title: Derivation of induced pluripotent stem cells by lentiviral transduction.
abstract: This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodeficiency virus (HIV) and provide an effective means for the delivery, integration, and expression of exogenous genes in mammalian cells. Lentiviruses are attractive gene delivery vehicles as they are able to infect both proliferating and nonproliferating cells. Lentiviruses stably integrate into the genome without incurring cellular toxicity and can maintain sustained transgene expression during prolonged host cell proliferation and differentiation. In this protocol, we describe how to prepare lentiviruses, stably transduce human fibroblasts, and identify bona fide iPSC colonies based on morphological similarity to human embryonic stem cell (ESC) colonies and live-cell immunological staining using cell-surface markers of human PSCs such as Tra-1-60 and Tra-1-81.
authors.affiliation: Neuroscience Laboratories, Centers for Neuroscience and Translational Research, CHOC Research Institute, Children's Hospital of Orange County Research Institute, Orange, CA, USA.
authors.forename: Hubert E
authors.initials: HE
authors.lastname: Nethercott
authors.forename: David J
authors.initials: DJ
authors.lastname: Brick
authors.forename: Philip H
authors.initials: PH
authors.lastname: Schwartz
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival; D065150:Cellular Reprogramming / Q000235:genetics; D003114:Colony-Forming Units Assay; D020405:Dermis / Q000166:cytology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D013194:Staining and Labeling; D014161:Transduction, Genetic