Human iPS Cell - The Michele Pagano Laboratory.pdf

Human iPS Cell UNIT 4A.1 Derivation/Reprogramming In-Hyun Park1 and George Q. Daley1, 2 1Children’s Hospital Boston and Dana-Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts 2Brigham and Women’s Hospital, Howard Hughes Medical Institute, Boston, Massachusetts ABSTRACT This unit describes a protocol for deriving induced pluripotent stem (iPS) cells from hu- man fibroblast cells. Human fibroblast cells are infected with retroviral vectors expressing four transcription factors (Oct4, Sox2, Klf4, and Myc) and selected for 3 to 4 weeks under human embryonic stem (hES) cell culture conditions. iPS cell colonies are mechanically isolated using a dissection microscope and handled like hES cells thereafter. Human iPS cells share similarities with hES cells including the expression of pluripotency genes, and differentiation as embryoid bodies in vitro into three germ layers (EB) and in vivo C as teratomas. Curr. Protoc. Stem Cell Biol. 8:4A.1.1-4A.1.8. 2009 by John Wiley Sons, Inc. Keywords: human induced pluripotent stem (iPS) cells human embryonic stem (hES) cells reprogramming retroviral vectors INTRODUCTION This unit describes a protocol for deriving induced pluripotent stem (iPS) cells from human fibroblasts. hES cells have the property of self-renewal and pluripotency that pro- vides an unlimited resource for research and medical applications. Recently, terminally differentiated murine and human somatic cells were induced to become pluripotent stem (iPS) cells by use of a four-transcription-factor cocktail (Oct-4, Sox-2, Klf4, and Myc; Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2007; Park et al., 2008). In this unit, the production of retrovirus-expressing reprogramming factors, infection of human fibro