Differentiation of Mouse Embryonic Stem Cells to Spinal Motor Neurons

Hynek Wichterle1, Mirza Peljto1

1 Departments of Pathology, Neurology and Neuroscience, Columbia University, New York, New York
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.1
DOI:  10.1002/9780470151808.sc01h01s5
Online Posting Date:  May, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Controlled differentiation of embryonic stem (ES) cells into clinically relevant cell types is a fundamental goal of stem cell research. This unit describes one of the most efficient protocols for conversion of mouse ES cells into a defined type of nerve cells, the spinal motor neurons. ES cells are separated from feeder mouse embryonic fibroblasts and aggregated to form embryoid bodies (EBs). Two days after the withdrawal of growth factors, EBs reach a stage at which they are responsive to patterning signals and can be effectively induced with retinoic acid (RA) to differentiate into spinal nerve cells. Nascent neural cells become responsive to the ventralizing signal sonic hedgehog (Hh) that controls expression of ventral spinal progenitor markers and initiates the genetic program of motor neuron differentiation. Curr. Protoc. Stem Cell Biol. 5:1H.1.1‐1H.1.9. © 2008 by John Wiley & Sons, Inc.

Keywords: controlled differentiation; mouse; embryonic stem cells; neural differentiation; spinal motor neurons; retinoic acid; sonic hedgehog

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Differentiation of ES Cells into Motor Neurons
  • Support Protocol 1: Preparation of Embryonic Stem Cells for Differentiation
  • Support Protocol 2: Fixation, Cryosectioning, and Immunostaining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Differentiation of ES Cells into Motor Neurons

  Materials
  • Dissociated ES cells suspended in ADFNK medium (see protocol 2)
  • ADFNK medium optimized for ES cell differentiation to HB9+ motor neurons (see recipe)
  • 1 mM all‐trans retinoic acid (RA; Sigma, cat. no. R2625)
  • Sonic hedgehog (Shh) protein (R&D Systems); Purmorphamine (Calbiochem, cat. no. 540220); or Hh agonist HhAg1.3 (Curis, Inc.)
  • Recombinant Rat GDNF (Glial cell line‐derived neurotrophic factor; R&D Systems, 512‐GF‐050)
  • 10‐cm Nunc tissue culture dishes (Nunc, cat. no. 150679)
  • Tissue culture microscope
  • 15‐ml tube
  • Suspension culture dishes (10‐cm; Corning, cat. no. 430591)
  • 100‐µm strainer (Falcon/Fisher Scientific, cat. no. 352360), optional
  • 200‐µl pipettor
  • Large orifice pipet tips (Fisher Scientific, 21‐197‐2A)
  • Additional reagents and equipment for fixation and immunocytochemistry ( protocol 3)

Support Protocol 1: Preparation of Embryonic Stem Cells for Differentiation

  Materials
  • Mouse ES cell line (for a better consistency it is recommended to start with a frozen aliquot of ES cells stored in liquid nitrogen)
  • 70% ethanol
  • ES medium (see recipe)
  • ADFNK medium (see recipe)
  • 0.05% (w/v) trypsin‐EDTA (Invitrogen, cat. no. 05300‐054)
  • Gelatinized 25‐cm2 culture flask (see recipe)
  • 37°C water bath
  • 15‐ml tube
  • Swinging‐bucket tabletop centrifuge (Eppendorf, cat. no. 5702)
  • Tissue culture microscope
  • Gelatinized 75‐cm2 culture flask, optional
  • 10‐cm tissue culture dish, optional
  • Additional reagents and equipment for cell counting (Phelan, )

Support Protocol 2: Fixation, Cryosectioning, and Immunostaining

  Materials
  • Cultures of differentiating EBs ( protocol 1)
  • Phosphate‐buffered saline (PBS; Cellgro, cat. no. 21‐030‐CV)
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • 30% (w/v) sucrose in PBS
  • Tissue‐Tek OCT (Electron Microscopy Diatome, Fisher Scientific, 62550‐12)
  • Dry ice
  • Blocking solution: 10% horse serum, 0.2% Triton X‐100 in PBS
  • Primary antibodies (see Table 1.1.1)
  • Secondary antibodies, fluorophore conjugated
  • Aqua‐Poly/Mount solution (Polysciences, cat. no. 18606)
  • Wide‐orifice pipet tip
  • 1.5‐ml microcentrifuge tubes
  • Swinging‐bucket tabletop centrifuge (Eppendorf, 5702)
  • Embedding molds
  • Superfrost Plus slides
  • ImmunoPen (Calbiochem/EMD, cat. no. 402176)
  • Humidified chamber
  • Additional reagents and equipment for cutting sections using a cryostat (Watkins, ) and immunohistochemistry including slide processing (Hoffman, )
    Table 1.0.1   MaterialsAntibodies Used for Screening Cells in Motor Neuron Differentiation

    Antigen Expression Supplier Catalog number Recommended dilutions
    Oct4 Expressed in ES cells Abcam ab19857 1:2000
    Sox2 Expressed in ES cells‐ day 4 Chemicon AB5603 1:2000
    Pax6 Expressed day 3 – 4 DSHB PAX6 1:30
    Olig2 Expressed day 4 – 5 Chemicon AB9610 1:20,000
    Hb9 Expressed day 5 – 7 DSHB 81.5C10 1:100
    Isl1 Expressed day 5 – 7 DSHB 39.4D5 1:100
    Lhx3 Expressed day 5 – 7 DSHB 67.4E12 1:100

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bain, G., Ray, W.J., Yao, M., and Gottlieb, D.I. 1996. Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochem. Biophys. Res. Commun. 223: 691‐694.
   Burdon, T., Stracey, C., Chambers, I., Nichols, J., and Smith, A. 1999. Suppression of SHP‐2 and ERK signalling promotes self‐renewal of mouse embryonic stem cells. Dev. Biol 210: 30‐43.
   Elkabetz, Y., Panagiotakos, G., Al Shamy, G., Socci, N.D., Tabar, V., and Studer, L. 2008. Human ES cell‐derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 22: 152‐165.
   Hofman, F. 2002. Immuohistochemistry. Curr. Protoc. Immunol. 49: 21.4.1‐21.4.23.
   Jessell, T.M. 2000. Neuronal specification in the spinal cord: Inductive signals and transcriptional codes. Nat. Rev. Genet. 1: L20‐29.
   Lee, H., Shamy, G.A., Elkabetz, Y., Schofield, C.M., Harrsion, N.L., Panagiotakos, G., Socci, N.D., Tabar, V., and Studer, L. 2007. Directed differentiation and transplantation of human embryonic stem cell‐derived motoneurons. Stem Cells 25: 1931‐1939.
   Li, X.J., Du, Z.W., Zarnowska, E.D., Pankratz, M., Hansen, L.O., Pearce, R.A., and Zhang, S.C. 2005. Specification of motoneurons from human embryonic stem cells. Nat. Biotechnol. 23: 215‐221.
   Li, X.J., Hu, B.Y., Jones, S.A., Zhang, Y.S., Lavaute, T., Du, Z.W., and Zhang, S.C. 2008. Directed Differentiation of Ventral Spinal Progenitors and Motor Neurons from Human Embryonic Stem Cells by Small Molecules. Stem Cells Jan 31, Epub ahead of print.
   Muhr, J., Graziano, E., Wilson, S., Jessell, T.M., and Edlund, T. 1999. Convergent inductive signals specify midbrain, hindbrain, and spinal cord identity in gastrula stage chick embryos. Neuron 23: 689‐702.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74: A.3F.1‐A.3F.18.
   Qi, X., Li, T.G., Hao, J., Hu, J., Wang, J., Simmons, H., Miura, S., Mishina, Y., and Zhao, G.Q. 2004. BMP4 supports self‐renewal of embryonic stem cells by inhibiting mitogen‐activated protein kinase pathways. Proc. Natl. Acad. Sci. U.S.A 101: 6027‐6032.
   Schulz, T.C., Palmarini, G.M., Noggle, S.A., Weiler, D.A., Mitalipova, M.M., and Condie, B.G. 2003. Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci. 4: 27.
   Shin, S., Dalton, S., and Stice, S.L. 2005. Human motor neuron differentiation from human embryonic stem cells. Stem Cells Dev. 14: 266‐269.
   Watkins, S. 1989. Cryosectioning. Curr. Protoc. Mol. Biol. 7: 14.2.1‐14.2.8.
   Wichterle, H., Lieberam, I., Porter, J.A., and Jessell, T.M. 2002. Directed differentiation of embryonic stem cells into motor neurons. Cell 110: 385‐397.
   Wilson, P.G. and Stice, S.S. 2006. Development and differentiation of neural rosettes derived from human embryonic stem cells. Stem Cell Rev. 2: 67‐77.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library