【作者】 奚佳捷；

【导师】 张素春；

【作者基本信息】 复旦大学 ， 疾病蛋白质组学， 2012， 博士

【摘要】 位于中脑腹侧黑质的多巴胺能神经元的退行性病变会导致帕金森氏症。目前对于帕金森氏症仍无有效的治疗方法,因此,寻求新型疗法成为当务之急。人多能干细胞,包括人胚胎干细胞和人诱导多能干细胞,具有分化的全能性,在特定条件下能定向分化为人体任何一种细胞,包括特种神经元。这些人类神经细胞被进一步修饰后可用来研究神经退行性疾病的病理发生过程,从而可以被用来进一步探索神经疾病的治疗方式。他们也很有可能被用于细胞替代治疗,从而达到治愈神经退行性疾病治疗的目的。病人特异性诱导多能干细胞(human induced pluripotent stem cells, hiPSCs)取自病人本身,一般认为在移植回病人本身后不会被免疫排斥,可以被用来作为自体细胞移植。为了模拟将来对帕金森病人的细胞替代疗法,我们探索从帕金森恒河猴获取的iPSCs在体外定向分化为多巴胺能神经元,然后将这些体外分化而来的多巴胺能神经元移植回恒河猴脑内。用SHH和FGF8进行处理后,猴iPSCs分化的神经上皮细胞进一步分化为多巴胺能神经元,表达酪氨酸羟化酶(TH),而其中一些表达中脑的标记物Enl和Foxa2。在不使用免疫抑制剂的情况下,恒河猴iPS来源的多巴胺能神经前体细胞被移植到MPTP造模的恒河猴脑内纹状体后,发现移植的细胞能够存活,并进一步分化成为神经元和胶质细胞,且未激活宿主的免疫系统。这为将来可能的个体化细胞移植疗法的开展带来了希望。在恒河猴iPS来源多巴胺能神经前体细胞被移植到恒河猴帕金森模型脑内后,虽然移植细胞得以存活并成熟,但由于分化所得的多巴胺能神经元不具备中脑的特性,接受细胞移植的猴子的症状并未缓解。遵循体内中脑多巴胺能神经元的发育,使用SHH和CHIR99021诱导多能干细胞产生中脑底板细胞,并在FGF8的作用下,进一步分化成为中脑多巴胺能神经元。我们发现在神经分化过程中,在特定时间段使用特定浓度的CHIR99021能特异性地诱导中脑底板细胞的生成,这些底板细胞表达Corin, Enl, FoxA2and Lmxla。FGF8能够促进中脑底板细胞分化成为多巴胺能神经元,这些分化所得的多巴胺能神经元表达中脑的标志物Lmxla/b、FoxA2、FoxP1、Nurr1以及Enl,并且具有典型的中脑多巴胺能神经元的电生理特征。这些中脑多巴胺能神经元中,有超过一半的细胞表达中脑A9亚群的标志物Girk2和ALDHlal。运用这一新的分化策略能够有效地从人/恒河猴多能干细胞分化得到大量的中脑多巴胺能神经元,为中脑多巴胺能神经元的发育研究、帕金森氏症发病机制的研究以及细胞移植治疗的开展奠定了基础。更多还原

【Abstract】 Degeneration of dopamine(DA)-producing neurons in the midbrain, especially the substantia nigra, underlies the pathophysiology of Parkinson’s disease(PD). At present, there has been no effective treatment for such disease. Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), can differentiate to any cell types of the human body, including DA neurons. These DA neurons may be used to study pathological processes of neural degeneration, which in turn may become a target for therapeutic discovery. These DA procursors may also become a source of cell based therapy for the Parkinson’s diseases.Autologous transplantation via induced pluripotent stem cells (iPSCs) is a potential strategy to have readily available matching donor cells and therefore, minimize host immune response. A recent study with iPSC transplants showed rejection of teratomas by syngenic host mice, casting doubt on the utility of reprogrammed cells for autologous transplant therapy. Clinical translation of cell replacement strategies will rely upon iPSC-differentiated progenies instead of iPSCs themselves. Here we show that transplantation of rhesus monkey iPSC-derived neural progenitors survive for up to6months without immune suppression and differentiate into neurons, astrocytes, and oligodendrocytes in the striatum of MPTP-induced hemiparkinsonian rhesus monkeys with minimal presence of inflammatory cells. This finding raises hopes of personalized regenerative therapies.By sequentially applying sonic hedgehog (C25II) and CHIR99021(GSK3□inhibitor) to induce the midbrain floor plate progenitors and fibroblast growth factor8(FGF8) to promote dopaminergic differentiation in a chemically defined medium, we have established a robust system for generation of midbrain dopamine (DA) neurons from human and rhesus monkey embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).We found that CHIR99021specifies diencephalon to hindbrain fates in a concentration-dependent manner and only a narrow concentration range of CHIR99021at a particular window is necessary to induce the midbrain floor plate progenitors, expressing Corin, Enl, FoxA2and Lmxla. FGF8enhances the dopaminergic fate of the progenitors, thus generating DA neurons with midbrain characteristics, including expression of TH, Lmxla/b, FoxA2, FoxPl, Nurr1and Enl as well as typical electrophysiological properties. More than half of these DA neurons expressed A9DA neuron markers Girk2and ALDHlal. The new strategy will allow generation of enriched populations of functional midbrain DA neurons from both human and monkey PSCs for disease modeling, drug testing, and potential cell therapy.更多还原