IN VITRO MODEL DEVELOPMENT OF SEROTONIN NEURONS

血清素神经元的体外模型开发

• 批准号: 8173186
• 负责人: CYNTHIA Louise BETHEA
• 金额: $ 4.76万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173186
• 关键词: Autoreceptors; Brain; Cell Cycle; Cell Line; Computer Retrieval of Information on Scientific Projects Database; Cues; Development; ES Cell Line; Erinaceidae; Estrogen Receptor beta; Funding; Gene Expression Profile; Genes; Grant; In Vitro; Institution; Macaca; Macaca mulatta; Membrane; Modeling; Neurons; PTCH gene; Physiological; Primates; Progesterone Receptors; Property; Protocols documentation; Research; Research Personnel; Resources; Serotonin; Signaling Molecule; Source; Staging; Tryptophan 5-monooxygenase; United States National Institutes of Health; axon guidance; embryonic stem cell; immunocytochemistry; improved; in vitro Model; model development; proto-oncogene protein kfgf; reuptake; transcription factor

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们寻找血清素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，开发了一种方案，根据色氨酸羟化酶和血清素再摄取转运蛋白的免疫细胞化学测定，该方案产生高百分比的血清素神经元。此外，ESC衍生的神经元以与猕猴大脑中的血清素神经元类似的方式表达雌激素受体β（ERβ）和孕激素受体（PR）。我们表征了这些神经元的生理功能，并检查了每个分化阶段的基因表达谱。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) 与音刺猬 (SHH)，提高了血清素神经元的产量。这些 ESC 衍生的血清素神经元具有神经元膜特性，并表达特定的功能性血清素再摄取转运蛋白 (SERT) 和 5HT1A 自身受体，从而使它们成为有用的体外模型。我们新的优化方案的可行性通过使用另一个恒河猴 ESC 系 ORMES-22 得到了证明，该系可有效分化为血清素神经元。在血清素发育级联中，音猬因子 (Shh)、PTCH (Shh-R) 和 Fev1 转录因子的表达与 N1 选择过程中血清素特异性标记基因的诱导一致。然而，在 ESC 衍生的神经元中，与细胞周期、轴突引导和背腹轴形成相关的探针组明显过多。该分析表明，366.4 细胞系在分化的早期阶段具有血清素分化的线索，但 ESC 衍生的血清素神经元仍不成熟。