Uncovering the therapeutic potential of adipose tissue derived neural stem cells for Hirschsprung's disease.

项目总结 我们研究的首要目标是从皮下脂肪组织中开发一种干细胞疗法。 (Sat)替代先天性巨结肠(HSCR)中先天缺失的肠道神经系统(ENS)。 手术切除受影响的结肠是目前唯一可行的治疗HSCR的方法。这是必要的 救命手术；尽管50%以上的患者仍有术后并发症，包括 便秘、大便失禁和小肠结肠炎。为了克服这些病态，对 可以保留直肠及其功能的治疗是有必要的。通过以下途径更换缺席的ENS 干细胞疗法被吹捧为实现这一目标最有希望的治疗策略。我们的团队已经 证明了从肠道中获取神经干细胞(NSCs)进行干细胞治疗的可行性 移植到HSCR小鼠体内后，可移植、迁移并分化为神经元网络。用于临床 应用：这需要外科手术切除一段肠道。防止不必要的切除 手术，其他来源的神经干细胞是感兴趣的。人类脂肪(脂肪)组织含有干细胞储存库。 容易接触到的东西。这些细胞已经在270多项针对多种疾病的临床试验中进行了检验。 这支持了有利的患者安全状况。在我们的初步数据中，我们还发现了神经纤维 来自小鼠脂肪沉积的束状脂肪-皮下脂肪组织(SAT)-含有内源性的 未被探索用于治疗神经疾病的神经干细胞。我们预测SAT可能会提供一个有用的 神经干细胞的来源用于治疗HSCR中的结肠无神经节细胞增多症；然而，SAT-NSCs是否可以治疗HSCR仍不确定 在肠道的无神经节(缺少ENS)环境中进行神经再生，目前没有 方法纯化扩增人SAT-NSCs。在这项研究的第一个目标中，我们将确定是否纯化了 来自小鼠的SAT-NSCs能够在无神经节细胞的肠道中再生神经。黑帮成员是 出生后由神经干细胞补充，神经干细胞从外源性神经纤维束迁移到肠道 根据来自肠道的环境提示分化为肠道神经元。我们将解决是否 当神经节细胞和神经节细胞提供信号信号时，SAT-NSCs也可以进行肠道神经发生。 体外无神经节细胞共培养系统和体内显微外科SAT-NSC植入。至 确定如何分离和扩展人类SAT-NSC，我们将解决有关 这些细胞的起源。为了做到这一点，从人类SAT神经纤维束中分离出来的细胞将被无偏见地 以干细胞培养过程前后的单核RNA-Seq为特征。表达神经干细胞的细胞 标记物将通过荧光激活的细胞分选纯化，其分化潜力将 在体外培养和体外移植到肠道平滑肌中进行评估。这些研究的结果 研究将建立分离SAT-NSCs的程序并评估其治疗先天性心脏病的潜力 HSCR中的神经病。

PROJECT SUMMARY The overarching objective of our research is to develop a stem cell therapy from subcutaneous fat tissue (SAT) to replace the congenitally absent enteric nervous system (ENS) in Hirschsprung disease (HSCR). Surgical resection of the affected colon is currently the only viable treatment for HSCR. This is a necessary life-saving procedure; albeit, more than 50% of patients still suffer from postoperative complications including constipation, fecal incontinence, and enterocolitis. To overcome these morbidities, investigations into treatments that can preserve the rectum and its functions are warranted. Replacement of the absent ENS via stem cell therapies is touted as the most promising treatment strategy to achieve this goal. Our group has demonstrated the feasibility of stem cell treatments by harvesting neural stem cells (NSCs) from the gut which engraft, migrate and differentiate into neuronal networks when transplanted into mice with HSCR. For clinical application this would require surgical resection of a piece of intestine. To prevent unnecessary resection surgery, other sources of NSCs are of interest. Human fat (adipose) tissue contains a reservoir of stem cells that are readily accessible. These cells have been examined in over 270 clinical trials for numerous diseases that support favourable patient safety profiles. In our preliminary data we have also identified that nerve fiber bundles from murine fat deposits – subcutaneous adipose tissue (SAT) - harbor an endogenous source of NSCs that are unexplored for the treatment of neuropathies. We predict that the SAT could provide a useful source of NSCs to treat colonic aganglionosis in HSCR; however, it remains undetermined if SAT-NSCs can undergo neurogenesis in the aganglionic (absent ENS) environment of the gut and there are currently no methods to purify and expand human SAT-NSCs. In the first aim of this study, we will determine if purified SAT-NSCs from mice are capable of neurogenesis in aganglionic intestine. The ganglionated ENS is supplemented postnatally by NSCs that migrate into the gut from extrinsic nerve fiber bundles and differentiate into enteric neurons in response to environmental cues from the gut. We will address whether SAT-NSCs can also undergo enteric neurogenesis when provided signalling cues from the ganglionic and aganglionic gut in in vitro coculture systems and via microsurgical SAT-NSC implantation in vivo. To determine how to isolate and expand human SAT-NSCs we will address the paucity of knowledge on the origin of these cells. To accomplish this, cells isolated from human SAT nerve fiber bundles will be unbiasedly characterised by single nuclei RNA-Seq before and after stem cell culture procedures. Cells expressing NSC markers will be purified by fluorescence activated cell sorting and their differentiation potential will be assessed in in vitro culture and in ex vivo transplants to the smooth muscle of the gut. The results of these studies will establish procedures to isolate SAT-NSCs and assess their potential to treat the congenital neuropathy in HSCR.