Assessment of a potential application of endogenous stem cells to treat congenital disorders

由于基因突变的毒性作用导致干细胞功能受损，先天性疾病往往导致器官发生不完全。虽然手术干预是这些器官缺陷的主要治疗方法，但其效果仍不令人满意。然而，随着基因组编辑技术的进步，现在可以在体内对干细胞中的基因突变进行校正。为了评估患病干细胞通过基因修复恢复其形成自主组织的能力的潜力，我们成功地开发了巨结肠病（HSCR）的小鼠模型，作为先天性干细胞疾病的模型。该模型通过cre - loxp介导的基因重组促进了致病突变的去除。杂合小鼠表现出类似HSCR病理的肠神经系统缺陷。通过将该小鼠与Phox2b-Cre小鼠杂交，我们证实在迷走神经嵴源性ENS干细胞中删除RET S811F可避免肠神经缺陷表型。此外，HSCR小鼠与Dhh-Cre小鼠杂交消除了雪旺细胞前体的显性阴性突变，这对出生后肠道神经发生至关重要。我们观察到，在缺乏适当神经功能的区域，mccherry阳性肠道神经元增加，表明显性阴性突变被移除的细胞进行了补偿。在不同发育时期（胚胎期10.5天）去除迷走神经嵴来源细胞致病突变的分析。E11.5和E12.5)在不同时间段间差异不显著。

Congenital diseases often result in incomplete organogenesis due to impaired stem cells function caused by toxic effects of gene mutations. Although surgical interventions are the primary treatment for these organ defects, their effectiveness remains unsatisfactory. However, advancements in genome editing technology now enable the in vivo correction of gene mutations in stem cells. To assess the potential of diseased stem cells to restore their ability to form autonomous tissues through gene repair, we successfully developed a mouse model of Hirschsprung disease (HSCR), serving as a model for congenital stem cell disorders. This model facilitates the removal of the disease-causing mutation through Cre-loxP-mediated gene recombination. The heterozygous mouse exhibited enteric nervous system defects resembling HSCR pathology. By crossing this mouse with Phox2b-Cre mice, we confirmed that deleting RET S811F in vagal neural crest-derived ENS stem cells evaded the enteric nerve defect phenotype. Moreover, crossing the HSCR mouse with Dhh-Cre mice eliminated the dominant negative mutation in Schwann cell precursors, which crucial for postnatal intestinal neurogenesis. We observed an increase in mCherry-positive intestinal neurons in regions lacking proper nerve function, suggesting compensation by cells where dominant-negative mutations were removed. The analysis of removing the disease-causing mutation in vagal neural crest-derived cells at different developmental time periods (Embryonic day 10.5. E11.5, and E12.5) revealed no notable differences among the observed time periods.