Modelling Hirschsprung’s disease using patient-specific induced pluripotent stem cells

Abstract

Hirschsprung’s (HSCR) disease is a congenital disorder in which the enteric nerve cells are absent in the bowel, causing chronic constipation. The incomplete colonization of bowel with enteric neural crest (NC) cells is the main cause of the disease. RET gene encodes for a tyrosine kinase receptor and is highly implicated in the neural crest development. Mutations or genetic variants in RET are accounted for most of the HSCR cases. In particular, a single nucleotide polymorphisms (SNP, rs2435362) residing in the intron one of RET gene is predominantly found in HSCR, which may cause a reduced RET expression in patient, is significantly associated with HSCR susceptibility. In this study, a HSCR patient carrying a risk allele T in rs2435362 of RET gene, exhibiting a short segment aganglionosis and atrial/ventricular septal defects (ASD/VSD) was selected to establish a human model for HSCR. The patient’s skin fibroblast cells were reprogrammed into iPS cells by ectopic expression of four reprogramming factors. Three patient-specific iPS cell lines were currently obtained. They were ES-like in morphology, expressing the pluripotency markers and showing low DNA methylation levels of CpG sites in the promoter regions of NANOG and OCT3/4. Importantly, they could generate teratoma comprising all three germ layers when they were injected in SCID mice, further corroborating the cells had acquired pluripotency. Subsequent differentiation experiments revealed that these HSCR iPS cells were able to differentiate into NC cells of a comparable capacity as that of the control iPS cells (IMR90). In addition, these iPS-derived NC cells were multipotent and could commit to both neurogenic and smooth muscle lineages under defined differentiation conditions. Nevertheless, in general, all the HSCR-iPS cells showed a lower competency to form neurons and smooth muscle cells, suggesting that differentiation defects of NC may represent a cause of HSCR and other NC-associated disorders. Taken together, these results substantiate the potential use of our patient-specific model to study the etiology of HSCR and other NC-associated diseases.