Autologous cell transplantation for the treatment of colorectal aganglionosis

项目总结 先天性巨结肠是一种具有潜在致命性的先天性疾病，其特征是缺乏 肠神经系统(ENS)沿不同长度的远端肠管 神经沟来源的细胞在发育过程中定植于整个肠道。外科手术 先天性巨结肠症的治疗包括切除无神经节段。虽然这就是生活- 手术后通常会出现严重的并发症，包括便秘、大便 大小便失禁和小肠结肠炎。肠神经脊源性细胞(ENCC)的移植 提供了一种有希望的新方法来替换丢失或异常的肠神经元。 先天性巨结肠和其他神经性肠道疾病。最近的研究，包括来自我们的 实验室证实，ENCCs可以从出生后的肠道中分离出来，在 文化，并移植到肠壁中。然而，主要挑战仍然存在：(1)数字 所产生的神经元的数量有限，(2)它们整合成神经胶质网络的能力 能否恢复肠道功能尚未得到证实，(3)患有先天性巨结肠的小鼠 疾病在4-6周大时死亡，限制了细胞后可用于分析的时间窗口 移植。为了克服这些挑战，我们提出了以下目标：(1) 采用一种创新的方法分离和扩大出生后肠道来源的ENCC 这产生了比以前的方法更多的祖细胞ENCC和(2)移植 一种新的非致死性大肠无神经节细胞增生症模型 由局部注射人白喉毒素(DT)产生的转基因小鼠，其神经 CREST来源的细胞表达DT受体。为了优化组合药物ENCC的扩张 将使用筛选方法。ENCC将与候选分子一起培养，以及 已知的生长因子，以确定祖细胞扩增的最佳“鸡尾酒”。单细胞 将进行RNAseq以表征细胞的转录组图谱 移植。自体来源的供体细胞将被移植到实验中- 产生了结肠的无神经节段。分析将包括定量测定 ENCC的存活、增殖和神经元分化及其功能特征 神经元活动。所获得的结果将建立ENCC分离的优化方法， 扩增和移植，并展示了基于细胞的治疗恢复 GI在先天性巨结肠和其他神经性肠道疾病中的作用。

PROJECT SUMMARY Hirschsprung disease is a potentially lethal congenital disorder characterized by the absence of enteric nervous system (ENS) along variable lengths of distal intestine due to the failure of neural crest-derived cells to colonize the entire intestine during development. The surgical treatment of Hirschsprung disease involves removing the aganglionic segment. While this is life- saving, significant complications commonly occur after surgery, including constipation, fecal incontinence, and enterocolitis. Transplantation of enteric neural crest-derived cells (ENCCs) offers a promising new approach to replacing missing or abnormal enteric neurons in Hirschsprung disease and other neurointestinal diseases. Recent studies, including from our laboratory, demonstrate that ENCCs can be isolated from the postnatal intestine, propagated in culture, and transplanted into the gut wall. However, major challenges remain: (1) the numbers of neurons generated have been limited, (2) their ability to integrate into neuroglial networks capable of restoring gut function has yet to be demonstrated, and (3) mice with Hirschsprung disease die at 4-6 weeks of age, limiting the time window available for analysis after cell transplantation. To overcome these challenges, we propose the following aims: (1) to incorporate an innovative approach to isolating and expanding postnatal gut-derived ENCCs that generates greater numbers of progenitor ENCCs than prior methods and (2) to transplant autologously-derived ENCCs into a novel non-lethal model of colorectal aganglionosis generated by local injection of human diphtheria toxin (DT) into transgenic mice whose neural crest-derived cells express DT receptor. To optimize ENCC expansion, a combinatorial drug screen approach will be used. ENCCs will be cultured with candidate molecules, together with known growth factors, to identify the optimal “cocktail” for progenitor cell expansion. Single cell RNAseq will be performed to characterize the transcriptome profile of cells prior to transplantation. Autologously-derived donor cells will be delivered into the experimentally- generated aganglionic segment of colon. Analyses will include quantitative determination of ENCC survival, proliferation, and neuronal differentiation, and functional characterization of neuronal activity. The results obtained will establish an optimized approach to ENCC isolation, expansion, and transplantation, and demonstrate the potential of cell-based therapy to restore GI function in Hirschsprung disease and other neurointestinal diseases.