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

Fabia Febbraro, Muwan Chen, Mark Denham*

*Corresponding author for this work

Research output: Contribution to book/anthology/report/proceedingBook chapterResearchpeer-review

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.

Original languageEnglish
Title of host publicationNuclear reprogramming : Methods and protocols
EditorsKejin Hu
Number of pages17
Place of publicationNew York
PublisherHumana Press
Publication date2021
Pages135-151
Chapter9
ISBN (Print)978-1-0716-1083-1
ISBN (Electronic)978-1-0716-1084-8
DOIs
Publication statusPublished - 2021
SeriesMethods in Molecular Biology
Volume2239
ISSN1064-3745

Keywords

  • Episomal reprogramming
  • Human peripheral mononuclear cells
  • Induced pluripotent stem cells
  • iPSC
  • Stem cells

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