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.
authors.affiliation: Dr. Lorenz Studer Laboratory, Developmental Biology Department, Gerstner Sloan-Kettering Graduate School, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Justine D
authors.initials: JD
authors.lastname: Miller
authors.forename: Thorsten M
authors.initials: TM
authors.lastname: Schlaeger
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D049109:Cell Proliferation; D065150:Cellular Reprogramming / Q000235:genetics; D002999:Clone Cells; D003114:Colony-Forming Units Assay; D005347:Fibroblasts / Q000166:cytology* / Q000821:virology*; D018014:Gene Transfer Techniques; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D013194:Staining and Labeling; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014779:Virus Replication / Q000502:physiology
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors