背景:肠神经病,由肠神经系统异常引起的,与显著的发病率和高昂的医疗费用有关,但目前的治疗方法并不令人满意。基于细胞的疗法提供了一种创新的方法来替代缺失或异常的肠神经元,从而恢复肠道功能。
方法:从Wnt1-Cre;R26td番茄小鼠的胃肠道中分离肠神经元干细胞(ENSC)并产生神经球(NS)。NS移植是通过注射到nNOS-/-小鼠的中结肠间质进行的,结肠运动障碍的模型,使用1次(n=12)或3次(n=12)注射(每次注射30NS),纵向间隔1-2mm。6周后使用肌电图(EMG)评估功能结果,电场刺激(EFS),光遗传学,通过测量结肠直肠的运动。
结果:移植的ENSC在nNOS-/-受体结肠中形成了硝能神经元。与单独单次注射相比,多次注射ENSC导致明显更大的覆盖面积,并且与结肠功能的显着改善有关。通过(1)通过肌电图记录增加结肠肌肉活动,(2)更快的直肠珠排出,和(3)增加体内粪便颗粒产量。器官浴研究揭示了通过光遗传学刺激表达通道视紫红质的ENSC和响应EFS而恢复平滑肌松弛的直接神经肌肉通讯。
结论:这些结果表明,移植的ENSC可以在结肠运动障碍模型中形成有效的神经肌肉连接并改善结肠运动功能,并另外揭示了多个细胞递送位点导致改善的反应,为优化临床试验设计铺平了道路。
Enteric neuropathies, which result from abnormalities of the enteric nervous system, are associated with significant morbidity and high health-care costs, but current treatments are unsatisfactory. Cell-based therapy offers an innovative approach to replace the absent or abnormal enteric neurons and thereby restore gut function.
Enteric neuronal stem cells (ENSCs) were isolated from the gastrointestinal tract of Wnt1-Cre;R26tdTomato mice and generated neurospheres (NS). NS transplants were performed via injection into the mid-colon mesenchyme of nNOS-/- mouse, a model of colonic dysmotility, using either 1 (n = 12) or 3 (n = 12) injections (30 NS per injection) targeted longitudinally 1-2 mm apart. Functional outcomes were assessed up to 6 weeks later using electromyography (EMG), electrical field stimulation (EFS), optogenetics, and by measuring colorectal motility.
Transplanted ENSCs formed nitrergic neurons in the nNOS-/- recipient colon. Multiple injections of ENSCs resulted in a significantly larger area of coverage compared to single injection alone and were associated with a marked improvement in colonic function, demonstrated by (1) increased colonic muscle activity by EMG recording, (2) faster rectal bead expulsion, and (3) increased fecal pellet output in vivo. Organ bath studies revealed direct neuromuscular communication by optogenetic stimulation of channelrhodopsin-expressing ENSCs and restoration of smooth muscle relaxation in response to EFS.
These results demonstrate that transplanted ENSCs can form effective neuromuscular connections and improve colonic motor function in a model of colonic dysmotility, and additionally reveal that multiple sites of cell delivery led to an improved response, paving the way for optimized clinical trial design.