Phenotypic shift of human amniotic epithelial cells in culture is associated with reduced osteogenic differentiation in vitro

Background: Amniotic membrane is a highly promising cell source for tissue engineering. Being part of the placenta, this tissue is abundantly available. It can be processed easily to yield large amounts of epithelial and mesenchymal cells that have shown broad differentiation potential. For tissue-engineering purposes, cells may be applied either directly after isolation from the tissue or after a period of in vitro expansion to obtain higher cell numbers. In order to investigate the advantages and drawbacks of these strategies we compared freshly isolated and cultivated human amniotic epithelial cells (hAEC) regarding their surface antigen (Ag) expression profile and osteogenic differentiation capacity. Methods: Expression of surface Ag that are characteristic for mesenchymal stromal and embryonic stem cells was analyzed by flow cytometry. Different protocols for osteogenic and adipogenic differentiation were compared. Results: We have demonstrated that expression of surface Ag changes dramatically during cultivation of hAEC. While not or only weakly expressed on primary isolates, the mesenchymal markers CD13, CD44, CD49e, CD54, CD90 and CD105 are strongly up-regulated during in vitro propagation. In contrast, expression of the embryonic markers TRA-1-60 and TRA-1-81, but not SSEA-4, rapidly decreases upon cultivation. This phenotypic shift is associated with a reduction in osteogenic differentiation. Discussion: Our results suggest that phenotypic alterations of hAEC during in vitro cultivation might be responsible for a functional reduction of the differentiation potential, which has to be considered for the potential application of these cells in regenerative medicine.