RRC ID 44223
Author Belinsky GS, Sirois CL, Rich MT, Short SM, Moore AR, Gilbert SE, Antic SD.
Title Dopamine receptors in human embryonic stem cell neurodifferentiation.
Journal Stem Cells Dev
Abstract We tested whether dopaminergic drugs can improve the protocol for in vitro differentiation of H9 human embryonic stem cells (hESCs) into dopaminergic neurons. The expression of 5 dopamine (DA) receptor subtypes (mRNA and protein) was analyzed at each protocol stage (1, undifferentiated hESCs; 2, embryoid bodies [EBs]; 3, neuroepithelial rosettes; 4, expanding neuroepithelium; and 5, differentiating neurons) and compared to human fetal brain (gestational week 17-19). D2-like DA receptors (D2, D3, and D4) predominate over the D1-like receptors (D1 and D5) during derivation of neurons from hESCs. D1 was the receptor subtype with the lowest representation in each protocol stage (Stages 1-5). D1/D5-agonist SKF38393 and D2/D3/D4-agonist quinpirole (either alone or combined) evoked Ca(2+) responses, indicating functional receptors in hESCs. To identify when receptor activation causes a striking effect on hESC neurodifferentiation, and what ligands and endpoints are most interesting, we varied the timing, duration, and drug in the culture media. Dopaminergic agonists or antagonists were administered either early (Stages 1-3) or late (Stages 4-5). Early DA exposure resulted in more neuroepithelial colonies, more neuronal clusters, and more TH(+) clusters. The D1/D5 antagonist SKF83566 had a strong effect on EB morphology and the expression of midbrain markers. Late exposure to DA resulted in a modest increase in TH(+) neuron clusters (∼75%). The increase caused by DA did not occur in the presence of dibutyryl cAMP (dbcAMP), suggesting that DA acts through the cAMP pathway. However, a D2-antagonist (L741) decreased TH(+) cluster counts. Electrophysiological parameters of the postmitotic neurons were not significantly affected by late DA treatment (Stages 4-5). The mRNA of mature neurons (VGLUT1 and GAD1) and the midbrain markers (GIRK2, LMX1A, and MSX1) were lower in hESCs treated by DA or a D2-antagonist. When hESCs were neurodifferentiated on PA6 stromal cells, DA also increased expression of tyrosine hydroxylase. Although these results are consistent with DA's role in potentiating DA neurodifferentiation, dopaminergic treatments are generally less efficient than dbcAMP alone.
Volume 22(10)
Pages 1522-40
Published 2013-5-15
DOI 10.1089/scd.2012.0150
PMID 23286225
PMC PMC3653401
MeSH Adult Biomarkers / metabolism Blotting, Western Brain / metabolism Bucladesine / pharmacology Calcium / metabolism Cell Differentiation* / drug effects Cell Differentiation* / genetics Coculture Techniques Culture Media / pharmacology Dopamine / pharmacology Dopamine Agonists / pharmacology Dopamine Antagonists / pharmacology Dopaminergic Neurons / cytology Dopaminergic Neurons / drug effects Dopaminergic Neurons / metabolism Electrophysiological Phenomena / drug effects Embryoid Bodies / cytology Embryoid Bodies / drug effects Embryoid Bodies / metabolism Embryonic Stem Cells / cytology* Embryonic Stem Cells / drug effects Embryonic Stem Cells / metabolism* Humans Neurons / cytology* Neurons / drug effects Neurons / metabolism RNA, Messenger / genetics RNA, Messenger / metabolism Receptors, Dopamine / genetics Receptors, Dopamine / metabolism*
IF 3.153
Times Cited 18
Human and Animal Cells MC3T3-G2/PA6(RCB1127)