An improved method for generating and identifying human induced pluripotent stem cells

This chapter describes the methods we use to derive and characterize human induced pluripotent stem (iPS) cells. We describe in order, first our culture techniques for the starting fibroblast populations and methods for retrovirus preparation and concentration. Subsequently, a detailed iPS derivatio...

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Published inMethods in molecular biology (Clifton, N.J.) Vol. 636; p. 191
Main Authors Mali, Prashant, Ye, Zhaohui, Chou, Bin-Kuan, Yen, Jonathan, Cheng, Linzhao
Format Journal Article
LanguageEnglish
Published United States 01.01.2010
Subjects
Animals
Cell Culture Techniques - instrumentation
Cell Culture Techniques - methods
Cell Separation - methods
Cells, Cultured
Cellular Reprogramming
Fibroblasts - cytology
Fibroblasts - physiology
Genetic Vectors - genetics
Genetic Vectors - metabolism
Humans
Induced Pluripotent Stem Cells - cytology
Induced Pluripotent Stem Cells - physiology
Mice
Retroviridae - genetics
Retroviridae - metabolism
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Summary:This chapter describes the methods we use to derive and characterize human induced pluripotent stem (iPS) cells. We describe in order, first our culture techniques for the starting fibroblast populations and methods for retrovirus preparation and concentration. Subsequently, a detailed iPS derivation protocol suitable for human fibroblast populations is discussed using standard retroviral vectors expressing the classic four or three reprogramming genes. Finally, we elaborate a robust technique for monitoring and identification of potential iPS cells through live staining of reprogrammed cells. We also outline steps for characterization of the resulting iPS cell lines.
ISSN:1940-6029
DOI:10.1007/978-1-60761-691-7_12