Neural Crest Cells from Dual SMAD Inhibition

Faranak Fattahi1, Lorenz Studer2, Mark J. Tomishima2

1 Developmental Biology Program, Sloan Kettering Institute, New York, New York, 2 Center for Stem Cell Biology, Sloan Kettering Institute, New York, New York
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.9
DOI:  10.1002/9780470151808.sc01h09s33
Online Posting Date:  May, 2015
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Abstract

Neural crest (NC) cells are migratory multipotent progenitors that delaminate from the neural tube during embryonic development and give rise to various cell types in different organs. These cells are a transient embryonic cell population and therefore difficult to obtain from primary sources. Deriving NC from human pluripotent stem cells offers an alternative way to provide large‐scale human NC cells for developmental and disease‐related studies. In recent years, the protocols to make these cells have matured, incorporating the efficient conversion of pluripotent stem cells to neural cells through dual SMAD inhibition and early Wnt activation to increase the yield of NC cells. Here, we provide a minor variation to this NC protocol that has been successful for many in our laboratories. © 2015 by John Wiley & Sons, Inc.

Keywords: neural crest; human pluripotent stem cells; dual SMAD inhibition

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Maintenance and Preparation of Pluripotent Stem Cells for Differentiation
  • Basic Protocol 2: NC Differentiation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Maintenance and Preparation of Pluripotent Stem Cells for Differentiation

  Materials
  • 0.1% (w/v) gelatin: prepare by dissolving the gelatin powder in CMF‐DPBS; autoclave to sterilize
  • Calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; Life Technologies, cat. no. 14190)
  • MEF CF‐1 mitomycin C–treated mouse embryonic fibroblasts (MEFs; Applied StemCell, Inc., cat. no. ASF‐1223)
  • MEF medium (see recipe)
  • Human embryonic stem cells (unit 1.5; Peura et al., ) or induced pluripotent stem cells (see appropriate units in Chapter 4, Section A) growing in culture
  • 1 mg/ml collagenase IV (Life Technologies, cat. no. 17104‐019) in DMEM/F12 medium (Life Technologies, cat. no. 11330‐032); filter sterilized
  • ES medium (see recipe)
  • KSR medium (see recipe)
  • 10‐cm culture dishes
  • Cell scraper

Basic Protocol 2: NC Differentiation

  Materials
  • Matrigel frozen stock (see recipe)
  • DMEM/F12 medium (Life Technologies, cat. no. 11330‐032)
  • Pluripotent stem cells growing in culture ( protocol 1)
  • Calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; Life Technologies, cat. no. 14190)
  • 0.05% (w/v) trypsin (Life Technologies, cat. no. 25300‐054)
  • ES medium (see recipe)
  • 10 mM (1000×) Y‐27632 dihydrochloride (Tocris Bioscience, cat. no. 1254) stock in sterile water
  • KSR medium (see recipe)
  • 10 mM (1000×) SB‐431542 (Tocris Bioscience, cat. no. 1614) stock in 100% ethanol
  • 5 mM (5000×) LDN‐193189 (Stemgent, cat. no. 04‐0074) stock in DMSO
  • 6 mM (2000×) CHIR 99021 (Tocris Bioscience, cat. no. 44230) stock in DMSO
  • N2 medium (see recipe)
  • 10‐cm culture dishes
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Figures

Videos

Literature Cited

Literature Cited
  Chambers, S.M., Mica, Y., Studer, L., and Tomishima, M.J. 2011. Converting human pluripotent stem cells to neural tissue and neurons to model neurodegeneration. Methods Mol. Biol. 793:87‐97.
  Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and Studer, L. 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27:275‐280.
  Chambers, S.M., Qi, Y., Mica, Y., Lee, G., Zhang, X.J., Niu, L., Bilsland, J., Cao, L., Stevens, E., Whiting, P., Shi, S.H., and Studer, L. 2012. Combined small‐molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat. Biotechnol. 30:715‐720.
  Chambers, S.M., Mica, Y., Lee, G., Studer, L., and Tomishima, M.J. 2013. Dual‐SMAD inhibition/WNT activation‐based methods to induce neural crest and derivatives from human pluripotent stem cells. Methods Mol. Biol.
  Lee, G., Kim, H., Elkabetz, Y., Al Shamy, G., Panagiotakos, G., Barberi, T., Tabar, V., and Studer, L. 2007. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat. Biotechnol. 25:1468‐1475.
  Mica, Y., Lee, G., Chambers, S.M., Tomishima, M.J., and Studer, L. 2013. Modeling neural crest induction, melanocyte specification, and disease‐related pigmentation defects in hESCs and patient‐specific iPSCs. Cell Rep. 3:1140‐1152.
  Peura, T., Schaft, J., Dumevska, B., and Stojanov, T. 2011. Generation of human embryonic stem cells. Curr. Protoc. Stem Cell Biol. 16:1A.5.1‐1A.5.19.
  Zeltner, N., Lafaille, F.G., Fattahi, F., and Studer, L. 2014. Feeder‐free derivation of neural crest progenitor cells from human pluripotent stem cells. J. Vis. Exp. 82. doi: 10.3791/51609.
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