IN VITRO MODEL DEVELOPMENT OF SEROTONIN NEURONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们寻求一种5-羟色胺神经元的体外灵长类动物模型。利用恒河猴胚胎干细胞系366.4，开发了一种通过免疫细胞化学检测色氨酸羟基酶和5-羟色胺再摄取转运蛋白来产生高比例5-羟色胺神经元的方法。此外，胚胎干细胞来源的神经元表达雌激素受体β(ERbeta)和孕激素受体(PR)的方式类似于猕猴大脑中的5-羟色胺神经元。我们描述了这些神经元的生理功能，并检测了分化每个阶段的基因表达谱。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(FGF4)和声波刺激物(SHH)，提高了5-羟色胺神经元的产量。这些ESC来源的5-羟色胺神经元具有神经细胞膜的特性，并表达特定的功能性5-羟色胺再摄取转运体(SERT)和5HT1A自身受体，因此它们是一个有用的体外模型。通过使用另一株高效分化为5-羟色胺神经元的恒河猴胚胎干细胞系Ormes-22，证明了我们新优化方案的可行性。在5-羟色胺发育级联反应中，Sonic hedgehog(Shh)、PTCH(Shh-R)和FEV1转录因子的表达与N1选择过程中5-羟色胺特异标记基因的诱导相吻合。然而，在胚胎干细胞来源的神经元中，与细胞周期、轴突引导和背腹轴形成相关的探针组显著过度表达。这一分析表明，366.4细胞系在分化的早期阶段就具有5-羟色胺分化的迹象，但胚胎干细胞来源的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.