327 PORCINE AMNION: A SOURCE OF EPITHELIAL STEM CELLS

Abstract

The use of pig models for preclinical testing is well established, and the availability of stem cells from this species would open the way to preclinical studies for application of cell therapy. 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 their clinical application seems to be hindered by the high tumorigenic rate after transplantation. Mesenchymal stem cells (MSC) derived from adult tissues are considered to be more limited in their potential and the risk of the immunological rejection of the transplanted stem cells by the recipient is an important limiting factor. The MSC derived from extra-fetal tissues could overcome many of these restrictions. Indeed, in veterinary medicine, MSC isolated from equine term placenta were the ideal candidates for tendon disease treatment, specifically for their plasticity and their reduced immunogenicity compared to bone marrow-derived cells. Extra-fetal derived MSC in porcine have been isolated from the umbilical cord matrix and amniotic fluid. The aim of this work was to provide, for the first time, an isolation protocol and the characterisation of stem cells from porcine amniotic membrane, which could hold potential uses in regenerative medicine. The amnion is a thin, avascular membrane made of an epithelial layer and an outer layer of connective tissue. From 3 samples of allanto-amnion retrieved at delivery, each amniotic membrane was stripped from the overlying allantois and, for isolation of the epithelial cells, it was digested with trypsin. After removal of epithelial cells, the stromal layer was digested with collagenase to obtain amniotic mesenchymal cells. The cellular yield from term amnion resulted only in epithelial cells (AEC) at a concentration of 10 × 10⁶ for 1 g of digested tissue while no MSC were obtained. Histology, indeed, revealed very few cells in the stromal layer. The AEC readily attached to plastic culture dishes. Culture was established in DMEM-HG medium, supplemented with 10% serum and 10 ng mL^–1 of EGF where the cells proliferated robustly. The AEC displayed typical cuboidal morphology. These cells showed a mean of 31 ± 0.24 cell population doublings after 31 days. The mean frequency of colony-forming unit fibroblasts was 1 for each of the 75 plated cells. The AEC expressed MSC mRNA markers (CD29, CD166, CD90, CD73, CD117) and pluripotent markers (Nanog and Oct4), while were negative for CD34 and MHC-II. Osteogenic, adipogenic, and neurogenic differentiations were confirmed by von Kossa, Red Oil, and Nissle stains, respectively, and by expression of specific markers (osteocalcin and osteopontin for osteogenic differentiation, adiponectin and leptin for adipogenic differentiation, and glial fibrillary acid protein and nestin for neurogenic differentiation). We conclude that porcine amnion contain unique and primitive cells whose potential is as yet undefined. Ease of collection and propagation of AEC make this tissue an attractive candidate as a resource for stem cell biotechnology and biomedical research.