Neural Differentiation of Human ES Cells

Malkiel A. Cohen1, Pavey Itsykson1, Benjamin E. Reubinoff1

1 Hadassah University Medical Center, Ein‐Kerem, Jerusalem
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 23.7
DOI:  10.1002/0471143030.cb2307s36
Online Posting Date:  September, 2007
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Abstract

Human embryonic stem cells (hESCs) may be converted into highly enriched cultures of neural precursors under defined culture conditions. The neural precursors can proliferate in culture for prolonged periods of time, and can differentiate in vitro into mature neurons, astrocytes, and oligodendrocytes. The neurons are functional and have normal electrophysiological properties. After transplantation to the developing rodent brain, the neural precursors migrate extensively into the host brain parenchyma, respond to host brain signals, and differentiate in a region‐specific manner to progeny of the three neural lineages. The establishment of neuroectodermal precursors from hESCs allows the study of human neurogenesis in vitro and is an aid in drug discovery. In addition, the neural precursors may potentially serve as a platform for the development of specific functional neural cells for transplantation and gene therapy of neurological disorders. In this unit, we introduce methods for the derivation, propagation and characterization of hESC‐derived neural precursors. Curr. Protoc. Cell Biol. 36:23.7.1‐23.7.20. © 2007 by John Wiley & Sons, Inc.

Keywords: neural induction; human embryonic stem cells; neural precursors; noggin; neural differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Controlled Derivation of Neural Precursors from hESCs
  • Support Protocol 1: Characterization of the Neural Precursors by Immunostaining
  • Support Protocol 2: Characterization of Neural Differentiation by Flow Cytometric Analysis
  • Support Protocol 3: Spontaneous Differentiation of hESC‐Derived NPs
  • Support Protocol 4: Culturing Human Embryonic Stem Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Controlled Derivation of Neural Precursors from hESCs

  Materials
  • 0.1% (w/v) low melting temperature (LMT) agarose (see recipe)
  • hESCs cultured on a feeder layer (see protocol 5) in 6‐well plates
  • 1 mg/ml collagenase type IV (see recipe)
  • Neural precursor medium (NPM; see recipe)
  • 20 µg/ml bFGF (see recipe)
  • 100 µg/ml noggin (see recipe)
  • NPM (see recipe) containing 20 ng/ml bFGF (added from 20 µg/ml bFGF stock; see recipe) and 500 ng/ml noggin (add from 100 µg/ml noggin stock; see recipe)
  • NPM (see recipe) containing 20 ng/ml bFGF (added from 20 µg/ml bFGF stock; see recipe)
  • 50‐ml conical polypropylene centrifuge tubes
  • Centrifuge
  • 24‐well tissue culture plates
  • 50‐ml centrifuge tubes
  • 20‐G surgical blades
  • Additional reagents and equipment for culturing hESCs ( protocol 5)

Support Protocol 1: Characterization of the Neural Precursors by Immunostaining

  Materials
  • 10 µg/ml poly‐D‐lysine (see recipe)
  • Tissue culture–grade distilled H 2O
  • 4 µg/ml laminin (see recipe)
  • NPs derived from hESCs (see protocol 1)
  • 0.008% (w/v) trypsin/2.4 mM EDTA (see recipe)
  • Phosphate‐buffered saline (PBS; containing calcium and magnesium; Invitrogen, cat. no. 14040)
  • 2.35 mg/ml DNase (see recipe)
  • 4% (w/v) paraformaldehyde (see recipe)
  • FACS buffer (see recipe)
  • 0.2% (v/v) Triton X‐100 (see recipe)
  • Blocking solution (see recipe)
  • Appropriate primary antibody (Table 23.7.1)
  • Secondary antibody: fluorophore‐conjugated antibody against IgG of species from which primary antibody was derived
  • Mounting medium with 4′‐6‐diamidino‐2‐phenylindole (DAPI; e.g., Vectashield from Vector Laboratories)
  • 16‐mm‐diameter round glass coverslips, sterile (Paul Marienfeld & Co.; http://www.marienfeld‐superior.com)
  • Center‐well organ culture dish (Falcon)
  • Glass microscope slides
    Table 3.7.1   Materials   Primary Antibodies for In Vitro Immunostaining and Flow Cytometry a   Primary Antibodies for In Vitro Immunostaining and Flow Cytometry

    Antigen/antibody Species and type Clonality Purpose Dilution Source
    ESC pluripotency Oct‐4 Mouse IgG Monoclonal Staining FACS 1:100 1:20 Santa Cruz Biotech.
    SSEA4 Mouse IgG Monoclonal Staining FACS 1:100 1:100 DSHB
    SSEA3 Rat IgM Monoclonal Staining FACS 1:100 Chemicon
    Tra‐1‐60 Mouse IgM Monoclonal Staining FACS 1:20‐50 1:100 Chemicon
    Tra‐1‐81 Mouse IgM Monoclonal Staining FACS 1:10 1:100 Chemicon
    Neural precursors NCAM Mouse IgG Monoclonal Staining 1:10 Dako
    PSA‐NCAM Mouse IgM Monoclonal Staining FACS 1:200 1:250 Chemicon
    Nestin Rabbit Polyclonal Staining 1:200 Chemicon
    Pax6 Mouse IgG Monoclonal Staining 1:100 DSHB
    Sox1 Chicken Polyclonal Staining 1:1000 Chemicon
    Musashi Rabbit Polyclonal Staining 1:100 Chemicon
    Neurons β‐tubulin III Mouse IgG Monoclonal Staining 1:2000 Sigma
    MAP2ab Rabbit Polyclonal Staining 1:500 Chemicon
    NF 70 kDa Mouse IgG Monoclonal Staining 1:100 Dako
    NF 160 kDa Mouse IgG Monoclonal Staining 1:50 Chemicon
    NF 200 kDa Rabbit Polyclonal Staining 1:5000 Sigma
    NeuN Mouse IgG Monoclonal Staining 1:100 Chemicon
    Neuron‐specific enolase (NSE) Rabbit Polyclonal Staining 1:200 Zymed
    Synaptophysin Mouse IgG Monoclonal Staining 1:50 Dako
    GABA Rabbit Polyclonal Staining 1:1000 Sigma
    Glutamate Rabbit Polyclonal Staining 1:1000 Sigma
    Serotonin Rabbit Polyclonal Staining 1:1000 Sigma
    Tyrosine hydroxylase (TH) Mouse IgG Monoclonal Staining 1:500 Sigma
    Glia GFAP Rabbit Polyclonal Staining 1:200 Dako
    O4 Mouse IgM Monoclonal Staining 1:30 Chemicon

     aAbbreviations: DSHB, Developmental Studies Hybridoma Bank, University of Iowa (http://dshb.biology.uiowa.edu/); GABA, γ‐aminobutyric acid; GFAP, glial fibrillary acid protein; NCAM, neural cell adhesion molecule; PSA‐NCAM, polysialylated form of NCAM; MAP2, microtubule‐associated protein 2; NF, neurofilament; NeuN, neuronal nuclei.

Support Protocol 2: Characterization of Neural Differentiation by Flow Cytometric Analysis

  Materials
  • hESCs cultured on a feeder layer (see protocol 5) or NPs derived from hESCs (see protocol 1)
  • 0.05% (w/v) disodium EDTA
  • 0.008% (w/v) trypsin/2.4 mM EDTA (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen) containing calcium and magnesium (Invitrogen, cat. no. 14040)
  • 2.35 mg/ml DNase (see recipe)
  • FACS buffer (see recipe)
  • 100% ethanol, –20°C
  • Permeabilization buffer (see recipe)
  • Appropriate primary antibody (Table 23.7.1)
  • Goat anti–mouse immunoglobulin conjugated with fluorescein isothiocyanate (FITC; Dako)
  • 2 µg/ml propidium iodide (see recipe)
  • 35‐µm nylon mesh
  • 15‐ml centrifuge tubes
  • 5‐ml polystyrene round‐bottom tubes (Falcon)
  • Refrigerated centrifuge
  • Flow cytometer (also see Robinson et al., )
  • Additional reagents and equipment for counting cells (unit 1.1) and flow cytometry (Robinson et al., )

Support Protocol 3: Spontaneous Differentiation of hESC‐Derived NPs

  Materials
  • 10 µg/ml poly‐D‐lysine (see recipe)
  • Tissue culture–grade distilled H 2O
  • 4 µg/ml laminin (see recipe)
  • NPs derived from hESCs (see protocol 1)
  • Neural precursor medium (NPM; see recipe)
  • 16‐mm‐diameter round glass coverslips, sterile (Paul Marienfeld & Co.; http://www.marienfeld‐superior.com)
  • Center‐well organ culture dish (Falcon)

Support Protocol 4: Culturing Human Embryonic Stem Cells

  Materials
  • Cultures of human foreskin fibroblasts (ATCC # SCRC‐1041)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 14190), prewarmed to 37°C
  • 0.04% (w/v) trypsin/0.16 mM EDTA (see recipe)
  • Feeder cell medium (see recipe)
  • 2 mg/ml mitomycin C (see recipe)
  • 0.1% (w/v) gelatin (see recipe)
  • Cultures of hESCs
  • 0.05% (w/v) disodium EDTA
  • KnockOut (KO) medium (see recipe)
  • 1 mg/ml collagenase type IV (see recipe)
  • 175‐cm2 tissue culture flasks with 2‐µm vent caps
  • Inverted microscope
  • 15‐ and 50‐ml conical polypropylene centrifuge
  • 6‐well tissue culture plates
  • Additional reagents and equipment for counting cells (unit 1.1)
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Figures

Videos

Literature Cited

Literature Cited
   Bain, G., Kitchens, D., Yao, M., Huettner, J.E., and Gottlieb, D.I. 1995. Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168:342‐357.
   Brustle, O., Spiro, A.C., Karram, K., Choudhary, K., Okabe, S., and McKay, R.D. 1997. In vitro‐generated neural precursors participate in mammalian brain development. Proc. Natl. Acad. Sci. U.S.A. 94:14809‐14814.
   Brustle, O., Jones, K.N., Learish, R.D., Karram, K., Choudhary, K., Wiestler, O.D., Duncan, I.D., and McKay, R.D. 1999. Embryonic stem cell‐derived glial precursors: A source of myelinating transplants. Science 285:754‐756.
   Carpenter, M.K., Inokuma, M.S., Denham, J., Mujtaba, T., Chiu, C.P., and Rao, M.S. 2001. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp. Neurol. 172:383‐397.
   Cebria, F., Kobayashi, C., Umesono, Y., Nakazawa, M., Mineta, K., Ikeo, K., Gojobori, T., Itoh, M., Taira, M., Sanchez Alvarado, A., and Agata, K. 2002. FGFR‐related gene nou‐darake restricts brain tissues to the head region of planarians. Nature 419:620‐624.
   Conti, L., Pollard, S.M., Gorba, T., Reitano, E., Toselli, M., Biella, G., Sun, Y., Sanzone, S., Ying, Q.L., Cattaneo, E., and Smith, A. 2005. Niche‐independent symmetrical self‐renewal of a mammalian tissue stem cell. PloS Biol 3:283.
   Encha‐Razavi, F. and Sonigo, P. 2003. Features of the developing brain. Child's Nerv. Syst. 19:426‐428.
   Fujita, S. 2003. The discovery of the matrix cell, the identification of the multipotent neural stem cell and the development of the central nervous system, Cell Struct. Funct. 28:205‐228.
   Gerrard, L., Rodgers, L., and Cui, W. 2005. Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cells 23:1234‐1241.
   Ginis, I., Luo, Y., Miura, T., Thies, S., Brandenberger, R., Gerecht‐Nir, S., Amit, M., Hoke, A., Carpenter, M.K., Itskovitz Eldor, J., and Rao, M.S., 2004. Differences between human and mouse embryonic stem cells. Dev. Biol. 269:360‐380.
   Guillaume, D.J., Johnson, M.A., Li, X.J., and Zhang, S.C. 2006. Human embryonic stem cell‐derived neural precursors develop into neurons and integrate into the host brain. J. Neurosci. Res. 84:1165‐1176.
   Itsykson, P., Ilouz, N., Turetsky, T., Goldstein, R.S., Pera, M.F., Fishbein, I., Segal, M., and Reubinoff, B.E. 2005. Derivation of neural precursors from human embryonic stem cells in the presence of noggin. Mol. Cell Neurosci. 30:24‐36.
   Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa, S.I., and Sasai, Y. 2000. Induction of midbrain dopaminergic neurons from ES cells by stromal cell‐derived inducing activity. Neuron 28:31‐40.
   Kim, J.H., Auerbach, J.M., Rodriguez‐Gomez, J.A., Velasco, I., Gavin, D., Lumelsky, N., Lee, S.H., Nguyen, J., Sanchez‐Pernaute, R., Bankiewicz, K., and McKay, R.D. 2002. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 418:50‐56.
   Launay, C., Fromentoux, V., Shi, D.L., and Boucaut, J.C. 1996. A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers. Development 122:869‐880.
   Lowell, S., Benchoua, A., Heavey, B., and Smith, A.G., 2006. Notch promotes neural lineage entry by pluripotent embryonic stem cells. PLoS Biol. 4:e121.
   Li, X.J. and Zhang, S.C. 2006. In vitro differentiation of neural precursors from human embryonic stem cells. Methods Mol. Biol. 331:169‐177.
   Li, M., Pevny, L., Lovell‐Badge, R., and Smith, A. 1998. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr. Biol. 8:971‐974.
   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. 23:215‐21.
   Mitalipova, M.M., Rao, R.R., Hoyer, D.M., Johnson, J.A., Meisner, L.F., Jones, K.L., Dalton, S., and Stice, S.L. 2005. Preserving the genetic integrity of human embryonic stem cells. Nat. Biotechnol. 23:19‐20.
   Mujtaba, T., Piper, D.R., Kalyani, A., Groves, A.K., Lucero, M.T., and Rao, M.S. 1999. Lineage‐restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells. Dev. Biol. 214:113‐127.
   Munoz‐Sanjuan, I. and Brivanlou, A.H. 2002. Neural induction, the default model and embryonic stem cells. Nat. Rev. Neurosci. 3:271‐280.
   Muotri, A.R., Nakashima, K., Toni, N., Sandler, V.M., and Gage, F.H. 2005. Development of functional human embryonic stem cell‐derived neurons in mouse brain. PNAS 102:1864‐18648.
   Nakai, J. and Fujita, S. 1994. Early events in the histo‐ and cytogenesis of the vertebrate CNS. Int. J. Dev. Biol. 38:175‐183.
   Okabe, S., Forsberg‐Nilsson, K., Spiro, A.C., Segal, M., and McKay, R.D. 1996. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech. Dev. 59:89‐102.
   Rathjen, J., Haines, B.P., Hudson, K.M., Nesci, A., Dunn, S., and Rathjen, P.D. 2002. Directed differentiation of pluripotent cells to neural lineages: Homogeneous formation and differentiation of a neurectoderm population. Development 129:2649‐2661.
   Renoncourt, Y., Carroll, P., Filippi, P., Arce, V., and Alonso, S. 1998. Neurons derived in vitro from ES cells express homeoproteins characteristic of motoneurons and interneurons. Mech. Dev. 79:185‐197.
   Reubinoff, B.E., Itsykson, P., Turetsky, T., Pera, M.F., Reinhartz, E., Itzik, A., and Ben‐Hur, T. 2001. Neural progenitors from human embryonic stem cells. Nat. Biotechnol. 19:1134‐1140.
   Robinson, J.P., Darzynkiewicz, Z., Hoffman, R., Nolan, J.P., Orfao, A., Rabinovitch, P.S., and Watkins, S,(eds.). 2007. Current Protocols in Cytometry. John Wiley & Sons Hoboken, N.J.
   Roy, N.S., Cleren, C., Singh, S.K., Yang, L., Beal, M.F., and Goldman, S.A., 2006. Functional engraftment of human ES cell‐derived dopaminergic neurons enriched by coculture with telomerase‐immortalized midbrain astrocytes. Nat. Med. 12:1259‐1268.
   Shin, S., Mitalipova, M., Noggle, S., Tibbitts, D., Venable, A., Rao, R., and Stice, S.L. 2006. Long‐term proliferation of human embryonic stem cell‐derived neuroepithelial cells using defined adherent culture conditions. Stem Cells 24:125‐138.
   Smukler, S.R., Runciman, S.B., Xu, S., and Van der Kooy, D. 2006. Embryonic stem cells assume a primitive neural stem cell fate in absence of extrinsic influences. J. Cell Biol. 172:79‐90.
   Stavridis, M.P. and Smith, A.G. 2003. Neural differentiation of mouse embryonic stem cells. Biochem. Soc. Trans. 31:45‐49.
   Streit, A., Berliner, A.J., Papanayotou, C., Sirulnik, A., and Stern, C.D. 2000. Initiation of neural induction by FGF signalling before gastrulation. Nature 406:74‐78.
   Tabar, V., Panagiotakos, G., Greenberg, E.D., Chan, B.K., Sadelain, M., Gutin, P.H., and Studer, L. 2005. Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain. Nat. Biotechnol. 23:601‐606.
   Thomson, J.A. and Odorico, J.S. 2000. Human embryonic stem cell and embryonic germ cell lines. Trends Biotechnol. 18:53‐57.
   Tropepe, V., Hitoshi, S., Sirard, C., Mak, T.W., Rossant, J., and Van der Kooy, D. 2001. Direct neural fate specification from embryonic stem cells: A primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 30:65‐78.
   Ueno, M., Matsumura, M., Watanabe, K., Nakamura, T., Osakada, F., Takahashi, M., Kawasaki, H., Kinoshita, S., and Sasai, Y. 2006. Neural conversion of ES cells by an inductive activity on human amniotic membrane matrix. Proc. Natl. Acad. Sci. U.S.A. 103:9554‐9559.
   Wiles, M.V. and Johansson, B.M. 1999. Embryonic stem cell development in a chemically defined medium. Exp. Cell. Res. 247:241‐248.
   Wilson, S.I. and Edlund, T. 2001. Neural induction: Toward a unifying mechanism. Nature Neurosci. 4:1161‐1168.
   Wilson, S.I., Graziano, E., Harland, R., Jessell, T.M., and Edlund, T. 2000. An early requirement for FGF signaling in the acquisition of neural cell fate in the chick embryo. Curr. Biol. 10:421‐429.
   Yan, Y., Yang, D., Zarnowska, E.D., Du, Z., Werbel, B., Valliere, C., Pearce, R.A., Thomson, J.A., and Zhang, S.C. 2005. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23:781‐790.
   Ying, Q.L., Stavridis, M., Griffiths, D., Li, M., and Smith, A., 2003. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21:183‐186.
   Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O., and Thomson, J.A. 2001. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19:1129‐1133.
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