成纤维细胞直接重编程为胃肠神经元的实验研究

The gastrointestinal motility disorders are some of the most challenging gastrointestinal disorders to manage, including Hirschsprung disease (HSCR), gastroparesis, and so on. Currently, therapeutic options for many gastrointestinal motility conditions remain inadequate and are limited to palliative interventions such as surgery and the provision of artificial nutrition. This highlights the fact that although current treatments aim to prevent the mortality and limit morbidity associated with the most significant complications of the disease, they are not curative. The identification and study of stem cells over the past decade opens up the possibility of definitive curative therapies for these disorders. The enteric nervous system has many potential advantages that favour the success of such cellular transplantation therapies. These advantages include ease of delivery of the transplant, as well as the possibility of minimizing immunological rejection by enteric neural crest stem cells (ENCSCs) obtained from unaffected regions of the patient’s own intestine for autologous transplantation. However, the invasive derivation, the small quantity and limited proliferation ability of ENCSCs restrain their clinical application. Previous studies showed that introduction of lineage specific-transcription factors can transform one mature somatic cell into another (also known as transdifferentiation), which makes the transdifferentiated cells an ideal seed cell of autologous cellular transplantation therapies for gastrointestinal motility disorders. We previously induced human iPSCs to differentiate to enteric neurons with high efficiency and gene expression profiles of differentiated cells in different days were detected by RNA-Seq. In this study, human embryonic and adult fibroblasts (HEFs and HAFs) will be induced to transdifferentiate to enteric neurons or enteric neural crest stem cells and different assays, including RNA-Seq, immunofluorescence, and electrophysiology, will be carried out to evaluate their gene expression profile and the characteristics of transdifferentiated cells. The migration, proliferation and differentiation capacity of transdifferentiated cells in gastrointestinal tissues of HSCR patients cultured in vitro and RET knockout mice in vivo were detected in detail. Our results will provide an ideal cell resource for autologous cell replacement therapies of gastrointestinal motility disorders and help to establish in vitro disease models of gastrointestinal motility disorders for mechanism studies.

以胃肠神经元缺失或病损导致的如先天性巨结肠、胃轻瘫等胃肠动力障碍性疾病，目前临床无有效治疗手段。胃肠神经元移植可能是该类疾病的有效治疗方法。然而成体组织中胃肠神经元数量少、取材与扩增困难，制约了临床应用。转分化技术有利于快速获得适合自体移植、安全性较高的种子细胞来源，在细胞替代治疗和疾病体外模型的建立中有良好的应用前景。申请人前期建立了人多能干细胞向胃肠神经元的高效分化体系，深入分析了不同分化阶段细胞的基因表达谱。在此基础上，本研究拟将人成纤维细胞直接重编程为胃肠神经元或其干细胞即胃肠神经嵴干细胞，通过转录组测序、电生理检测等方法鉴定转分化细胞的生物学特性，并探讨转分化细胞在体外对先天性巨结肠患者肠道组织，及在RET基因敲除的巨结肠小鼠模型中的神经修复再生能力。通过本研究的实施，不仅可以为胃肠动力障碍性疾病的治疗提供新的思路和技术手段，也有助于建立胃肠动力障碍性疾病的细胞模型探索发病机制。

以胃肠神经元缺失或病损导致的先天性巨结肠（HSCR）等胃肠动力障碍性疾病（GI），目前临床无有效治疗手段，而胃肠神经元的移植可能是一种有效的治疗方法。本研究拟建立将人成纤维细胞直接重编程为胃肠神经嵴干细胞或胃肠神经元的转分化体系，通过转录组测序、电生理检测等方法鉴定转分化细胞的生物学特性，并探讨转分化细胞在巨结肠小鼠模型中的神经修复再生能力。为胃肠神经元的移植提供新的细胞来源。.申请团队利用基因过表达的方法，通过逆转录病毒的方法结合四环素可调控载体技术，成功建立了将人皮肤成纤维细胞（HDF）转分化为胃肠神经嵴干细胞（iENCSCs）和胃肠神经元（iENs）的体系。我们首先通过TUJ1染色筛选实现胃肠神经元转分化的基因组合，发现APSH四因子组合可以较为高效的诱导得到转分化胃肠神经元；进一步检测转分化过程中不同时间点的细胞特性，发现转分化第14天，约有5-10%的细胞表达胃肠神经嵴干细胞的特异标志物SOX10、AP2α、p75和HNK1等，即为转分化胃肠神经嵴干细胞（iENCSCs）；iENCSCs可在神经元分化体系中进一步诱导分化具有典型神经元形态的细胞，免疫荧光染色显示存在不同亚型（VIP, ChAT, NOS, VIP等）的肠道神经元iENs，且具有成熟神经元的特征包括钙通道活性和动作电位等；表达谱测序的结果提示iENs与成体组织分离的肠道神经元其基因表达谱相似度较高（R2＞0.90）；进一步利用无神经的先天性巨结肠病人肠道组织和化学诱导的先天性巨结肠免疫缺陷小鼠模型证实，iENCSCs能在小鼠肠道组织中有效的存活、迁移和分化。.综上，本项目成功建立了个体特异的胃肠神经元转分化体系，利用该技术有望获得病人特异的转分化胃肠神经元用于GI等疾病的自体移植。通过本研究的实施，不仅可以为胃肠动力障碍性疾病的治疗提供新的思路和技术手段，也有助于建立胃肠动力障碍性疾病的细胞模型探索发病机制。