Dopamine Receptors in Human Embryonic Stem Cell Neurodifferentiation

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.

Dopamine receptor agonist bromocriptine restrains the follicular development, hatchling success and puberty in Gambusia affinis

“…Whereas the presence of D2-like DA receptors was shown in the human embryonic stem cells (Belinsky et al, 2013), cocaineinduced dysfunction of DA receptor signalling lead to abnormal differentiation of cerebral cortical neurons in rabbits (Jones et al, 2000). Reimer et al (2013) demonstrated that dopaminergic signal was essential during neurogenesis in the zebrafish D. rerio.…”

Characterization of Transcriptional Repressor Gene MSX1 Variations for Possible Associations with Congenital Heart Diseases

“…MSX1 seems to be an active gene in the HES cells as implicated by its up-regulated expression when the HES cells are co-cultured with PA6 cells [ 37 ], and, conversely, the expression levels of MSX1 become lower when the HES cells are treated with dopamine [ 38 ]. In a genome-wide methylation-gene expression study between the epigenetic modifications of retinoic acid treated and undifferentiated HES cells, the author uncovered 166 differentially methylated CpG sites and 2,013 differentially expressed genes, in which 19 genes including MSX1 are highly correlated with each other [ 39 ].…”

Calcium signaling in human pluripotent stem cells

“…While cells and tissues differentiated from PSCs have been widely used for calcium signaling studies, we found only a limited number of data about such signaling in pluripotent stem cells, and even these data are mainly from mouse cell studies. Calcium signals in hESC-derived cardiomyocytes and neural cell types have been measured in most cases by calcium sensitive dyes: for cardiomyocytes Fura-2/AM [25][26][27][28], Fluo-4/AM [29,30] or dsRed+ [27], while for neural cell types Fluo-3/AM [8,31] Fura-2/AM [8,32] or Oregon green 488 Bapta-1 AM [33] have been applied. In pluripotent cells the application of Fluo-3/AM (mESC- [34], hESC- [8]) or Fluo-4/AM (mESC- [35][36][37], and hESC [12]) has been reported.…”