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Methods Mol Biol. 2010;636:191-205. doi: 10.1007/978-1-60761-691-7_12.

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

Author information

  • 1Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Abstract

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.

PMID:
20336524
[PubMed - indexed for MEDLINE]
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