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Episomal Reprogramming of Human Amniotic Mesenchymal Stem Cells: Another Step towards Translational Application

Sunday, October 21, 2012: 8:26 AM
Versailles Ballroom (Hilton Riverside)
Fabienne L. Gray, MD1, Elliot C. Pennington, MD1, Azra Ahmed, BS1, Alexander L. DeVine, BS2, Kelly Fitzgerald, BA2, George Q. Daley, MD, PhD2 and Dario O. Fauza, MD, PhD, FAAP1, (1)Department of Surgery, Children's Hospital Boston, Harvard Medical School, Boston, MA, (2)Department of Hematology & Oncology, Children's Hospital Boston, Harvard Medical School, Boston, MA

Purpose: Amniotic mesenchymal stem cells (aMSCs) have shown experimentally to be a valuable resource for cell and tissue replacement strategies in the perinatal period, as well as for the establishment of unique models of structural development and surgical disease. Such capabilities can be magnified by submitting aMSCs to induced pluripotent stem cell (iPS) reprogramming. However, to date, aMSC-iPS reprogramming has only been achieved via viral transfection methods, which carry a variety of risks stemming from the integration of the viral vector into the host cell's genome, thus limiting its translational potential. In this study, we aimed at determining whether aMSCs could be reprogrammed in the absence of a viral vector and at comparing this methodology with a conventional viral-based protocol. Methods: After IRB approval, expanded human aMSCs were characterized by comprehensive flow cytometry and then divided in two groups, depending on the reprogramming method to which they were exposed. One group was reprogrammed by ectopic expression of the transcription factors Oct-4, Klf4, Sox-2, and c-Myc via conventional transfection utilizing a lentivirus. The other group was reprogrammed by ectopic expression of Sox2, Klf4, Lin28A, L-Myc, Oct-3/4, and a short hairpin for p53 knockdown via concomitant episomal transfection of 3 plasmids containing these factors. Cells from both groups were cultured on a mouse embryonic fibroblast (MEF) feeding layer with standard human embryonic stem cell media through 7-9 passages. Prior to phenotypic characterization, cells were transitioned to a MEF-free environment, on plates pre-coated with a solubilized basement membrane preparation containing numerous extracellular matrix proteins, cytokines, and growth factors (Matrigel). They were then characterized by immunohistochemistry for multiple markers of a primitive pluripotent state shared with human embryonic stem cells. Results: On average, reprogrammed colonies began to appear on day 10 after lentiviral reprogramming, while after episomal transfection this happened on day 20. However, once reprogrammed, aMSCs-iPS from both groups displayed similar morphology and expansion kinetics in vitro. Similarly, reprogrammed cells from both groups remained viable on the MEF-free environment and consistently expressed all of the markers of an embryonic stem cell-like state, namely Tra-1-81, Tra-1-60, SSEA3, SSEA4, Oct-4, and NANOG. Conclusions: Amniotic mesenchymal stem cells can be successfully reprogrammed in the absence of a viral vector via episomal transfection. These cells can be maintained in culture in an undifferentiated, embryonic-like pluripotent state. These findings lend further support to the eventual applicability of reprogrammed amniotic mesenchymal stem cells in clinically viable regenerative therapies.