Abstract
This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.
Keywords:
Erythroblast; Human induced pluripotent stem cells; Peripheral blood mononuclear cells; Reprogramming; Sendai virus.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cell Culture Techniques / methods*
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Cell Separation / methods
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Cells, Cultured
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Cellular Reprogramming Techniques / methods*
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Cellular Reprogramming*
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Culture Media
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Cytokines / pharmacology
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Erythroblasts / cytology
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Erythroblasts / drug effects
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Erythroblasts / virology
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Fibroblasts / cytology
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Genetic Vectors / genetics*
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Humans
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Induced Pluripotent Stem Cells / cytology*
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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 / physiology
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Leukocytes, Mononuclear / cytology*
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Leukocytes, Mononuclear / drug effects
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Leukocytes, Mononuclear / virology
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Mice
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Octamer Transcription Factor-3 / genetics
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Octamer Transcription Factor-3 / physiology
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Proto-Oncogene Proteins c-myc / genetics
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Proto-Oncogene Proteins c-myc / physiology
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Recombinant Proteins / genetics
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SOXB1 Transcription Factors / genetics
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SOXB1 Transcription Factors / physiology
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Sendai virus / genetics*
Substances
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Culture Media
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Cytokines
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KLF4 protein, human
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Klf4 protein, mouse
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Kruppel-Like Factor 4
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Kruppel-Like Transcription Factors
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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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Recombinant Proteins
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SOX2 protein, human
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SOXB1 Transcription Factors