Developing a human pluripotent stem cell-based strategy for treating Hirschsprung disease

先天性巨结肠症是一种危及生命的肠道疾病，由最远端胃肠道缺乏内在神经细胞（神经节细胞缺乏症）引起。它发生在大约1/5000的活产婴儿中，使其成为影响肠道的最常见的先天性疾病之一。由于肠内神经细胞（肠神经元）介导正常肠道功能所需的收缩，因此先天性巨结肠患者缺乏这些细胞会导致严重的便秘或肠梗阻。唯一可用的治疗方法是手术切除受影响的肠道部分，并结合“拉通”程序，这需要将肠道的健康部分连接到肛门。然而，手术需要保留部分异常肠道，包括肛门括约肌，这可能是大多数先天性巨结肠病患者长期（往往是终身）胃肠道问题和生活质量差的部分原因。管理这种疾病所需的手术、再入院和门诊预约给医疗保健系统带来了沉重的负担。消化道内分泌系统发育的研究进展（或肠）神经系统和疾病的发病机制，以及再生医学的相当大的进展，已经突出了替代治疗的潜力，例如细胞替代疗法。肠神经系统来源于称为神经嵴细胞的瞬时细胞群，道。在先天性巨结肠病中，这些细胞不能在远端肠道定植，留下可变的无神经节细胞节段。动物模型中的实验已经证明，肠神经系统祖细胞/干细胞（原始神经嵴细胞的衍生物，并且从小鼠肠外植体或小鼠胚胎干细胞收获）的移植具有分化成肠神经元的能力，并且当移植到无神经节肠外植体中时能够定殖。类似的神经元祖细胞从人类出生后的肠道外植体已被证明有效地殖民和分化的无神经节肠外植体的先天性巨结肠患者。然而，从肠道收获这种人类神经祖细胞是困难的，并且随着出生后年龄的增加变得更加困难。因此，尽管这样的临床前试验已经证明细胞疗法应该是治愈先天性巨结肠病的可行选择，但来自出生后肠道的人肠神经元的可用性仍然是开发基于细胞的疗法的瓶颈。其中在数量和针对该任务定制细胞性质方面存在产生用于治疗的合适细胞的显著潜力。我们最近开发了有效的协议，以产生人类神经嵴细胞和肠神经系统祖细胞从人类多能干细胞。在这个项目中，我们将测试这些祖细胞的能力，以纠正缺乏肠神经元的动物和患者来源的模型先天性巨结肠症移植后。我们还将优化目前从多能干细胞中产生和纯化人类肠神经系统祖细胞的方法，并将开发一种新的改进的先天性巨结肠病动物模型。通过这种方式，我们将建立再生医学方法的临床前基础，通过移植来自人类多能干细胞的细胞来重新支配肠道的受影响部分来治疗先天性巨结肠病。首先，我们的目标是通过常规手术后剩余的短节段的神经支配来改善目前治疗的结果。

Hirschsprung disease is a life-threatening intestinal disorder caused by an absence of intrinsic nerve cells (aganglionosis) in the most distal gastrointestinal tract. It occurs in approximately 1 in 5000 live births, making it one of the most common congenital diseases affecting the gut. Given that intrinsic gut nerve cells (enteric neurons) mediate the contractions necessary for normal gut function, their absence in Hirschsprung patients causes severe constipation or intestinal obstruction. The only treatment available is surgical removal of the affected part of the bowel combined with a 'pull through' procedure, which entails connecting the healthy part of the gut to the anus. However, the surgery necessitates retention of part of the abnormal gut including the anal sphincter, which is likely to account in part for the long-term, often life-long, gastrointestinal problems and poor quality of life suffered by the majority of patients with Hirschsprung disease. Surgery, readmissions and outpatients hospital appointments required for management of this condition present a significant burden for the healthcare system. Recent advances in the understanding of development of the gut's intrinsic (or enteric) nervous system and pathogenesis of the disease, as well as considerable progress in regenerative medicine, have highlighted potential for alternative treatments, such as cell replacement therapy.During normal embryonic development, the enteric nervous system is derived from a transient population of cells termed neural crest cells that migrate from the neural tube to innervate the gastrointestinal tract. In Hirschsprung disease these cells fail to colonize the distal gut leaving a variable segment of aganglionosis. Experiments in animal models have demonstrated that transplantation of enteric nervous system progenitor/stem cells (derivatives of the original neural crest cells and harvested from mouse gut explants or mouse embryonic stem cells) have the ability to differentiate into enteric neurons and colonize when transplanted into aganglionic gut explants. Similar neuronal progenitor cells obtained from human postnatal gut explants have been shown to effectively colonize and differentiate within aganglionic gut explants of Hirschsprung patients. However, the harvesting of such human nerve progenitor cells from gut is difficult and becomes more so with increasing post-natal age. Therefore, although such preclinical testing has demonstrated that cell therapy should be a viable option for curing Hirschsprung disease, the availability of human enteric neurons from post-natal gut remains a bottleneck for development of cell-based therapies.An attractive alternative source is offered by pluripotent stem cells, where there is significant potential of generating appropriate cells for therapy both in terms of numbers and tailoring cell properties for the task. We have recently developed efficient protocols to generate human neural crest cells and enteric nervous system progenitors from human pluripotent stem cells. In this project, we will test the ability of these progenitors to correct for the lack of enteric neurons in animal and patient-derived models of Hirschsprung disease following transplantation. We will also optimise the current methods of generating and purifying human enteric nervous system progenitors from pluripotent stem cellsand will develop a new improved animal model of Hirschsprung disease. In this way, we will establish the pre-clinical basis for a regenerative medicine approach for treating Hirschsprung disease by transplantation of cells derived from human pluripotent stem cells to re-innervate the affected part of the gut. In the first instance our goal will be to improve the outcome of current treatments by innervation of the short section remaining after routine surgery.