Pancreas Differentiation of Mouse ES Cells

Suzanne J. Micallef1, Xueling Li1, Andrew G. Elefanty1, Edouard G. Stanley1

1 Monash University, Victoria, Australia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1G.2
DOI:  10.1002/9780470151808.sc01g02s2
Online Posting Date:  August, 2007
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Abstract

This unit describes the derivation of pancreatic cells from mouse embryonic stem cells (ESCs). Mouse ESCs are pluripotent immortal cells derived from the inner cell mass of pre‐implantation blastocyst‐stage embryos that possess the ability to differentiate into any cell type within the adult animal. In vitro, ESCs can be differentiated into a variety of cell types representing derivatives of the three embryonic germ layers, mesoderm, endoderm, and ectoderm. Successfully differentiating ES cells to pancreatic cells has the potential to provide an alternative to cadaver‐derived cells for treatment of type I diabetes. This unit outlines a method for the differentiation of ESCs toward pancreatic endoderm in serum‐free medium from embryoid bodies (EBs) formed in suspension or spin EBs. In addition there is a protocol for maintaining ESC. Curr. Protoc. Stem Cell Biol. 2:1G.2.1‐1G.2.8. © 2007 by John Wiley & Sons, Inc.

Keywords: ES cells; differentiation; pancreatic endoderm

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differentiation of Mouse ES Cells to Pancreatic Cells from Embryoid Bodies Formed in Suspension
  • Alternate Protocol 1: Differentiation of ES Cells to Pancreatic Cells from Spin EBs
  • Support Protocol 1: Maintenance of Embryonic Stem Cells In Vitro
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Differentiation of Mouse ES Cells to Pancreatic Cells from Embryoid Bodies Formed in Suspension

  Materials
  • Mouse ESCs ( protocol 3)
  • Mouse ES cell medium with LIF (see recipe)
  • Phosphate‐buffered saline without CaCl 2 and MgCl 2 (CMF‐PBS)
  • Trypsin‐Versine chicken serum (TVCS; Invitrogen)
  • Feeder depletion medium (see recipe)
  • Chemically defined medium (CDM; see recipe)
  • Recombinant human BMP4 (rhBMP4; R & D Systems)
  • 10 mM all‐trans retinoic acid (ATRA; Sigma‐Aldrich)
  • Trypan blue
  • 1 M nicotinamide
  • 15‐ml conical centrifuge tube
  • Refrigerated benchtop centrifuge (e.g., Sigma, 4K15; www.sigma‐zentrifugen.de)
  • 10‐cm tissue culture dishes
  • 6‐cm low‐adherent tissue culture dishes (Phoenix Biomedical).
  • Adherent 96‐well tissue culture plates
  • 50‐ml tubes
  • Dissecting microscope
  • Pulled, glass capillaries
  • Additional reagents and equipment for cell counting (Phelan, )

Alternate Protocol 1: Differentiation of ES Cells to Pancreatic Cells from Spin EBs

  • Gelatin solution
  • Multichannel pipet
  • Low‐adherent, round‐bottomed 96‐well tissue culture plates

Support Protocol 1: Maintenance of Embryonic Stem Cells In Vitro

  Materials
  • Mitotically inactivated PMEFs
  • PMEF medium (see recipe)
  • Mouse ESC cultures grown on feeder cells in 25‐cm2 tissue culture flasks
  • Phosphate‐buffered saline without CaCl 2 and MgCl 2 (CMF‐PBS)
  • Mouse ES cell medium with LIF (see recipe)
  • Trypsin‐Versine chicken serum (TVCS, Invitrogen)
  • 25‐cm2 gelatinized‐tissue culture flask with vented cap (see recipe)
  • 15‐ml conical centrifuge tube
  • Benchtop centrifuge (Sigma, 4K15; www.sigma‐zentrifugen.de)
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Figures

Videos

Literature Cited

Literature Cited
   Barnett, L.D and Kontgen, F. 2001. Gene targeting in a centralized facility. Methods Mol. Biol. 158:65‐82.
   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.
   Merani, S. and Shapiro, A.M. 2006. Current status of pancreatic islet transplantation. Clin Sci (Lond) 110:611‐625.
   Micallef, S.J., Janes, M.E., Knezevic, K., Davis, R.P., Elefanty, A.G., and Stanley, E.G. 2004. Retinoic acid induces pdx1‐positive endoderm in differentiating mouse embryonic stem cells. Diabetes 54:301‐305.
   Micallef, S.J., Li, X., Janes, M.E., Jackson, S.A., Sutherland, R.M., Lew, A.M., Harrison, L.C., Elefanty, A.G., and Stanley, E.G. 2007. Endocrine cells develop within pancreatic bud‐like structures derived from mouse ES cells differentiated in response to BMP4 and retinoic acid. Stem Cell Res. In press.
   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., Marina, G., Vintersten, K., and Behringer, R. 2003. Manipulating the Mouse Embryo: A Laboratory Manual (3rd edition). Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
   Ng, E.S., Azzola, L., Sourris, K., Robb, L., Stanley, E.G., and Elefanty, A.G. 2005a. The primitive streak gene Mixl1 is required for efficient haematopoiesis and BMP4‐induced ventral mesoderm patterning in differentiating ES cells. Development 132:873‐884.
   Ng, E.S., Davis, R.P., Azzola, L., Stanley, E.G., and Elefanty, A.G. 2005b. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood 106:1601‐1603.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A3F.1‐A.3F.18.
   Stafford, D. and Prince, V.E. 2002. Retinoic acid signaling is required for a critical early step in zebrafish pancreatic development. Curr. Biol. 12:1215‐1220.
   Stafford, D., Hornbruch, A., Mueller, P.R., and Prince, V.E. 2004. A conserved role for retinoid signaling in vertebrate pancreas development. Dev. Genes Evol. 214:432‐441.
   Wiles, M.V. and Johansson, B.M. 1999. Embryonic stem cell development in a chemically defined medium. Exp. Cell Res. 247:241‐248.
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