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Autologous cell transplantation for the treatment of colorectal aganglionosis

自体细胞移植治疗结直肠无神经节细胞病

基本信息

批准号：
10308113
负责人：
Ryo Hotta
金额：
$ 7.75万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10308113
关键词：
Action Potentials Address Affect Age Area Autologous Autologous Transplantation Cell Separation Cell Survival Cell Therapy Cell Transplantation Cells Child Childhood Chronic Clinical Colon Colorectal Congenital Disorders Congenital Megacolon Constipation Data Development Diphtheria Toxin Disease Distal Drug Screening Electrophysiology (science)Embryo Enteral Enteric Nervous System Enterocolitis Environment Esophageal achalasia Excision Exhibits Failure Fecal Incontinence Functional Gastrointestinal Disorders Functional disorder Gastrointestinal Diseases Gastrointestinal tract structure Gastroparesis Goals Growth Factor Healthcare Human Immunosuppression In Vitro Injections Intestinal Pseudo-Obstruction Intestines Irritable Bowel Syndrome Laboratories Length Life Mediating Methods Modeling Morbidity - disease rate Mus Nerve Neural Crest Neuroglia Neuronal Differentiation Neuronal Injury Neurons Only Child Operative Surgical Procedures Proliferating Rodent Savings Stem cell transplant Synapses Testing Time Transgenic Mice Transplantation base cell motility clinical application clinically significant combinatorial cost diphtheria toxin receptor effective therapy enteric neuropathy experimental study ganglion cell gastrointestinal gastrointestinal function in vivo induced pluripotent stem cell innovation migration mouse model nerve stem cell novel novel strategies postnatal progenitor reduce symptoms single-cell RNA sequencing stem stem cells transcriptome transcriptomics transplantation therapy

项目摘要

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.

项目摘要先天性巨结肠是一种潜在的致命性先天性疾病，其特征是缺乏肠神经系统（ENS）沿着可变长度的远端肠，由于失败的神经嵴衍生的细胞在发育期间定殖于整个肠。手术先天性巨结肠的治疗包括切除无神经节段。如果这就是生活- 保存，手术后通常会发生重大并发症，包括便秘，粪便失禁和小肠结肠炎。肠神经嵴源性细胞（ENCC）的移植提供了一种很有前途的新方法来替代缺失或异常的肠神经元，先天性巨结肠及其他神经肠道疾病。最近的研究，包括我们的实验室，证明ENCC可以从出生后的肠道中分离，在肠道中繁殖，培养，并移植到肠壁中。然而，主要挑战仍然存在：（1）数字的神经元生成有限，（2）它们整合到神经胶质网络中的能力能够恢复肠道功能尚未得到证实，以及（3）患有先天性巨结肠的小鼠疾病在4 - 6周龄时死亡，限制了细胞后分析可用的时间窗。移植为了克服这些挑战，我们提出以下目标：（1）采用创新方法分离和扩增出生后肠源性ENCC 与现有方法相比，该方法产生更多的祖细胞ENCC;（2）用于移植将自体来源的ENCC转化为结直肠无神经节细胞症的新型非致死性模型通过将人白喉毒素（DT）局部注射到转基因小鼠中产生，嵴源性细胞表达DT受体。为了优化ENCC扩增，将使用屏幕方法。ENCC将与候选分子一起培养，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谱，移植自体来源的供体细胞将被输送到实验中- 产生无神经节结肠段。分析将包括定量测定 ENCC的存活、增殖和神经元分化，以及ENCC的功能表征。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，并证明基于细胞的治疗恢复的潜力，先天性巨结肠和其他神经肠道疾病的胃肠道功能。