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Generation of induced pluripotent stem cells from fetal human hepatocytes in feeder-free conditions

Marc C. Hansel¹, Roberto Gramignoli¹, Veysel Tahan¹, Kristen Skvorak¹, Kenneth Dorko¹, William Blake², Julio Davila², Alejandro Soto-Gutierrez³, Ira Fox³, Stephen C. Strom¹

¹Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA; ²Genetically Modified Models Center of Emphasis, Pfizer, Groton, CT; ³Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States

Hepatocyte transplantation has been used as cellular therapy for liver disease. If iPS cells could be produced and induced to differentiate to hepatocytes, they might be useful for this cellular therapy. We report the generation of iPS cell lines from primary human fetal hepatocytes in feeder-free conditions following exposure to lentiviral constructs carrying the reprogramming factors OCT4, SOX2, KLF4, c-MYC or OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, NANOG. A profile was conducted pre- and post-reprogramming by Flow Cytometry for 18 surface markers commonly found on different types of stem cells. The percentage of SSEA-3, SSEA-4, TRA1-60 and TRA1-81 positive cells after reprogramming was dramatically increased in iPS cells (up to 90%). The putative liver stem cell marker, EpCAM, was expressed in approximately 50% of the cells prior to, and in nearly all cells following reprogramming. Similar observations were made with three additional stem cell markers, CD24, CD133/2 and c-Kit, which increased from near zero to 95%, 35% and 70%, respectively after reprogramming. Reprogrammed colonies have morphological features and the surface marker and gene expression profile typical of fully reprogrammed cells and they also form teratomas upon transplantation into NOD/SCID mice. Fetal human hepatocytes are more easily reprogrammed than adult hepatocytes. Reprogramming of adult hepatocytes was quite inefficient (<1/10 ⁶ cells) while, with four factors, reprogramming efficiency of fetal hepatocytes was approximately 1/5x10⁴ cells. Moreover, iPS cells were also created following exposure to only 3 reprogramming factors (OCT4, SOX, and NANOG). Protocols to differentiate these cells back to a hepatic phenotype are in progress. These studies demonstrate that iPS cell lines can be efficiently generated from primary human fetal hepatoblasts/hepatocytes and suggest that this source of stem cells might be useful for regenerative medicine.