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

TY - CHAP

T1 - Generation of Human iPSCs by Episomal Reprogramming of Skin Fibroblasts and Peripheral Blood Mononuclear Cells

AU - Febbraro, Fabia

AU - Chen, Muwan

AU - Denham, Mark

N1 - Funding Information: 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. Publisher Copyright: © 2021, Springer Science+Business Media, LLC, part of Springer Nature. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.

PY - 2021

Y1 - 2021

N2 - 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.

AB - 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.

KW - Episomal reprogramming

KW - Human peripheral mononuclear cells

KW - Induced pluripotent stem cells

KW - iPSC

KW - Stem cells

UR - http://www.scopus.com/inward/record.url?scp=85096753076&partnerID=8YFLogxK

U2 - 10.1007/978-1-0716-1084-8_9

DO - 10.1007/978-1-0716-1084-8_9

M3 - Book chapter

C2 - 33226617

AN - SCOPUS:85096753076

SN - 978-1-0716-1083-1

T3 - Methods in Molecular Biology

SP - 135

EP - 151

BT - Nuclear reprogramming

A2 - Hu, Kejin

PB - Humana Press

CY - New York

ER -