Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1357; p. 23
• Main Authors: Soares, Filipa A C, Pedersen, Roger A, Vallier, Ludovic
• Format: Journal Article
• Language: English
• Published: United States 01.01.2016
• Subjects: Animals; Cell Culture Techniques - methods; Cell Separation - methods; Cells, Cultured; Cellular Reprogramming; Cellular Reprogramming Techniques - methods; Culture Media; Cytokines - pharmacology; Erythroblasts - cytology; Erythroblasts - drug effects; Erythroblasts - virology; Fibroblasts - cytology; Genetic Vectors - genetics; Humans; Induced Pluripotent Stem Cells - cytology; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors - genetics; Kruppel-Like Transcription Factors - physiology; Leukocytes, Mononuclear - cytology; Leukocytes, Mononuclear - drug effects; Leukocytes, Mononuclear - virology; Mice; Octamer Transcription Factor-3 - genetics; Octamer Transcription Factor-3 - physiology; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - physiology; Recombinant Proteins - genetics; Sendai virus - genetics; SOXB1 Transcription Factors - genetics; SOXB1 Transcription Factors - physiology; Human induced pluripotent stem cells; Peripheral blood mononuclear cells; Reprogramming; Sendai virus; Erythroblast
• ISSN: 1940-6029
• DOI: 10.1007/7651_2015_202

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.