Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture

Thomas C. Schulz, Scott A. Noggle, Gail M. Palmarini, Deb A. Weiler, Ian G. Lyons, Kate A. Pensa, Adrian C.B. Meedeniya, Bruce P. Davidson, Nevin A Lambert, Brian G. Condie

Research output: Contribution to journalArticle

201 Citations (Scopus)

Abstract

The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.

Original languageEnglish (US)
Pages (from-to)1218-1238
Number of pages21
JournalStem Cells
Volume22
Issue number7
DOIs
StatePublished - Dec 24 2004

Fingerprint

Dopaminergic Neurons
Suspensions
Oxidopamine
Neurons
Serum
Tyrosine 3-Monooxygenase
Catecholamines
Cell Differentiation
Bone and Bones
Aptitude
Cell Transplantation
Cell Lineage
Cell- and Tissue-Based Therapy
Mesencephalon
Coculture Techniques
Cues
Parkinson Disease
Real-Time Polymerase Chain Reaction
Dopamine
Proteins

Keywords

  • Differentiation
  • ES cells
  • Embryoid bodies
  • Neural differentiation Serum-free medium
  • Real-time RT-PCR

ASJC Scopus subject areas

  • Molecular Medicine
  • Developmental Biology
  • Cell Biology

Cite this

Schulz, T. C., Noggle, S. A., Palmarini, G. M., Weiler, D. A., Lyons, I. G., Pensa, K. A., ... Condie, B. G. (2004). Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. Stem Cells, 22(7), 1218-1238. https://doi.org/10.1634/stemcells.2004-0114

Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. / Schulz, Thomas C.; Noggle, Scott A.; Palmarini, Gail M.; Weiler, Deb A.; Lyons, Ian G.; Pensa, Kate A.; Meedeniya, Adrian C.B.; Davidson, Bruce P.; Lambert, Nevin A; Condie, Brian G.

In: Stem Cells, Vol. 22, No. 7, 24.12.2004, p. 1218-1238.

Research output: Contribution to journalArticle

Schulz, TC, Noggle, SA, Palmarini, GM, Weiler, DA, Lyons, IG, Pensa, KA, Meedeniya, ACB, Davidson, BP, Lambert, NA & Condie, BG 2004, 'Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture', Stem Cells, vol. 22, no. 7, pp. 1218-1238. https://doi.org/10.1634/stemcells.2004-0114
Schulz TC, Noggle SA, Palmarini GM, Weiler DA, Lyons IG, Pensa KA et al. Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. Stem Cells. 2004 Dec 24;22(7):1218-1238. https://doi.org/10.1634/stemcells.2004-0114
Schulz, Thomas C. ; Noggle, Scott A. ; Palmarini, Gail M. ; Weiler, Deb A. ; Lyons, Ian G. ; Pensa, Kate A. ; Meedeniya, Adrian C.B. ; Davidson, Bruce P. ; Lambert, Nevin A ; Condie, Brian G. / Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. In: Stem Cells. 2004 ; Vol. 22, No. 7. pp. 1218-1238.
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