通过直接转分化技术高效地获得多巴胺能神经元并将其应用于帕金森病的细胞替代治疗

Parkinson’s disease (PD) is the second most common neurodegenerative disease in the aged populations, which results from extensive and specific loss of dopaminergic (DA) neurons in the substantia nigra par compacta (SNpc) of the midbrain. Although a number of medication, there are currently no effective therapeutic interventions to regenerate the lost DA neurons in the midbrain of PD patients. Therefore, cell replacement therapy through surgical grafting has long been suggested as a promising strategy to cure PD. A variety of candidate cell sources have been used for the cell replacement for PD, including embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, neural progenitor cells (NPCs), mesenchymal stem cells (MSCs), and DA neurons either derived from the above stem cells or directly reprogrammed from somatic cells. Indeed, ES and iPS cells have been efficiently differentiated into DA neurons and various studies have demonstrated that transplantation of differentiated DA neurons significantly improve the behavior impairment in animal models of PD. However, DA neurons derived from ES and iPS cells may contain undifferentiated pluripotent cells, which contributed to the teratoma formation after cell transplantation. .Recently, the direct cell reprogramming technology has provided a novel avenue to generate patient- and disease-specific cells for transplantation in PD patients. Two cell types (induced neural progenitor cells (iNPCs) and induced dopaminergic (iDA) neurons) have been reprogrammed from somatic cells. The iDA neurons can be directly converted from somatic cells by ectopic expression of transcription factors. However, the post-mitotic DAs are not able to self-renew and proliferate, restricting its further clinical application. More recently, functional and renewable dopaminergic progenitors (DPs) have been identified by coexpression of Otx2 and Corin from differentiated ES cells. The purified Otx2+Corin+ DPs can proliferate without spontaneous differentiation and generate well-integrated DA neurons after transplantation in a rat model of PD. Nonetheless, the authentic DPs only exist in the embryo stage and it’s unrealistic to obtain these cells from PD patients for cell replacement therapy. Our previous studies have demonstrated that mouse dermal fibroblasts can be directly reprogrammed into iPS cells or iNPCs by a cocktail of defined transcriptions factors. In the current proposal, we seek to highly efficiently generate dopaminergic neurons through two strategies: strategy I is to overexpress Lmx1a and Foxa2 in iNPCs to different into more dopaminergic neurons; strategy II is to directly convert mouse somatic cells into induced dopaminergic progenitors (iDPs) by ectopic expression of transcription factors and investigate its therapeutic application in a murine model of PD.

帕金森病是一种常见的主要影响老年人的神经退行性变，目前的治疗手段仅能改善患者的症状，无法阻止病情的进展。因其由中脑多巴胺能神经元的丢失而引起，细胞替代治疗被认为是一种极有前途的治疗手段，有望临床治愈帕金森病。而目前获得足够用于治疗的多巴胺能神经元细胞是及其困难的，本课题紧密围绕在如何高效地获得多巴胺能神经元，拟采取两种策略：策略一，前期工作中我们已将成纤维细胞直接转分化成神经干细胞，但其多巴胺能神经元分化效率极低，将通过高表达Lmx1a及Foxa2以大幅提高多巴胺能神经元的分化效率；策略二，因多巴胺能神经前体细胞既能自我更新并增殖，而且能够定向分化成多巴胺能神经元，被认为是用于帕金森病替代治疗的最理想候选细胞，因此，我们将应用Sox2、Brn2、Lmx1a及Foxa2将成纤维细胞直接转分化成多巴胺能神经前体细胞，并进一步探索其在MPTP诱导的帕金森小鼠模型中治疗的可行性。

帕金森病（Parkinson’s disease, PD）是一种常见的主要影响老年人的神经退行性变，目前的治疗手段仅能改善患者的症状，无法阻止病情的进展。因其由中脑多巴胺能神经元的丢失而引起，细胞替代治疗被认为是一种极有前途的治疗手段，有望临床治愈帕金森病。而目前获得足够用于治疗的多巴胺能神经元细胞是及其困难的，本课题紧密围绕如何高效地获得多巴胺能神经元这一科学问题，通过组合特异的转录因子（Brn2, Sox2 and Foxa2）将成纤维细胞直接转分化成可诱导性的多巴胺能神经前体细胞（induced Dopaminergic Progenitors，iDPs），体外实验中证实iDPs细胞具有高效地多巴胺能神经元分化能力，分化成的神经元呈现典型的双极神经元形态，电生理检测结果证实分化的神经元在电压钳模式下，K+和Na+离子通道电流可被监测到，在电流钳模式下，可监测到动作电位。体内实验中将iDPs细胞注射至MPTP诱导的PD小鼠纹状体内，结果证实能够显著减轻运动功能损伤，且注射至体内的iDPs细胞可分化成Th+的多巴胺能神经元，尚未检测到iDPs细胞分化成GFAP+的小胶质细胞。本项目的顺利开展证实组合特异性的转录因子可将成纤维细胞转化为功能性的iDPs细胞，为探索临床治疗PD的替代细胞开辟了新的途径。