Expansion of Human Embryonic Stem Cells In Vitro

Magdaline Costa1, Koula Sourris1, Tanya Hatzistavrou1, Andrew G. Elefanty1, Edouard G. Stanley1

1 Monash University, Clayton, Victoria
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.1
DOI:  10.1002/9780470151808.sc01c01s5
Online Posting Date:  May, 2008
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Abstract

This unit describes a protocol for the large‐scale expansion of karyotypically normal human embryonic stem cells (hESCs). hESCs can be maintained indefinitely as dense colonies that are mechanically cut into pieces, which are subsequently transferred to fresh organ culture dishes seeded with primary mouse embryonic fibroblasts (MEFs). hESCs can also be enzymatically passaged (bulk culture); however, over time, this style of culturing may lead to the acquisition of chromosomal abnormalities. Nevertheless, enzymatic passaging can be used for short periods (up to 25 passages) without the appearance of cells with abnormal karyotypes. Curr. Protoc. Stem Cell Biol. 5:1C.1.1‐1C.1.7. © 2008 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells (hESCs); mechanical passaging; enzymatic passaging; hESC expansion

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Maintenance of hESC Cultures by Mechanical Passaging (Maintenance Culture)
  • Basic Protocol 2: Expansion of hESC in Bulk Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Maintenance of hESC Cultures by Mechanical Passaging (Maintenance Culture)

  Materials
  • hESCs, starting from macroscopic colonies (∼1‐mm diameter) grown on MEFs (unit 1.2)
  • Mitotically inactivated (irradiation‐ or mitomycin C–treated; unit 1.3) mouse embryonic fibroblasts (MEFs; Conner, ; Nagy et al., )
  • hESC medium (see recipe)
  • Stereomicroscope
  • 26‐G × ½‐in. (0.45 × 13–mm) needles
  • 1‐ml syringe
  • 60 × 15–mm center‐well organ culture dishes
  • Additional reagents and equipment for hESCs grown on feeder cells (unit 1.2) and mitotically inactivated mouse embryonic fibroblasts (Conner, ; unit 1.3)

Basic Protocol 2: Expansion of hESC in Bulk Culture

  Materials
  • hESCs in organ culture dishes ( protocol 1)
  • hESC medium (see recipe)
  • Trypsin (see recipe) or TrypLE Select (Invitrogen)
  • Phosphate‐buffered saline without CaCl 2, without MgCl 2 (CMF‐PBS)
  • Soybean Trypsin Inhibitor (Invitrogen), optional
  • 26‐G × ½‐in. (0.45 × 13–mm) needles
  • 1‐ml syringe
  • 200‐µl Gilson pipet (optional)
  • 75‐cm2 flask containing preseeded mitotically inactivated MEFs at a density of 4 × 104 cells/cm2 , 2 × 104 cells/cm2, and 1 × 104 cells/cm2 (Connor, ; unit 1.3)
  • 150‐cm2 tissue culture flask with vented cap
  • Stereomicroscope
  • Additional reagents and equipment for preparing mitotically inactivated mouse embryonic fibroblasts (Connor, ; unit 1.3), growing hESCs in organ culture dishes ( protocol 1), cell counting (Phelan, ; unit 1.3), and electroporation (Costa et al., )
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Figures

Videos

Literature Cited

Literature Cited
   Amit, M., Carpenter, M.K., Inokuma, M.S., Chiu, C.P., Harris, C.P., Waknitz, M.A., Itskovitz‐Eldor, J., and Thomson, J.A. 2000. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227: 271‐278.
   Conner, D.A. 2000. Mouse embryo fibroblast (MEF) feeder cell preparation. Curr. Protoc. Mol. Biol. 51: 23.2.1‐23.2.7.
   Costa, M., Dottori, M., Sourris, K., Jamshidi, P., Hatzistavrou, T., Davis, R., Azzola, L., Jackson, S., Lim, S‐M., Pera, M., Elefanty, A.G., and Stanley, E.G. 2007. A method for genetic modification of human embryonic stem cells using electroporation. Nat. Protoc. 2: 792‐796.
   Draper, J.S., Smith, K., Gokhale, P., Moore, H.D., Maltby, E., Johnson, J., Meisner, L., Zwaka, T.P., Thomson, J.A., and Andrews, P.W. 2004. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22: 53‐54.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154‐156.
   Martin, G.R. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. U.S.A. 78: 7634‐7638.
   McNeish, J. 2004. Embryonic stem cells in drug discovery. Nat. Rev. Drug Discov. 3: 70‐80.
   Menendez, P., Wang, L., and Bhatia, M. 2005. Genetic manipulation of human embryonic stem cells: A system to study early human development and potential therapeutic applications. Curr. Gene Ther. 5: 375‐385.
   Nagy, A., Rossant, J., Nagy, R., Abramow‐Newerly, W., and Roder, J.C. 1993. Derivation of completely cell culture‐derived mice from early‐passage embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 90: 8424‐8428.
   Nagy, A., Gertsenstein, M., and Vintersten, K. 2003. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74: A3F.1‐A.3F.18.
   Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. 2000. Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nat. Biotechnol. 18: 399‐404.
   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282: 1145‐1147.
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