Isolation and Immunophenotypjng of human amniotic epithelial cells

Human amniotic epithelial cells (hAECs) have been introduced as a valuable source of stem cells with therapeutic applications. Despite the extensive study and emerging evidence، cell surface marker signature of hAECs remained controversial. The aim of the present study was to establish an efficient and optimized isolation procedure of hAECs and their characterization in terms of proliferation and stem cell markers. Four human placentas were collected from healthy women undergoing elective caesarian delivery at term labor under sterile condition. Amniotic membranes were carefully isolated and subjected to trypsin digestion. Single cell suspensions were obtained and the epithelial origin was confirmed by assessment of cytokeratin expression. Expression of CD9، CD10، CD29، CD34، CD38، CD44، CD45، CD73، CD105، CD133، MHC-I، HLA-DR، HLA-G، SSEA-4، STRO-1 and OCT-4 was then evaluated by flow cytometry. The overall yield of hAEC ranged from 80-130×106 cells per placenta with more than 98% viability. Microscopic examination revealed that hAECs are large and refractive cells with great capacity to adhere to plastic surfaces. The cells substantially expressed cytokeratin implying their epithelial origin. Flow cytometry data revealed that hAECs are a heterogenic population consisting of immune phenotypically mixed cells. Nearly all purified cells expressed mesenchymal markers، CD9، CD10، CD29، and CD73. A large proportion of cells also expressed HLA-I، HLA-G، STRO-1، OCT-4 and SSEA-4. Isolated cells، however، failed to express CD34، CD38، CD44، CD45، CD105، CD133 and HLA-DR. The procedure presented here is a simple and cost-effective protocol for isolation of a large numbers of viable hAECs. Surface markers and such features as proliferation capacity of hAECs are among the characteristics that are strongly dependent upon isolation and cell culture conditions. Thus such variables should be taken in to consideration when stem cell properties of hAECs are to be interpreted.