According to the developmental stage from which they are obtained, stem cells are classified as being embryonic, fetal, or adult. Embryonic stem cells have unlimited self-renewing capacity and multilineage differentiation potential, but the separation of these cells requires destruction of the embryo. Moreover, their clinical application seems to be hindered by the high tumorigenic rate after transplantation. Stem cells derived from adult tissues are considered to be more limited in their potential; although, they are currently the more versatile cells in the clinical field. However, the risk of the immunological rejection of the transplanted stem cells by the recipient is an important limiting factor. In human medicine, stem cells isolated from term placenta are the ideal candidates for disease treatment, specifically because of their plasticity and reduced immunogenicity. The aim of this work was to provide, for the first time, an isolation protocol and the characteristics of the stem cells from horse amniotic membrane, which hold potential uses in equine clinical regenerative medicine. Minimal criteria for stemness definition are adherence to plastic culture dish, formation of fibroblast colony forming units (CFU-F), specific pattern of surface antigen expression, and differentiation potential toward one or more lineages. The amnion is a thin, avascular membrane composed of an epithelial layer and an outer layer of connective tissue. From 3 samples of allantoamnion retrieved at delivery, each amniotic membrane was stripped from the overlying allantois and, for isolation of the epithelial cells, digested with trypsin. After removal of epithelial cells, the AMSC population was obtained by digestion with collagenase and DNase. The cellular yield from term amnion was 10-fold more epithelial cells than AMSC. Isolated cells readily attached to plastic culture dishes. Culture was established in DMEM-HG medium, supplemented with 10% serum and EGF, where the cells proliferated robustly. Epithelial cells displayed typical cuboidal morphology, whereas AMSC were fibroblast-like. Normally, 5 to 6 passages were achieved before proliferation decreased, with a mean of 13.08 and 26.5 cell population doublings after 31 days, respectively, for epithelial cells and AMSC. The mean frequency of CFU-F was, respectively, 1 : 283 and 1:111 for epithelial cells and AMSC. The 2 cellular lines expressed MSC mRNA markers (CD29, CD105, CD44) and were negative for CD34, which was expressed at the fifth passage in both cellular types. Osteogenic differentiation of epithelial stem cells and AMSC was confirmed by von Kossa stain and by an increased expression of osteocalcin and osteopontin. Our preliminary data showed that equine amnion holds apparent potential as a source of presumptive stem cells, which might have widespread clinical applications, but aspects including immunohistochemical study, preclinical experimentation, and immunological properties must be studied.
Long-term results of limbal conjunctival autograft transplantation associated with an amniotic membrane in patients with total limbal stem cells deficiency