Human embryonic stem cell-derived neural crest stem cells and Hirschsprung disea

巨结肠病，又称神经节性巨结肠，是一种先天性缺陷，每5000个活产儿中就有1个受其影响，其特征是在可变长度的后肠肠管中不能形成肠神经系统（ENS）。这种潜在的致命疾病导致无法协调肠道蠕动运动，最常见的原因是通过神经胶质细胞系衍生的神经营养因子（GDNF）或内皮素-3 （EDN3）信号通路[2]的信号减少突变。神经嵴干细胞（NCSCs）表达GDNF受体Ret和EDN3受体内皮素受体B (EDNRB)，从而产生ENS ADDIN EN。引用[3]。这些信号通路相互作用，调节NCSCs和其他定植肠道的神经嵴祖细胞的增殖和迁移，尽管EDN3信号的主要作用是抑制过早分化还是促进迁移仍然存在疑问。引用(3 - 10)。在Ret或Ednrb缺乏的动物中，神经嵴细胞从未迁移到肠道的神经节部分。引用(3、6)。这些观察结果提高了通过将传统手术方法与细胞疗法相结合来改善巨结肠疾病治疗的可能性，细胞疗法将NCSCs直接移植到肠道的神经节部分，绕过迁移/增殖缺陷产生肠神经节。引用(3、6、11、12)。与这种可能性相一致的是，我们和其他人已经证明，从胎儿啮齿动物肠道中分离的NCSCs在移植到巨结肠疾病啮齿动物模型肠道的神经节区后可以移植并形成肠神经元。引用(6,11 - 13)。尽管如此，在考虑对患者进行这种治疗之前，有必要获得足够数量的人类NCSCs以供临床使用。由于人类胎儿组织非常有限且质量不稳定，因此从人类胚胎干（hES）细胞中获得具有肠道特征的NCSCs是理想的。在对小鼠和大鼠肠道NCSCs进行广泛表征后，我们建议优化培养条件，从hES细胞中获得具有相似特性的人NCSCs。我们将把这些人类NCSCs注射到Ednrb突变大鼠的神经节后肠，以测试这些细胞在体内形成神经元和胶质细胞的能力。这些研究将测试具有特定区域特征的NCSCs是否可以从hES细胞中获得，以及这些细胞是否可以移植到巨结肠疾病动物模型的肠道中。

Hirschsprung disease, or aganglionic megacolon, is a congenital defect that affects 1 out of 5,000 live births and is characterized by a failure to form enteric nervous system (ENS) in a variable length of the hindgut [1]. This potentially fatal condition results in an inability to coordinate peristaltic movements of the bowel and is most commonly caused by mutations that reduce signaling through the glial cell line-derived neurotrophic factor (GDNF) or endothelin-3 (EDN3) signaling pathways [2]. Both the GDNF receptor, Ret, and the EDN3 receptor Endothelin receptor B (EDNRB) are expressed by the neural crest stem cells (NCSCs) that give rise to the ENS ADDIN EN.CITE [3]. These signaling pathways interact to regulate the proliferation and migration of NCSCs and other neural crest progenitors that colonize the gut, though questions remain about whether the primary role of EDN3 signaling is to inhibit premature differentiation or to promote migration ADDIN EN.CITE [3-10]. Neural crest cells never migrate into the aganglionic portion of the gut in animals affected by Ret or Ednrb deficiency ADDIN EN.CITE [3,6]. These observations raise the possibility of improving the treatment of Hirschsprung disease by combining traditional surgical approaches with cell therapy in which NCSCs are transplanted directly into the aganglionic portion of the gut to generate enteric ganglia by bypassing the migration/proliferation defects ADDIN EN.CITE [3,6,11,12]. Consistent with this possibility, we and others have shown that NCSCs isolated from the fetal rodent gut can engraft and form enteric neurons after transplantation into the aganglionic region of the gut from rodent models of Hirschsprung disease ADDIN EN.CITE [6,11-13]. Nonetheless, before such a therapy can be contemplated for patients it will be necessary to obtain human NCSCs in quantities adequate for clinical use. Since human fetal tissue is very limited and of inconsistent quality for clinical use [14], it would be ideal to derive NCSCs with enteric characteristics from human embryonic stem (hES) cells. Having extensively characterized mouse and rat enteric NCSCs, we propose to optimize culture conditions to derive human NCSCs with similar properties from hES cells. We will inject these human NCSCs into the aganglionic hindgut of Ednrb mutant rats to test the ability of these cells to form neurons and glia in vivo. These studies will test whether NCSCs with specific regional characteristics can be derived from hES cells and whether these cells engraft in the gut of an animal model of Hirschsprung disease.