Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus
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:Reprogramming Human induced pluripotent stem cells Sendai virus Peripheral blood mononuclear cells Erythroblast
The authors thank Dr. Thomas Moreau for expert advice and support. The authors also acknowledge the contribution of “Fundação para a Ciência e a Tecnologia” (SFRH/BD/69033/2010) and Cambridge Hospitals National Institute for Health Research Biomedical Research Center and ERC starting grant Relieve-IMDs.
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