Potential use of patient specific induced-pluriopotent stem cell (iPSC) to delineate the molecular pathogenesis of syndromic Hirschsprung (HSCR)

Abstract

Hirschsprung (HSCR) disease is a complex congenital disorder attributed to a failure of enteric neural crest cells (NCCs) to fully colonize the bowel and form ganglia in the hindgut, leading to bowel obstruction and megacolon. A significant number of HSCR patients also suffer from other disorders of NC origin, such as ventricular and atrial septal defects (VSD/ASD). The pathologies of these disorders are largely unclear. Recently, we have established iPSC lines from a syndromic HSCR patient with VSD/ASD. While these patient iPSCs showed similar capacity for generating NCCs (p75NTR+; HNK1+), their NCC derivatives exhibited severe differentiation defects in making neurons and cardiac smooth muscle cells (SMC). Intriguingly, the neural differentiation defects were restricted to NC lineage. The capacity of patient iPSC to make various types of CNS progenitors (PAX6+) and neurons was comparable to that of the control iPSC, nicely recapitulating the patient’s phenotype where only enteric neurons, but not CNS progenitors were affected. These observations prompted us to further delineate the causative genes/pathways that lead to failure of making neurons/glia or cardiac SMCs. By transcriptome sequencing, we revealed that FACS sorted p75NTR+; HNK1+ population express various NC markers (e.g. SNAI1, NGFR(p75NTR), TWIST, TFAP2A, SOX10, PHOX2B, SOX9, SOX10, RET and PAX3), a gene expression pattern conferring multipotency of NCCs. Subsequent comparative analysis of the transcriptomes of the control and patient iPSC-derived NCCs and their derivatives showed that pathway genes implicated in neural (ERBB4, NGFR, EDNRB, SOX10, NGF, EDN1/2) and SMC (e.g. GATA6, HAND1) differentiation are consistently dysregulated in the patient cells. More importantly, premature upregulation of glial differentiation markers was detected in the patient NCCs, suggesting that the neuronal differentiation defects of patient NCCs would be the result of differentiation bias towards glial lineage. Taken together, our data indicated that NCCs generated from patient iPSC could serve as a useful human disease model for a better understanding of molecular pathogenesis of neurocristopathies.