Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells.
How dopamine (DA) neuronal subtypes are specified remains unknown. In this study we show a robust generation of functional DA neurons from human embryonic stem cells (hESCs) through a specific sequence of application of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH). Treatment of hESC-derived Sox1+ neuroepithelial cells with FGF8 and SHH resulted in production of tyrosine hydroxylase (TH)-positive neurons that were mostly bipolar cells, coexpression with gamma-aminobutyric acid, and lack of midbrain marker engrailed 1 (En1) expression. However, FGF8 treatment of precursor cells before Sox1 expression led to the generation of a similar proportion of TH+ neurons characteristic of midbrain projection DA neurons with large cell bodies and complex processes and coexpression of En1. This suggests that one mechanism of generating neuronal subtypes is temporal availability of morphogens to a specific group of precursors. The in vitro-generated DA neurons were electrophysiologically active and released DA in an activity-dependent manner. They may thus provide a renewable source of functional human DA neurons for drug screening and development of sustainable therapeutics for disorders affecting the DA system.
Yan
Yiping
Y
Department of Anatomy, University of Wisconsin-Madison, 1500 Highland Avenue, Madison, Wisconsin 53705, USA.
Yang
Dali
D
Zarnowska
Ewa D
ED
Du
Zhongwei
Z
Werbel
Brian
B
Valliere
Chuck
C
Pearce
Robert A
RA
Thomson
James A
JA
Zhang
Su-Chun
SC
D000818:Animals; D001921:Brain / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D004594:Electrophysiology; D004622:Embryo, Mammalian / Q000166:cytology*; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors / Q000378:metabolism; D053823:Hedgehog Proteins; D006609:High Mobility Group Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008957:Models, Genetic; D046569:Neuroepithelial Cells / Q000166:cytology; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors; D013234:Stem Cells / Q000166:cytology*; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D004268:DNA-Binding Proteins; C513076:EN1 protein, human; C080330:En1 protein, mouse; C496456:FGF8 protein, human; C496457:Fgf8 protein, mouse; D053823:Hedgehog Proteins; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; C509239:SHH protein, human; C490906:SOX1 protein, human; D055748:SOXB1 Transcription Factors; D015534:Trans-Activators; D051524:Fibroblast Growth Factor 8; D005680:gamma-Aminobutyric Acid; D005346:Fibroblast Growth Factors; D004298:Dopamine