Abstract：
OBJECTIVE: Hirschsprung disease (HSCR) is a severe congenital disorder affecting 1:5000 live births. HSCR results from the failure of enteric nervous system (ENS) progenitors to fully colonise the gastrointestinal tract during embryonic development. This leads to aganglionosis in the distal bowel, resulting in disrupted motor activity and impaired peristalsis. Currently, the only viable treatment option is surgical resection of the aganglionic bowel. However, patients frequently suffer debilitating, lifelong symptoms, with multiple surgical procedures often necessary. Hence, alternative treatment options are crucial. An attractive strategy involves the transplantation of ENS progenitors generated from human pluripotent stem cells (hPSCs).
METHODS: ENS progenitors were generated from hPSCs using an accelerated protocol and characterised, in detail, through a combination of single-cell RNA sequencing, protein expression analysis and calcium imaging. We tested ENS progenitors\' capacity to integrate and affect functional responses in HSCR colon, after ex vivo transplantation to organotypically cultured patient-derived colonic tissue, using organ bath contractility.
RESULTS: We found that our protocol consistently gives rise to high yields of a cell population exhibiting transcriptional and functional hallmarks of early ENS progenitors. Following transplantation, hPSC-derived ENS progenitors integrate, migrate and form neurons/glia within explanted human HSCR colon samples. Importantly, the transplanted HSCR tissue displayed significantly increased basal contractile activity and increased responses to electrical stimulation compared with control tissue.
CONCLUSIONS: Our findings demonstrate, for the first time, the potential of hPSC-derived ENS progenitors to repopulate and increase functional responses in human HSCR patient colonic tissue.
METHODS: ENS progenitors were generated from hPSCs using an accelerated protocol and characterised, in detail, through a combination of single-cell RNA sequencing, protein expression analysis and calcium imaging. We tested ENS progenitors\' capacity to integrate and affect functional responses in HSCR colon, after ex vivo transplantation to organotypically cultured patient-derived colonic tissue, using organ bath contractility.
RESULTS: We found that our protocol consistently gives rise to high yields of a cell population exhibiting transcriptional and functional hallmarks of early ENS progenitors. Following transplantation, hPSC-derived ENS progenitors integrate, migrate and form neurons/glia within explanted human HSCR colon samples. Importantly, the transplanted HSCR tissue displayed significantly increased basal contractile activity and increased responses to electrical stimulation compared with control tissue.
CONCLUSIONS: Our findings demonstrate, for the first time, the potential of hPSC-derived ENS progenitors to repopulate and increase functional responses in human HSCR patient colonic tissue.
摘要：
目的：先天性巨结肠病（HSCR）是一种严重的先天性疾病，影响1:5000的活产。HSCR起因于肠神经系统(ENS)祖细胞在胚胎发育期间未能完全定植于胃肠道。这导致远端肠的神经节病，导致运动活动中断和蠕动受损。目前,唯一可行的治疗选择是手术切除神经节肠.然而,患者经常衰弱，终身症状，经常需要多个外科手术。因此，替代治疗方案至关重要。有吸引力的策略涉及移植由人多能干细胞(hPSC)产生的ENS祖细胞。
方法：使用加速方案从hPSC产生ENS祖细胞，在细节上,通过单细胞RNA测序的组合，蛋白质表达分析和钙成像。我们测试了ENS祖细胞整合和影响HSCR结肠功能反应的能力，在离体移植到器官型培养的患者来源的结肠组织后，使用器官浴收缩力。
结果:我们发现,我们的方案一致地产生具有早期ENS祖细胞的转录和功能标志的细胞群的高产量。移植后,HPSC衍生的ENS祖细胞整合，在外植的人HSCR结肠样品中迁移并形成神经元/神经胶质。重要的是,与对照组织相比,移植的HSCR组织显示出显著增加的基础收缩活动和增加的对电刺激的反应.
结论：我们的研究结果表明，第一次,hPSC衍生的ENS祖细胞在人类HSCR患者结肠组织中重新填充和增加功能反应的潜力。
方法：使用加速方案从hPSC产生ENS祖细胞，在细节上,通过单细胞RNA测序的组合，蛋白质表达分析和钙成像。我们测试了ENS祖细胞整合和影响HSCR结肠功能反应的能力，在离体移植到器官型培养的患者来源的结肠组织后，使用器官浴收缩力。
结果:我们发现,我们的方案一致地产生具有早期ENS祖细胞的转录和功能标志的细胞群的高产量。移植后,HPSC衍生的ENS祖细胞整合，在外植的人HSCR结肠样品中迁移并形成神经元/神经胶质。重要的是,与对照组织相比,移植的HSCR组织显示出显著增加的基础收缩活动和增加的对电刺激的反应.
结论：我们的研究结果表明，第一次,hPSC衍生的ENS祖细胞在人类HSCR患者结肠组织中重新填充和增加功能反应的潜力。
