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

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.