An in-vitro model derived from hESC for screening phytochemicals with dopaminergic differentiation-boosting potential for improving Parkinson’s disease
Date Issued
2014
Date
2014
Author(s)
Hsieh, Wen-Ting
Abstract
This study aimed to establish an in-vitro model using hESC (human embryonic stem cell) H9 cell line, which is expected to simulate the development of midbrain dopaminergic neurons (mDA neurons). Thus, through the model, we expected to screen phytochemicals with potential on stimulating mDA neurogenesis to improve Parkinson''s disease (PD). As a result, we successfully established a five-stage process which could differentiate hESC H9 cell line into mDA-like neurons, and the differentiation rate of TH+ cells was 32.8-53.6%. Moreover, the final differentiated DA neurons truly expressed mature DA neuron-related factors like En1, Lmx1a, TH (tyrosine hydroxylase), AADC (aromatic L-amino acid decarboxylase), GIRK2 and other factors. Through the analysis of immunefluorescence staining, the in-vitro model also showed specific protein expression such as nestin, TH, β-III tubulin and DAT (dopamine transporter). These results demonstrated these differentiated cells were certainly mDA-like neurons. Besides, these cells showed mRNA expression of several voltage-gated ion channels, especially the mDA-specific one, Kv 10.1. In addition, the dopamine release (7.28 ± 1.30 ng/ml) by the differentiated cells was also detected. After that, according to the qPCR analysis of two marker genes, TH and GIRK2, we determined bromocriptine and 7-OH-DPAT as positive control drugs and sulpiride as negative control suitable for this model. Their effective dosages are presented as the following: 20 μM bromocriptine, 10 μM 7-OH-DPAT and 10 μM sulpiride. Finally, we tested green tea catechins (GTCs), including EGCG, ECG, EGC and EC as well as ginsenoside Rg1, Rg3 and Rb1 using the established model. We found that the abilities of GTCs might be related to their differences in structure. EGCG, which possesses both galloyl moiety and tri-hydroxyl substitutes on the B-ring, significantly increased the TH and GIRK2 expression at 12.5 μM. Moreover, better than GTCs, at 6.25 μM, ginsenoside Rb1 greatly enhanced the TH and GIRK2 expression to 12 and 14 folds, respectively. Thus, it appeared that Rb1 was the most potent phtyochemical to have potential of stimulating the differentiation of mDA-like neurons. Its possible mechanism might include stimulating the BDNF (brain-derived neurotrophic factor) and NT-3 (neurotrophin-3) secretion from surrounding astrocytes and activting the Wnt-1/ frizzled-1/β-catenin signaling pathway.
Subjects
帕金森氏症
黑質體緻密部
A9型多巴胺神經細胞
人類胚胎幹細胞
綠茶兒茶素
人參皂苷
SDGs
Type
thesis
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