
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.
Staining and Labeling, Cell Survival, Genetic Vectors, Induced Pluripotent Stem Cells, Lentivirus, Cell Culture Techniques, Enzyme-Linked Immunosorbent Assay, Dermis, Fibroblasts, Cellular Reprogramming, Immunohistochemistry, Colony-Forming Units Assay, Kruppel-Like Factor 4, Imaging, Three-Dimensional, Transduction, Genetic, Escherichia coli, Humans, Plasmids
Staining and Labeling, Cell Survival, Genetic Vectors, Induced Pluripotent Stem Cells, Lentivirus, Cell Culture Techniques, Enzyme-Linked Immunosorbent Assay, Dermis, Fibroblasts, Cellular Reprogramming, Immunohistochemistry, Colony-Forming Units Assay, Kruppel-Like Factor 4, Imaging, Three-Dimensional, Transduction, Genetic, Escherichia coli, Humans, Plasmids
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| influence This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically). | Top 10% | |
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