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Reprogramming of Adipose Derived Stem cells (ADSCs) to iPS and Further Differentiation to Neuronal Cells for the Treatment of Peripheral Neuropathy
Dilip Dey, Jonathan Eichelsdoerfer, Sean Pham, Marko Tomov and Gregory R.D. Evans
Aesthetic and Plastic Surgery Institute, University of California, Irvine, Orange, CA
Introduction: Despite the intrinsic capacity of the peripheral nervous system to regenerate as well as advances in neurobiology, pathophysiology, intra- and post -operative management and microsurgery, functional recovery and clinical outcomes after peripheral nerve injuries are still poor. ADSCs are the most abundant in fat tissue, easy to collect autologously, making it an attractive source for adult stem cells. These adult cells are reprogrammed to induced pluripotent stem cells (iPSC) which is a great source of varying cell types and can be used for tissue regeneration or repair of peripheral nerve. The iPSCs can be used as an alternative to embryonic stem cells whose clinical application has been mired with ethical issues.
Method: ADSCs were isolated from routine lipoaspirate according to a standard protocol. The cells were cultured in DMEM-F12 media with 10% FBS and antibiotics. Cells were nucleofected with a “minicircle” vector that contains 4 reprogramming factors (Oct3/4, Sox2, Nanog, KLF4) plus a GFP marker. After nucleofection, cells were grown in stem cell media containing knockout serum. The iPS colonies were visible and were tested for dedifferentiation and the presence of pluripotent markers.
Results: We have been able to reprogram ADSCs to iPS by using 4 reprogramming factors. During reprogramming, cells expressed GFP marker and gradually lost the expression of GFP with the concomitant expression of pluripotent markers. ADSCs became undifferentiated that was confirmed by alkaline phosphatase (AP) assay. Now we are characterizing them for the presence pluripotent markers (Oct3/4, Sox2, Nanog and Klf4) by immunofluorescence.
Conclusion: The use of iPS may offer an ability to use a cell based technology to enhance peripheral nerve regeneration autologously. Our goal is the establishment of a tissue engineered conduit for clinical use in bridging nerve gaps.
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