CBL271 Sigma-AldrichAnti-Fibroblasts Antibody, clone TE-7

This paper describes a significant biotechnological advancement by creating a minimalist serum-free defined system to co-culture rat mammalian nerve and muscle cells in order to form functional neuromuscular junctions. To date, all the known in vitro nerve and muscle co-culture models use serum containing media; and while functional neuromuscular junctions (NMJ) are described, they failed to detail or quantify the minimum factors needed to recreate the NMJ in vitro. In this work, we demonstrate the development of a defined motoneuron and muscle co-culture system resulting in the formation of NMJs including: 1) a new culture technique, 2) a novel serum-free medium formulation and 3) a synthetic self-assembled monolayer (SAM) substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine (DETA). We characterized the culture by morphology, immunocytochemistry, electrophysiology and videography. This model system provides a better understanding of the minimal growth factor and substrate interactions necessary for NMJ formation and provides a basic system that can be utilized for nerve-muscle tissue engineering, regenerative medicine and development of limb prosthetics.

The human thymic microenvironment is important in promotion of T cell maturation, particularly during early stages of thymic ontogeny. Hassall's bodies (HB) are epithelial swirls in the human thymic medulla that are thought to be derived from endocrine medullary thymic epithelium. To study the ontogeny and function of various components of the human thymic microenvironment, we have produced four monoclonal antibodies (TE-8, TE-15, TE-16, and TE-19) that selectively reacted in thymus with HB. Antibodies TE-8 and TE-16 reacted with the cells forming the outer rim of the HB swirl. Antibody TE-19 reacted with the entire cellular portion of HB and with epithelial cells immediately surrounding HB. Granular foci in the cellular swirls of greater than 90% of HB reacted with antibody TE-15. During thymic ontogeny, the antigens defined by antibodies TE-8, TE-15, TE-16, and TE-19 were first detected in fetal thymus on HB beginning at 16 wk gestation, the age when HB morphologically appear in the thymus. Aberrant expression of the antigens corresponding to antibodies TE-8, TE-15, TE-16, and TE-19 was observed on thymic tissue from individuals with severe cellular immunodeficiency disease. In human skin, antibodies TE-8, TE-16, and TE-19 reacted with the stratum granulosum; antibody TE-15 reacted with the stratum corneum. Thus, with the use of antibodies TE-8, TE-15, TE-16, and TE-19, we have identified HB as antigenically distinct regions of endocrine thymic epithelium. Furthermore, we have shown that these anti-HB reagents also selectively react with epidermal keratinocytes in the terminal stages of keratinocyte maturation.

Several major points should be emphasized that provide directions for future research. First, using monoclonal reagents we have been able to phenotypically identify four major regions of the human thymus microenvironment: the thymic capsule, interlobular septae and stroma (TE-7+), the subcapsular cortex (TE-4+, Thy-1+, A2B5+, anti-p19+, BB TECS+, TE-3+), the cortex (TE-3+), and the medulla (TE-4+, A2B5+, anti-p19+, BB TECS+). TE-4+ and TE-3+ thymic epithelium constitute HLA+, Ia+ subsets of thymic epithelium that are candidates for cell types of the human thymic microenvironment that might participate in conferring MHC restriction to maturing T lymphocytes. TE-7+ stroma most likely represents the mesodermal-derived thymic component that early in development induces thymic epithelial differentiation. Second, whereas TE-4, anti-p19, and BB TECS antibodies may be thymic epithelial lineage markers, they all react with the basal layer of squamous epithelium of various organs. In particular, in the tonsil, A2B5+, TE-4+ epithelium splays out in the base of tonsillar crypts and morphologically appears similar to thymic medullary epithelial cells. Therefore, these markers of endocrine thymic epithelium may also identify extrathymic areas of T cell differentiation. Third, the concept that thymic epithelium is constantly differentiating in the developed thymus is suggested by the coexpression of TE-4, TE-8, TE-16, and TE-15 antigen by layers of squamous epithelial keratinocytes and by thymic epithelium. That there is a TE-4/TE-8/TE-15 keratinocyte maturation pathway in skin gives credence to the notion that a similar pathway exists from TE-4+, TE-8-, TE-15- endocrine medullary epithelial cells to TE-4-, TE-8+, TE-15+ Hassall's bodies. Fourth, from the literature and the work presented in this review, three phases of thymic microenvironment development can be defined. The first phase is during early fetal development (4 to 8 weeks in humans) when mesodermal-derived fibrous tissue induces endodermal and ectodermal-derived thymic epithelium to proliferate and mature. TE-7+ mesenchymal stroma invaginates TE-4+ thymic epithelium and effects thymic lobulation. The second phase occurs between 9 and 15 weeks fetal development when the thymic primordia is colonized by blood-borne thymocyte precursors. Presumably during this stage, thymic epithelium promotes bone marrow cell colonization of thymus by producing chemoattractant molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Using murine monoclonal antibodies TE-4 and TE-7 raised against human thymic stroma, we identified two distinct and mutually exclusive thymic microenvironment components: the thymic endocrine epithelium (TE-4+) and mesodermal-derived fibrous stroma (TE-7+). TE-4-reactive epithelium did not react with antibody TE-7, contained thymosin alpha 1 and keratin, and expressed other known markers of thymic endocrine epithelium (A2B5 and p19). Moreover, TE-4+ thymic epithelial cells strongly expressed class I (HLA-A, -B and -C) and class II (Ia-like) major histocompatibility complex (MHC) antigens. In contrast, TE-7+ thymic fibrous stroma did not react with antibody TE-4, did not contain thymosin alpha 1 nor keratin, and did not express the thymic endocrine epithelium markers A2B5 and p19. TE-7+ thymic stromal cells weakly expressed class I and did not express class II MHC antigens. Both TE-4+ and TE-7+ thymic microenvironment compartments were identifiable in thymus from 7 wk gestation through adult life. At 7 wk fetal gestation, TE-7+ stroma surrounded a cylindrical TE-4+, A2B5+ thymic epithelial rudiment. Between 10 and 15 wk fetal gestation, TE-7+ thymic stroma surrounded early thymic lobules. By 15 wk fetal gestation, antibody TE-4 defined subcapsular cortical and medullary zones of endocrine thymic epithelium, while antibody TE-7 bound to interlobular fibrous septae, vessels, and thymic fibrous capsule. While otherwise specific for endocrine thymic epithelium, antibody TE-4 reacted with the basal layer of squamous epithelium in skin, tonsil, conjunctiva, and upper esophagus.