Genetic Manipulation of Human Induced Pluripotent Stem Cells

Angela Wang1, Chee Gee Liew1

1 Stem Cell Core, Department of Cell Biology and Neuroscience, University of California Riverside, Riverside, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5B.2
DOI:  10.1002/9780470151808.sc05b02s23
Online Posting Date:  November, 2012
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Abstract

Human induced pluripotent stem cells (HIPSC) have tremendous value as a source of autologous cells for cellular transplantation in the treatment of degenerative diseases. The protocols described here address methods for large‐scale genetic modification of HIPSCs. The first is an optimized method for transfecting HIPSCs cultured in feeder‐free conditions. The second method allows nucleofection of trypsinized HIPSCs at an optimal cell density. Both methods enable robust generation of stable HIPSC transfectants within two weeks. Our protocols are highly reproducible and do not require optimization for individual HIPSC and human embryonic stem cell (HESC) lines. Curr. Protoc. Stem Cell Biol. 23:5B.2.1‐5B.2.9. © 2012 by John Wiley & Sons, Inc.

Keywords: human induced pluripotent stem cells (HIPSCs); human embryonic stem cells (HESCs); transfection; genetic manipulation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Establishment and Expansion of HIPSC Cultures on Feeder‐Free
  • Basic Protocol 2: Transfection of HIPSC on Monolayer
  • Basic Protocol 3: Nucleofection of HIPSC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Establishment and Expansion of HIPSC Cultures on Feeder‐Free

  Materials
  • Geltrex reduced growth factor basement membrane matrix (Invitrogen, cat. no. 12760‐021, A10480‐02) or BD Matrigel basement membrane matrix (BD Biosciences)
  • Dulbecco's modified Eagle medium (DMEM; Lonza, cat. no. 12‐741F)
  • Previously established HIPSC lines
  • Accutase (Innovative Cell Technologies, cat. no. AT 104‐500)
  • mTeSR1 medium (see recipe) or STEMPRO medium (see recipe)
  • 1.7‐ml microcentrifuge tubes
  • −20°C freezer
  • 15‐ml conical tubes
  • 6‐well plates
  • 10‐ml sterile serological pipet
  • 15‐mm glass beads
  • 1000‐µl sterile filtered pipet tips
  • Centrifuge
  • 37°C humidified incubator with 5% CO 2
NOTE: HIPSCs previously maintained on MEF feeder cells can be split, transferred onto Geltrex‐coated dish, and maintained as feeder‐free cultures for long‐term expansion.

Basic Protocol 2: Transfection of HIPSC on Monolayer

  Materials
  • Previously established feeder‐free HIPSC lines
  • Knockout‐DMEM/F12 (KO‐DMEM/F12; Invitrogen, cat. no. 12660‐012)
  • GeneJuice transfection reagent (Novagen, cat. no. 70967)
  • Plasmid DNA, e.g., pCAG‐eGFP (Liew et al., )
  • mTeSR1 medium (see recipe)
  • Appropriate antibiotics
  • 6‐well plates
  • 1.7‐ml microcentrifuge tubes
  • 37°C humidified incubator with 5% CO 2
  • Vortex mixer

Basic Protocol 3: Nucleofection of HIPSC

  Materials
  • 6‐well plate with feeder cells
  • HIPSC KnockOut serum replacement medium (see recipe)
  • ROCK inhibitor (Y27632; Reagents Direct, cat. no. 53‐B85)
  • Amaxa Human Stem Cell Nucleofection kit 1 (Lonza, cat. no. VVPH‐5012) containing:
    • Human stem cell nucleofactor solution
    • Supplement 1
  • Previously established feeder‐free HIPSC lines
  • Phosphate‐buffered serum, calcium‐ and magnesium‐free (CMF‐PBS; Lonza, cat. no. BE17‐512F)
  • 0.25% trypsin‐EDTA
  • 10% fetal bovine serum (FBS)‐containing MEF medium (see recipe)
  • Appropriate antibiotics
  • Plasmid DNA, e.g., pCAG‐eGFP (Liew et al., )
  • 1.7‐ml microcentrifuge tubes
  • 37°C humidified incubator with 5% CO 2
  • 1000‐µl sterile‐filtered pipet tips
  • 15‐ml conical tubes
  • Centrifuge
  • Amaxa nucleofector II device (Lonza)
  • 1‐ml pipettor
  • Nucleofection cuvette
  • Sterile Pasteur pipet
  • Microscope
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Figures

Videos

Literature Cited

Literature Cited
   Braam, S.R., Denning, C., Matsa, E., Young, L.E., Passier, R., and Mummery, C.L. 2008a. Feeder‐free culture of human embryonic stem cells in conditioned medium for efficient genetic modification. Nat. Protoc. 3:1435‐1443.
   Braam, S.R., Denning, C., van den Brink, S., Kats, P., Hochstenbach, R., Passier, R., and Mummery, C.L. 2008b. Improved genetic manipulation of human embryonic stem cells. Nat. Methods 5:389‐392.
   Chatterjee, P., Cheung, Y., and Liew, C.G. 2011. Transfecting and nucleofecting human induced pluripotent stem cells. J. Vis. Exp. e3110.
   Fischer, Y., Ganic, E., Ameri, J., Xian, X., Johannesson, M., and Semb, H. 2010. NANOG reporter cell lines generated by gene targeting in human embryonic stem cells. PLoS One 5:e12533.
   Irion, S., Luche, H., Gadue, P., Fehling, H.J., Kennedy, M., and Keller, G. 2007. Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat. Biotechnol. 25:1477‐1482.
   Liew, C.G., Draper, J.S., Walsh, J., Moore, H., and Andrews, P.W. 2007. Transient and stable transgene expression in human embryonic stem cells. Stem Cells 25:1521‐1528.
   Liew, C.G., Shah, N.N., Briston, S.J., Shepherd, R.M., Khoo, C.P., Dunne, M.J., Moore, H.D., Cosgrove, K.E., and Andrews, P.W. 2008. PAX4 enhances beta‐cell differentiation of human embryonic stem cells. PLoS One 3:e1783.
   Norrman, K., Fischer, Y., Bonnamy, B., Wolfhagen Sand, F., Ravassard, P., and Semb, H. 2010. Quantitative comparison of constitutive promoters in human ES cells. PLoS One 5:e12413.
   Schinzel, R.T., Ahfeldt, T., Lau, F.H., Lee, Y.K., Cowley, A., Shen, T., Peters, D., Lum, D.H., and Cowan, C.A. 2011. Efficient culturing and genetic manipulation of human pluripotent stem cells. PLoS One 6:e27495.
   Siemen, H., Nix, M., Endl, E., Koch, P., Itskovitz‐Eldor, J., and Brustle, O. 2005. Nucleofection of human embryonic stem cells. Stem Cells Dev. 14:378‐383.
   Sun, N., Panetta, N.J., Gupta, D.M., Wilson, K.D., Lee, A., Jia, F., Hu, S., Cherry, A.M., Robbins, R.C., Longaker, M.T., and Wu, J.C. 2009. Feeder‐free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc. Natl. Acad. Sci. U.S.A. 106:15720‐15725.
   Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J.B., Nishikawa, S., Muguruma, K., and Sasai, Y. 2007. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25:681‐686.
   Zwaka, T.P. and Thomson, J.A. 2003. Homologous recombination in human embryonic stem cells. Nat. Biotechnol. 21:319‐321.
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