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Generation of induced pluripotent stem cells from Amnion Epithelial cells

Roberto Gramignoli¹, Marc C. Hansel¹, Fabio Marongiu², William L. Blake³, Alejandro Soto-Gutierrez⁵, Kenneth Dorko¹, Julio C. Davila⁴, Stephen C. Strom¹

¹Pathology, University of Pittsburgh, Pittsburgh, PA, United States; ²Biomedical Sciences and Technologies, Università degli Studi di Cagliari, Cagliari, Italy; ³Genetically Modified Models Center of Emphasis, Pfizer, Groton, CT; ⁴Pfizer, St. Louis, MO; ⁵Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, United States

Placenta is a readily available and non-controversial source of cells that could be used in regenerative medicine. Human Amniotic Epithelial (AE) cells and induced pluripotent stem (iPS) cell technologies would seem to be devoid of most if not all of the ethical concerns associated with ES cells. We generated of iPS cell lines from primary human AE (AE-iPS). After exposure to lentiviral constructs carrying the reprogramming factors, cells with appropriate morphology were collected and were analyzed for markers of pluripotency including Oct4, Nanog, Sox2 and alkaline phosphatase. A profile of AE was conducted pre- and post-reprogramming by Flow Cytometry for surface markers including: SSEA-3, SSEA-4, TRA1-60, TRA1-81, HLA-ABC, CD24, CD29, CD34, CD44, CD49f, CD73, CD105, CD90, CD117, CD133/2, CD146, CD166, EpCAM and ABCG2. FACS analysis revealed a morphological difference in forward scatter characterized by a compartment with a higher side scatter in AE, not present in AE-iPS thought to indicate a different level of cellular complexity and granularity. The percentage of SSEA-3, SSEA-4, TRA1-60 and TRA1-81 positive cells after reprogramming was dramatically increased in AE-iPS cells (30-90%). We also observed that stem cell marker such as CD133/2 was highly expressed only after the reprogramming procedure. Colonies generated have morphological features and the surface marker and gene expression profile of fully reprogrammed cells and also form teratomas upon transplantation. These studies confirm that iPS lines can be generated from primary human amnion. Protocols to differentiate these cells as previously published by our group on AE cells toward a hepatic phenotype are in progress.