Kinetic Barriers of Transdifferentiation

摘要 肾上腺素能系统的功能障碍与多种疾病密切相关， 疾病，累计影响至少20%的人口。在我们的初步研究中， 我们鉴定了能将人成纤维细胞转化为诱导的肾上腺素能受体的转录因子， （i5 HT）神经元。p53基因敲除可显著提高细胞的效率， 细胞培养条件。在重编程的第12天，50%的细胞是Tuj 1+神经元， 25%为5 HT+神经元。这种表观遗传重编程依赖于泰特蛋白， 关键调节表观基因组的三种DNA羟化酶家族。打倒每一个 泰特基因消除了表观遗传转化。我们假设p53，泰特蛋白， 适当的细胞外环境对于人成纤维细胞直接转化为 15 HT神经元。为了验证这一假设，我们将确定最佳转录因子 研究p53敲低如何诱导泰特基因，并研究泰特蛋白如何 和重编程因子影响转录组，以促进 人成纤维细胞转化为15 HT神经元。我们还将研究细胞培养环境的影响 对大鼠i5-HT神经元移植后的转化及存活和功能的影响 大脑该提案将开发一种强大的方法，用于生成患者特异性和 亚型特异性i5 HT神经元，这将使基础研究和药物发现， 与降钙素有关的疾病。

Abstract Dysfunction of the serotonergic system is critically involved in a diverse range of diseases, which accumulatively affect at least 20% of the population. In our preliminary studies, we identified transcription factors that converted human fibroblasts to induced serotonergic (i5HT) neurons. The efficiency was significantly increased by p53 knockdown and appropriate cell culture conditions. At day 12 of reprogramming, 50% of the cells were Tuj1+ neurons and 25% were 5HT+ neurons. This epigenetic reprogramming was dependent on Tet proteins, a family of three DNA hydroxylases that critically regulate the epigenome. Knocking down each of the Tet genes abolished the epigenetic conversion. We hypothesize that p53, Tet proteins, and appropriate extracellular environment are critical for the direct conversion human fibroblasts to i5HT neurons. To test this hypothesis, we will identify the optimal transcription factor combinations, investigate how p53 knockdown induces Tet genes, and study how Tet proteins and reprogramming factors impact on the transcriptome to facilitate the transdifferentiation of human fibroblasts to i5HT neurons. We will also examine the impact of cell culture environment on the conversion and assess the survival and function of i5HT neurons transplanted in rat brains. The proposal will develop a robust method for the generation of patient-specific and subtype-specific i5HT neurons, which would enable basic research and drug discovery on serotonin-related disorders.