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

先天性巨结肠或无神经节巨结肠是一种先天性缺陷，每5，000例活产婴儿中就有1例受影响，其特征是在可变长度的后肠中未能形成肠神经系统（ENS）[1]。这种可能致命的疾病导致无法协调肠道蠕动，最常见的原因是通过胶质细胞源性神经营养因子（GDNF）或内皮素-3（EDN 3）信号传导途径减少信号传导的突变[2]。GDNF受体Ret和EDN 3受体内皮素受体B（EDNRB）均由产生ENS的神经嵴干细胞（NCSC）表达。这些信号传导途径相互作用以调节NCSC和其他定殖于肠道的神经嵴祖细胞的增殖和迁移，尽管关于EDN 3信号传导的主要作用是抑制过早分化还是促进迁移的问题仍然存在[3-10]。在受Ret或Ednrb缺乏症影响的动物中，神经嵴细胞从不迁移到肠道的无神经节部分[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先天性巨结肠病治疗的可能性，其中将NCSC直接移植到肠道的无神经节部分中，以通过绕过迁移/增殖缺陷来产生肠神经节。与这种可能性相一致，我们和其他人已经表明，从胎儿啮齿动物肠道分离的NCSC可以在移植到来自先天性巨结肠病啮齿动物模型的肠道无神经节区后植入并形成肠神经元[6，11 -13]。尽管如此，在可以为患者考虑这种疗法之前，将需要获得足以用于临床使用的量的人NCSC。由于人胎儿组织非常有限并且临床使用的质量不一致[14]，因此理想的是从人胚胎干（hES）细胞衍生具有肠特性的NCSC。在广泛表征小鼠和大鼠肠道NCSC后，我们建议优化培养条件以从hES细胞获得具有相似性质的人NCSC。我们将这些人NCSC注射到Ednrb突变大鼠的无神经节后肠中，以测试这些细胞在体内形成神经元和神经胶质的能力。这些研究将测试具有特定区域特征的NCSC是否可以从hES细胞中获得，以及这些细胞是否可以移植到先天性巨结肠动物模型的肠道中。

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.