Dopaminergic Differentiation of Human Pluripotent Cells

Leah F. Boyer1, Benjamin Campbell2, Samantha Larkin2, Yanling Mu2, Fred H. Gage2

1 Biomedical Sciences Graduate Program, School of Medicine, University of California, San Diego, California, 2 Salk Institute for Biological Studies, San Diego, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.6
DOI:  10.1002/9780470151808.sc01h06s22
Online Posting Date:  August, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Here we describe protocols for the dopaminergic differentiation of pluripotent stem cells. We have optimized and compared two distinct protocols, both of which are chemically defined and applicable to both embryonic and induced pluripotent stem cells. First, we present a five‐step method based on rosette formation (Basic Protocol 1); then we describe a monolayer paradigm based on inhibition of alternate developmental pathways (Basic Protocol 2). Directed differentiation of pluripotent cells into specific cell types is a crucial step towards understanding human development and realizing the biomedical relevance of these cells, whether for replacement therapy or disease modeling. Curr. Protoc. Stem Cell Biol. 22:1H.6.1‐1H.6.11. © 2012 by John Wiley & Sons, Inc.

Keywords: neural development; dopaminergic differentiation; pluripotent stem cells

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Traditional Differentiation Into DA Neurons
  • Basic Protocol 2: Adherent Differentiation Into DA Neurons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Traditional Differentiation Into DA Neurons

  Materials
  • Pluripotent cells growing on inactivated mouse embryonic fibroblasts (MEFs); see unit 1.1
  • Human pluripotent stem cell medium (see recipe)
  • Neural induction medium (NIM; see recipe)
  • DMEM/F12 medium base (Invitrogen, cat. no. 11330‐032)
  • NIM (see recipe) supplemented with 1 µg/ml laminin (Invitrogen, cat. no. 23017‐015)
  • Collagenase solution (see recipe)
  • Neural progenitor medium (NPM; see recipe)
  • 100× Antibiotic‐Antimycotic (“Anti‐Anti”; Invitrogen, cat. no. 15240)
  • TrypLE (Invitrogen, cat. no. 12604‐013)
  • Freezing medium: 90% (v/v) NPM/10% (v/v) DMSO
  • Neural differentiation medium (see recipe)
  • 10‐cm cell culture plates
  • 15‐ml conical centrifuge tube (e.g., BD Falcon)
  • Ultra‐low‐attachment plates (Corning, cat. no. 3262)
  • Polyornithine/laminin‐coated 10‐cm plates and 6‐well plates (see recipe)
  • Stereomicroscope or phase‐contrast microscope
  • Cryovials
  • Additional reagents and equipment for growing human embryonic stem cells on inactivated MEFs (unit 1.1)

Basic Protocol 2: Adherent Differentiation Into DA Neurons

  Materials
  • Pluripotent cells growing on Matrigel in MEF conditioned medium (MEF‐CM; see recipe); see unit 1.5 for culturing technique
  • 10 mM ROCK inhibitor Y27632 (Axxora, cat. no. ALX‐270‐333‐M025; http://www.axxora.com/)
  • MEF‐CM (see recipe) with and without 20 ng/ml FGF2 (Peprotech, cat. no. 100‐18B)
  • Accutase (Sigma, cat. no. A6964)
  • DMEM/F12 medium base (Invitrogen, cat. no.11330‐032)
  • TGF‐β inhibitor SB431542 (Tocris, cat. no. 1614; 10 mM stock)
  • Noggin (Peprotech, cat. no. 120‐10C; 500 µg/ml stock)
  • Neural induction medium (NIM)
  • Growth factors (Table 1.6.1)
  • Neural differentiation medium (see recipe)
  • Matrigel‐coated 6‐well plates (see recipe)
  • 15‐ml conical tubes (BD Falcon)
  • Additional reagents and equipment for growing human embryonic stem cells under feeder‐free MEF‐CM conditions (unit 1.5) and counting viable cells by trypan blue exclusion (unit 1.3)
    Table 1.0.1   Materials   Media and Growth Factors for Adherent Differentiation into DA Neurons a   Media and Growth Factors for Adherent Differentiation into DA Neurons

    Day Medium Growth factors
    1‐5 MEF‐CM ‐FGF2 SB431542 (10 µM), Noggin (500 ng/ml)
    6‐7 3:1 MEF‐CM:NIM SHH (R&D Systems, cat. no. 1314SH;200 ng/ml), FGF8 (Peprotech, cat. no. 100‐25;100 ng/ml)
    8‐9 1:1 MEF‐CM:NIM SHH (200 ng/ml), FGF8 (100 ng/ml)
    10‐11 1:3 MEF‐CM:NIM SHH (200 ng/ml), FGF8 (100 ng/ml), BDNF (Peprotech, cat. no. 450‐02;20 ng/ml), ascorbic acid (Sigma, cat. no. A0278;200 nM)
    12‐26 NIM BDNF (20 ng/ml), GDNF (Peprotech, cat. no. 450‐10;20 ng/ml), cAMP (Sigma, cat. no. D0627;1 mM) and ascorbic acid (200 nM)

     aSee protocol 2, step 10.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Alavian, K, Scholz, C., and Simon, H. 2008. Transcriptional regulation of mesencephalic dopaminergic DA neurons: The full circle of life and death. Movement Disorders 23:319‐328.
   Andersson, E., Tryggvason, U., Deng, Q., Frilling, S., Alekseenko, A., Robert, B., Perlmann, T., and Ericson, J. 2006. Identification of intrinsic determinants of midbrain dopaminergic neurons. Cell 124:393‐405.
   Chambers, S., Fasano, C., Papapetrou, E., 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.
   Hwang, K., Kim, J., Chang, W., Kim, D., Lim, S., Kang, S., Song, B., Ha, H., Huh, Y., Choi, I., Hwang, D., Song, H., Jang, Y., Kim, S., and Kim, D. 2008. Chemicals that modulate stem cell differentiation. Proc. Natl. Acad. Sci. U.S.A. 105:7467‐7471.
   Kawasaki, H., Mizuseki, , Nishikawa, S., Kaneko, S., Kuwana, Y, Nakanishi, S., Nishikawa, S., and Sasai, Y. 2000. Induction of midbrain dopaminergic neurons from ES cells by stromal cell‐derived inducing activity. Neuron 28:31‐40.
   Kriks, S., Shim, J., Piao, J., Ganat, Y., Wakeman, D., Xie, Z., Carrillo‐Reid, L., Auyeung, G., Anotonacci, C., Buch, A., Yang, L., Beal, M.F., Surmeier, D.J., Kordower, J.H., Tabar, W., and Studer, L. 2011. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480:547‐551.
   Okabe, S., Forsberg‐Nilsson, K., Spiro, A., Segal, M., and McKay, R. 1996. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech. Dev. 59:89‐102.
   Prakash, N. and Wurst, W. 2006. Genetic networks controlling the development of midbrain dopaminergic neurons. J. Physiol. 575:403‐410.
   Roy, S., Cleren, C., Singh, S., Yang, L., Beal, MF., and Goldman, SA. 2006. Functional engraftment of human ES cell derived dopaminergic neurons enriched by coculture with telomerase immortalized midbrain astrocytes. Nat. Med. 12:1259‐1268.
   Sachetti, P., Sousa, K.M., Hall, A.C., Liste, I., Steffensen, K.R., Theofilopoulos, S., Parish, C.L., Hazenburg, C., Richter, L.A., Hovatta, O., Gustafsson, J.A., and Arenas, E. 2009. Liver X receptors and oxysterols promote ventral midbrain neurogenesis in vivo and in human embryonic stem cells. Cell Stem Cell 5:409‐419.
   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.
   Yin, M., Liu, S., Yin, Y., Li, S., Li, Z., Wu, X., Zhang, B., Ang, S., Ding, Y., and Zhou, J. 2009. Ventral mesencephalon‐enriched genes that regulate the development of dopaminergic neurons in vivo. J. Neurosci. 29:5170‐5182.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library