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Episomal Reprogramming of Amniotic Mesenchymal Stem Cells: A Step Towards Translational Application
Elliot C Pennington, MD, Fabienne L Gray, MD, Azra Ahmed, MD, Alexander L. DeVine, MD, Kelly Fitzgerald, MD, George Q Daley, MD, Dario O Fauza, MD
Boston Children's Hospital, Boston MA

Background: The therapeutic potential of amniotic mesenchymal stem cells (aMSCs) can be magnified by induced pluripotent stem cell (iPS) reprogramming. To date, aMSC-iPS reprogramming has only been achieved via viral transfection, which carries various risks. In this study, we aimed to determine whether aMSCs could be reprogrammed without a viral vector and to compare this methodology with a conventional viral-based protocol.

Methods: Expanded human aMSCs were divided in two groups. One group was reprogrammed by ectopic expression of transcription factors Oct-4, Klf4, Sox-2, and c-Myc via lentiviral transfection. The other 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 eventually cultured on a mouse embryonic fibroblast (MEF)-free environment. 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 and on day 20 after episomal transfection. Reprogrammed cells from both groups 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 applicability of reprogrammed amniotic mesenchymal stem cells in clinically viable regenerative therapies.

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