Abstract
Human-induced pluripotent stem cells (iPSCs) can be generated from patient-specific somatic cells by forced expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Sustained expression of the transgenes during reprogramming is crucial for the successful derivation of iPSCs. Integrating retroviruses have been used to achieve the required prolonged expression; however, issues of undesirable transgene expression in the iPSC-derived cell types post reprogramming can occur. Alternative non-integrating approaches to reprogram somatic cells into pluripotency have been established. Here, we describe a detailed method for generating human iPSCs from fibroblasts and peripheral blood mononuclear cells (PBMCs) using the non-integrating episomal plasmids. The delivery of the episomal plasmids into the somatic cells is achieved using a nucleofection technique, and reprogramming is performed in chemically defined media. This process takes approximately 30 days to establish the iPSC colonies. We also describe a method for growing iPSCs on vitronectin as well as procedures for the long-term expansion of iPSCs on human fibroblast feeder cells.
Keywords:
Episomal reprogramming; Human peripheral mononuclear cells; Induced pluripotent stem cells; Stem cells; iPSC.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Culture Techniques / methods
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Cells, Cultured
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Cellular Reprogramming / genetics*
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Culture Media / chemistry*
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Electroporation / methods
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Feeder Cells
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Fibroblasts / cytology
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Fibroblasts / metabolism
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Genetic Vectors
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Humans
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Induced Pluripotent Stem Cells / cytology*
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Induced Pluripotent Stem Cells / metabolism
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Kruppel-Like Factor 4
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Kruppel-Like Transcription Factors / genetics
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Kruppel-Like Transcription Factors / metabolism
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Leukocytes, Mononuclear / cytology
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Leukocytes, Mononuclear / metabolism
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Octamer Transcription Factor-3 / genetics
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Octamer Transcription Factor-3 / metabolism
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Plasmids / genetics
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Plasmids / metabolism*
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Proto-Oncogene Proteins c-myc / genetics
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Proto-Oncogene Proteins c-myc / metabolism
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism
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SOXB1 Transcription Factors / genetics
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SOXB1 Transcription Factors / metabolism
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Vitronectin
Substances
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Culture Media
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KLF4 protein, human
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Kruppel-Like Factor 4
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Kruppel-Like Transcription Factors
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Lin28A protein, human
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MYC protein, human
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Octamer Transcription Factor-3
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POU5F1 protein, human
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Proto-Oncogene Proteins c-myc
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RNA-Binding Proteins
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SOX2 protein, human
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SOXB1 Transcription Factors
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Transcription Factors
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Vitronectin
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Green Fluorescent Proteins