Derivation of Cerebellar Neurons from Human Pluripotent Stem Cells

Slaven Erceg1, Dunja Lukovic2, Victoria Moreno‐Manzano3, Miodrag Stojkovic4, Shomi S. Bhattacharya2

1 Medical Genome Project, Sevilla, Spain, 2 CABIMER (Centro Andaluz de Biología Molecular y Medicina Regenerativa), Sevilla, Spain, 3 Neural Regeneration Lab, Valencia, Spain, 4 Human Genetics Department, University of Kragujevac, Serbia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.5
DOI:  10.1002/9780470151808.sc01h05s20
Online Posting Date:  March, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Here we provide a protocol for differentiation of human embryonic stem cells (hESC) into cerebellar neurons using a novel defined culture method. This protocol is based on the application of inductive signaling factors involved in the early patterning of the cerebellar region of the neural tube, followed by the application of factors responsible for cerebellar neuron specification. Human pluripotent stem cells are induced to form spherical embryonic‐like structures called embryoid bodies (EBs) and neuroepithelial tube‐like rosettes using defined chemical conditions. In the presence of FGF, Wnt, and RA signaling factors the rosettes were specified to OTX2‐expressing cells. Further specification of derived cells involves application of BMP factors involved in early development of granule cell progenitors, followed by mitogens and neurotrophins. It typically takes 5 weeks to generate the functional cerebellar granule neurons. This protocol is feeder‐free, applies human recombinant factors, and produces high yield of desired neurons. Curr. Protoc. Stem Cell Biol. 20:1H.5.1‐1H.5.10. © 2012 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cell; cerebellar neuron; human pluripotent stem cell; cerebellar granule neuron

PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1:

  • hESC lines H1 and H9 (National Stem Cell Bank, Wicell, cat. nos. WA01 and WA09)
  • Human fibroblast feeder (ATTC #CRL‐2429)
  • hESC medium (see recipe)
  • Fibroblast growth factor 2 (FGF‐2; see recipe)
  • Neural induction medium (MIM medium; see recipe)
  • Fibroblast growth factor 8 (FGF‐8; see recipe)
  • Retinoic acid (RA; see recipe)
  • Basal medium Eagle (BME; Invitrogen, cat. no. 41010‐026)
  • Insulin‐transferrin‐selenium supplement 100× (ITS; Gibco‐BRL, cat. no. 41400)
  • Laminin/fibronectin‐coated 6‐well plates (see recipe)
  • Fibroblast growth factor 4 (FGF4; see recipe)
  • WNT‐3A (see recipe)
  • WNT‐1 (see recipe)
  • N2 supplement 100× (Gibco‐BRL, cat. no. 17502‐048)
  • B27 supplement (Invitrogen, cat. no. 0080085‐SA)
  • Bone morphogenetic factor 6 (BMP‐6; see recipe)
  • Bone morphogenetic factor 7, (BMP‐7; see recipe)
  • Growth differentiation factor 7 (GDF‐7; see recipe)
  • Sonic hedgehog (Shh; see recipe)
  • Brain‐derived neurotrophic factor (BDNF; see recipe)
  • Recombinant human neurotrophin 3 (NT‐3; see recipe)
  • Ultra‐low attachment 6‐well plates (Corning Costar, cat. no.3471)
  • Humidified tissue culture incubator (37°C, 5% CO 2)
  • 25‐µl and 500‐µl pipet tips
  • 15‐ and 50‐ml conical tubes (BD Biosciences, cat. nos. 352095 and 352073)
  • 30‐mm petri dishes (Fisher Scientific, cat. no. 08‐757‐13A)
  • Pipet‐aid
  • 1000‐, 200‐,100‐, and 10‐µl pipets
  • Inverted phase contrast microscope (Nikon, ECLIPSE TS100)
  • Centrifuge
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Akazawa, C., Ishibashi, M., Shimizu, C., Nakanishi, S.,and Kageyama, R. 1995. A mammalian helix‐loop‐helix factor structurally related to the product of Drosophila proneural gene atonal is a positive transcriptional regulator expressed in the developing nervous system. J. Biol. Chem. 270:8730‐8738.
   Alder, J., Lee, K.J., Jessell, T.M., and Hatten, M.E. 1999. Generation of cerebellar granule neurons in vivo by transplantation of BMP‐treated neural progenitor cells. Nat. Neurosci. 2:535‐540.
   Ben‐Arie, N., McCall, A.E., Berkman, S., Eichele, G., Bellen, H.J., and Zoghbi, H.Y. 1996. Evolutionary conservation of sequence and expression of the bHLH protein Atonal suggests a conserved role in neurogenesis. Hum. Mol. Genet. 5:1207‐1216.
   Borghesani, P.R., Peyrin, J.M., Klein, R., Rubin, J., Carter, A.R., Schwartz, P.M., Luster, A., Corfas, G., and Segal, R.A. 2002. BDNF stimulates migration of cerebellar granule cells. Development 129:1435‐1442.
   Crossley, P.H., Martinez, S., and Martin, G.R. 1996. Midbrain development induced by FGF8 in the chick embryo. Nature 380:66‐68.
   Erceg, S., Ronaghi, M., Zipancic, I., Lainez, S., Roselló, M.G., Xiong, C., Moreno‐Manzano, V., Rodríguez‐Jiménez, F.J., Planells, R., Alvarez‐Dolado, M., Bhattacharya, S.S., and Stojkovic, M. 2010. Efficient differentiation of human embryonic stem cells into functional cerebellar‐like cells. Stem Cells Dev. 19:1745‐1756.
   Harding, A.E. 1982. The clinical features and classification of the late onset autosomal dominant cerebellar ataxias. A study of 11 families, including descendants of the ‘the Drew family of Walworth’. Brain 105:1‐28.
   Harding, A.E. 1993. Clinical features and classification of inherited ataxias. Adv. Neurol. 61:1‐14.
   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.
   Joyner, A.L. 1996. Engrailed, Wnt and Pax genes regulate midbrain–hindbrain development. Trends Genet. 12:15‐20.
   Joyner, A.L., Liu, A., and Millet, S. 2000. Otx2, Gbx2 and Fgf8 interact to position and maintain a mid‐hindbrain organizer. Curr. Opin. Cell Biol. 12:736‐741.
   Komine, O., Nagaoka, M., Watase, K., Gutmann, D.H., Tanigaki, K., Honjo, T., Radtke, F., Saito, T., Chiba, S., and Tanaka, K. 2007. The monolayer formation of Bergmann glial cells is regulated by Notch/RBP‐J signaling. Dev. Biol. 311:238‐250.
   Kurosawa, H. 2007. Methods for inducing embryoid body formation: In vitro differentiation system of embryonic stem cells. J. Biosci. Bioeng. 103:389‐398.
   McMahon, A.P. and Bradley, A. 1990. The Wnt‐1 (int‐1) proto‐oncogene is required for development of a large region of the mouse brain. Cell 62:1073‐1085.
   Middleton, F.A. and Strick, P.L. 1998. The cerebellum: An overview. Trends Neurosci. 21:367‐369.
   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.
   Salero, E. and Hatten, M.E. 2007. Differentiation of ES cells into cerebellar neurons. Proc. Natl. Acad. Sci. U.S.A. 104:2997‐3002.
   Su, H.L., Muguruma, K., Matsuo‐Takasaki, M., Kengaku, M., Watanabe, K., and Sasai, Y. 2006. Generation of cerebellar neuron precursors from embryonic stem cells. Dev. Biol. 290:287‐296.
   Yang, X.W., Zhong, R., and Heintz, N. 1996. Granule cell specification in the developing mouse brain as defined by expression of the zinc finger transcription factor RU49. Development 122:555‐566.
PDF or HTML at Wiley Online Library