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
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Abstract

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

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • 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
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Figures

Videos

Literature Cited

Literature Cited
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