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Generation of Human iPSCs by Episomal Reprogramming of Skin Fibroblasts and Peripheral Blood Mononuclear Cells

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DOI

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.

OriginalsprogEngelsk
TitelNuclear reprogramming : Methods and protocols
RedaktørerKejin Hu
Antal sider17
UdgivelsesstedNew York
ForlagHumana Press
Udgivelsesår2021
Sider135-151
Kapitel9
ISBN (trykt)978-1-0716-1083-1
ISBN (Elektronisk)978-1-0716-1084-8
DOI
StatusUdgivet - 2021
SerietitelMethods in Molecular Biology
Vol/bind2239
ISSN1064-3745

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