Human enteric nervous system progenitor transplantation restores functional responses in Hirschsprung Disease patient-derived tissue
Objective Hirschsprung disease (HSCR) is a severe congenital disorder affecting 1:5000 live births. HSCR results from 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).  Design 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 restore 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 cell populations exhibiting transcriptional and functional hallmarks of early ENS progenitors. Following transplantation, hPSC-derived ENS progenitors integrate, migrate and form neurons within explanted human HSCR colon samples. Importantly, the transplanted HSCR tissue displayed increased basal contractile activity and increased responses to electrical stimulation compared to control tissue.  Conclusion Our findings demonstrate, for the first time, the potential of hPSC-derived ENS progenitors to repopulate and restore functional responses in human HSCR patient colonic tissue. Overall design: ENS progenitors generated from Human embryonic stem cells were collected at day 0 (SHE-AT-g001), day 4 (SHE-AT-g006) and day 6 (SHE-AT-g007) of differentation and cryopreserved.  Cells were analysed by single cell RNA sequencing by single cell discoveries (Utrecht, The Netherlands)