Generation of induced pluripotent stem cell lines from human fibroblasts via retroviral gene transfer Journal Article
| Authors: | Miller, J. D.; Schlaeger, T. M. |
| Article Title: | Generation of induced pluripotent stem cell lines from human fibroblasts via retroviral gene transfer |
| Abstract: | This chapter describes a protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts. Human fibroblasts, cultured in fibroblast medium, are infected with a cocktail of retroviral vectors expressing the transcription factors OCT4, SOX2, KLF4, and MYC. The culture conditions are then switched to conditions that support human embryonic stem cell growth and emerging iPSC colonies that morphologically resemble human embryonic stem cell (hESC) colonies and have silenced the retroviral vectors (as evidenced by downregulation of retroviral GFP expression) that are mechanically isolated and subsequently cultured in identical fashion to hESCs. Putative iPSC lines are validated to be bona fide human iPSC lines by analyzing them for the expression of pluripotency markers and by differentiation in vitro and in vivo. © 2011 Springer Science+Business Media, LLC. |
| Keywords: | genetics; methodology; cell proliferation; reproducibility; reproducibility of results; cytology; metabolism; cell line; transcription factor; transfection; physiology; gene transfer; virology; transcription factors; genetic transfection; staining; fibroblast; cell culture techniques; fibroblasts; pluripotent stem cell; virus replication; cell clone; culture technique; clonogenic assay; colony-forming units assay; clone cells; retrovirus; retroviridae; nuclear reprogramming; induced pluripotent stem cells; staining and labeling; reprogramming; gene transfer techniques; human embryonic stem cells; pluripotency; human induced pluripotent stem cells; ipsc; retroviral transduction |
| Journal Title: | Methods in Molecular Biology |
| Volume: | 767 |
| ISSN: | 1064-3745 |
| Publisher: | Humana Press Inc |
| Date Published: | 2011-01-01 |
| Start Page: | 55 |
| End Page: | 65 |
| Language: | English |
| DOI: | 10.1007/978-1-61779-201-4_5 |
| PROVIDER: | scopus |
| PUBMED: | 21822867 |
| DOI/URL: | |
| Notes: | Chapter 5 in "Human Pluripotent Stem Cells" Methods and Protocols" (ISBN: 978-1-61779-200-7) --- "Export Date: 9 December 2011" - "Source: Scopus" |
