IN VITRO MODEL DEVELOPMENT OF SEROTONIN NEURONS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们寻求一种5-羟色胺神经元的体外灵长类动物模型。使用恒河猴胚胎干细胞（ESC）系366.4，开发了一种产生高百分比的5-羟色胺神经元的方案，如通过色氨酸羟化酶和5-羟色胺再摄取转运蛋白的免疫细胞化学所确定的。此外，ESC衍生的神经元以与猕猴脑中的5-羟色胺神经元类似的方式表达雌激素受体β（ER β）和孕酮受体（PR）。我们表征了这些神经元的生理功能，并在分化的每个阶段检查了基因表达谱。促有丝分裂信号分子的不同组合，特别是成纤维细胞生长因子4（FGF 4）与音速刺猬（SHH），提高了5-羟色胺神经元的产量。这些ESC衍生的5-羟色胺神经元具有神经元膜特性，并表达特定的功能性5-羟色胺再摄取转运蛋白（SERT）和5 HT 1A自身受体，从而使它们成为有用的体外模型。我们的新优化方案的可行性通过使用另一种恒河猴ESC系ORMES-22来显示，其有效地分化为5-羟色胺神经元。在5-羟色胺的发展级联，音刺猬（Shh），PTCH（Shh-R），和Fev 1转录因子的表达与5-羟色胺特异性标记基因的诱导在N1选择。然而，在ESC衍生的神经元中，与细胞周期、轴突导向和背腹轴形成相关的探针组显著过量表达。该分析表明，366.4细胞系在分化的早期阶段具有5-羟色胺分化的线索，但是ESC衍生的5-羟色胺神经元仍然不成熟。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We sought an in vitro primate model for serotonin neurons. Using the rhesus embryonic stem cell (ESC) line 366.4, a protocol was developed which generates a high percentage of serotonin neurons as determined by immunocytochemistry for tryptophan hydroxylase and the serotonin reuptake transporter. In addition, the ESC-derived neurons express estrogen receptor beta (ERbeta) and progesterone receptors (PR) in a manner similar to serotonin neurons in the macaque brain. We characterized physiological functions of these neurons and examined the gene expression profile during each stage of differentiation. Different combinations of mitogenic signaling molecules, specifically fibroblast growth factor 4 (FGF4) with sonic hedgehog (SHH), improved the yield of the serotonin neurons. These ESC-derived serotonin neurons have neuronal membrane properties and express specific functional serotonin reuptake transporters (SERT) and 5HT1A autoreceptors, thus making them a useful in vitro model. The feasibility of our new optimized protocol was shown by using another rhesus ESC line ORMES-22, which efficiently differentiated to serotonin neurons. In the serotonin developmental cascade, sonic hedgehog (Shh), PTCH (Shh-R), and Fev1 transcription factor expression coincided with the induction of serotonin specific marker genes during N1-selection. However, in the ESC-derived neurons, there was significant over-representation of probe sets related to cell cycle, axon guidance & dorso-ventral axis formation. This analysis suggests that the 366.4 cell line possesses cues for serotonin differentiation at early stages of differentiation, but that ESC-derived serotonin neurons are still immature.