Human iPS Cell Derivation/Reprogramming

In‐Hyun Park1, George Q. Daley2

1 Children's Hospital Boston and Dana‐Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts, 2 Brigham and Women's Hospital, Howard Hughes Medical Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.1
DOI:  10.1002/9780470151808.sc04a01s8
Online Posting Date:  January, 2009
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Abstract

This unit describes a protocol for deriving induced pluripotent stem (iPS) cells from human 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 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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of VSV‐G Pseudotyped Retrovirus
  • Basic Protocol 2: Infection of Fibroblasts and Isolation of iPS Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Production of VSV‐G Pseudotyped Retrovirus

  Materials
  • 293T cells (ATCC, cat. no. CRL11268)
  • Fugene 6 (Roche Applied Science, cat. no. 1181509001)
  • DMEM
  • DMEM/F12 (Invitrogen)
  • pMIG‐OCT4 (Addgene, clone 17225), pMIG‐SOX2 (Addgene, clone 17226), pMIG‐KLF4 (clone 17227), and pMIG‐MYC (requested from Dr. Cleveland from the Scripps Research Institute)
  • VSV‐G (Addgene, clone 8454), and Gag‐Pol (Addgene, clone 8455)
  • 293T cell medium (see recipe)
  • 10‐cm dishes
  • 0.45‐µm filter
  • 38.5‐ml polyallomer centrifuge tube (Beckman, cat. no. 326823)
  • Cryovials
  • Additional reagents and equipment for determining the titer of the virus (Sastry et al., ; Tiscornia et al., )

Basic Protocol 2: Infection of Fibroblasts and Isolation of iPS Cells

  Materials
  • Human fibroblasts, acquired from skin biopsy (refer to unit 1.1; split fibroblasts when they reach 70% confluency)
  • Human fibroblast medium (see recipe)
  • Protamine sulfate (see recipe)
  • Retroviral supernatants bearing the appropriate plasmids ( protocol 1)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Mediatech, cat. no. 21‐040‐CV)
  • MEF (mouse embryonic fibroblasts), CF‐1 strain, irradiated (Global Stem, cat. no. GSC‐6001G)
  • MEF medium (see recipe)
  • 0.1% (w/v) gelatin (see recipe)
  • 0.05% trypsin/EDTA
  • hESC medium (see recipe)
  • Gelatin‐coated 12‐well plate preplated with MEFs at a density of 1 × 104 cells/cm2
  • Collagenase IV (see recipe)
  • Gelatin‐coated 6‐well plate preplated with MEFs
  • Freezing medium (see recipe)
  • Liquid nitrogen
  • 6‐well plate
  • 10‐cm dish
  • Dissection microscope
  • 20‐ and 1000‐µl pipets
  • 21‐G needle or cell lifter (Corning, cat. no. CT‐3008)
  • 15‐ml conical tube
  • Cryovials
  • −80°C freezer
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Figures

Videos

Literature Cited

Literature Cited
   Hanna, J., Wernig, M., Markoulaki, S., Sun, C.W., Meissner, A., Cassady, J.P., Beard, C., Brambrink, T., Wu, L.C., Townes, T.M., and Jaenisch, R. 2007. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318:1920‐1923.
   Jaenisch, R. and Young, R. 2008. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132:567‐582.
   Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., Yachechko, R., Tcheiu, J., Jaenisch, R., Plath, K., and Hochedlinger, K. 2007. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1:55‐70.
   Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N., and Yamanaka, S. 2008. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26:101‐106.
   Okita, K., Ichisaka, T., and Yamanaka, S. 2007. Generation of germline‐competent induced pluripotent stem cells. Nature 448:313‐317.
   Park, I.H., Zhao, R., West, J.A., Yabuuchi, A., Huo, H., Ince, T.A., Lerou, P.H., Lensch, M.W., and Daley, G.Q. 2008. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141‐146.
   Sastry, L., Johnson, T., Hobson, M.J., Smucker, B., and Cornetta, K. 2002. Titering lentiviral vectors: Comparison of DNA, RNA and marker expression methods. Gene Ther. 9:1155‐1162.
   Shi, Y., Do, J.T., Desponts, C., Hahm, H.S., Scholer, H.R., and Ding, S. 2008. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2:525‐528.
   Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861‐872.
   Takahashi, K. and Yamanaka, S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663‐676.
   Tiscornia, G., Singer, O., and Verma, I.M. 2006. Production and purification of lentiviral vectors. Nat. Protoc. 1:241‐245.
   Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E., and Jaenisch, R. 2007. In vitro reprogramming of fibroblasts into a pluripotent ES‐cell‐like state. Nature 448:318‐324.
   Yu, J., Vodyanik, M.A., Smuga‐Otto, K., Antosiewicz‐Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.L., and Thomson, J.A. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917‐1920.
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