Abstract
Human-induced pluripotent stem cells (iPSCs) can be generated from patient-specific somatic cells by forced expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Sustained expression of the transgenes during reprogramming is crucial for the successful derivation of iPSCs. Integrating retroviruses have been used to achieve the required prolonged expression; however, issues of undesirable transgene expression in the iPSC-derived cell types post reprogramming can occur. Alternative non-integrating approaches to reprogram somatic cells into pluripotency have been established. Here, we describe a detailed method for generating human iPSCs from fibroblasts and peripheral blood mononuclear cells (PBMCs) using the non-integrating episomal plasmids. The delivery of the episomal plasmids into the somatic cells is achieved using a nucleofection technique, and reprogramming is performed in chemically defined media. This process takes approximately 30 days to establish the iPSC colonies. We also describe a method for growing iPSCs on vitronectin as well as procedures for the long-term expansion of iPSCs on human fibroblast feeder cells.
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Acknowledgment
This study was supported by Lundbeckfonden grant no. R248-2016-2518. MC is supported by a postdoctoral fellowship from the Lundbeckfonden grant no. R209-2015-3100. MD is a partner of BrainStem—Stem Cell Center of Excellence in Neurology, funded by Innovation Fund Denmark.
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Febbraro, F., Chen, M., Denham, M. (2021). Generation of Human iPSCs by Episomal Reprogramming of Skin Fibroblasts and Peripheral Blood Mononuclear Cells. In: Hu, K. (eds) Nuclear Reprogramming. Methods in Molecular Biology, vol 2239. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1084-8_9
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DOI: https://doi.org/10.1007/978-1-0716-1084-8_9
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