A Patient-Specific IPSC Model for Studying the Pathophysiology of Hirschsprung’s Disease

Abstract

Hirschsprung’s (HSCR) disease is a congenital neurocristopathy in which some enteric ganglia are absent due to incomplete colonization of neural crest cells (NCCs) in the hindgut, causing chronic constipation. A significant number of HSCR patients also clinically present with other NC-associated disorders, such as ventricular and atrial septal defects (VSD/ASD). A hypomorphic allele or SNP of a major gene, RET, causes or imparts susceptibility to HSCR. In particular, SNP (rs2435362) residing in the intron 1 of RET gene was found to be highly associated with HSCR and lead to reduced RET expression. However, the molecular basis of HSCR associated VSD/ASD is largely unclear. With the use of a iPSC-based HSCR model, we illustrate the pathophysiology of a specific HSCR patient. Three iPSC clones from a syndromic HSCR patient, carrying the RET risk allele in rs2435362 were generated. We used different caudalizing cues to differentiate iPSCs into NCCs with unique HOX expression patterns, corresponding to anterior cranial or posterior vagal NCCs. Consistently, the patient iPSCs displayed similar capacities in generating NCCs at all axial levels, marked by HNK1 and p75NTR. Nevertheless, the patient NCCs and their derivatives exhibited severe migration and/or differentiation defects in making neurons and smooth muscle cells. In particular, HNK1+p75NTR+ posterior NCCs derived from patient-iPSCs were less migratory compared to the control NCCs, while no obvious migration defect was observed in their cranial counterpart, indicating that the migration defect was only restricted to the more posterior NCCs. These patient NCCs were also less capable in generating neurons and readily biased toward generating glial cells, which pinpoints the fine balance between neurogenesis and gliogenesis. Intriguingly, the neural differentiation defects were restricted to NC lineage. The capacity of patient iPSCs to make various types of CNS progenitors and neurons was comparable to that of the control iPSCs, nicely recapitulating the patient’s phenotype where only enteric neurons, but not CNS progenitors were affected. Subsequent expression analysis revealed that patient NCCs express lower level of RET which is known to be regulating enteric NCC migration and differentiation. Together, our patient-specific model endow a reliable platform to decipher the underlying pathogenesis in other HSCR patients.