title: Reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) using retroviral vector with GFP.
abstract: Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine(1). It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)(2-4) . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone(4). Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.
authors.affiliation: Yale Stem Cell Center, Department of Genetics, Yale School of Medicine.
authors.forename: Kun-Yong
authors.initials: KY
authors.lastname: Kim
authors.forename: Eriona
authors.initials: E
authors.lastname: Hysolli
authors.forename: In-Hyun
authors.initials: IH
authors.lastname: Park
mesh_terms: D054337:Cell Dedifferentiation / Q000502:physiology; D065150:Cellular Reprogramming / Q000235:genetics / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D008856:Microscopy, Fluorescence / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D019076:Transgenes
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D049452:Green Fluorescent Proteins