Human enteric nervous system progenitor transplantation restores functional responses in Hirschsprung Disease patient-derived tissue

ABSTRACT 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. What is already known on this topic Hirschsprung disease is a devastating condition characterized by aganglionosis of the enteric nervous system (ENS) in the distal bowel, leading to dysmotility, severe constipation and enterocolitis. Stem cell therapy offers the potential to generate an enteric nervous system in aganglionic tissue and previous studies have described methods for generating ENS progenitors. However, the ability of these cells to establish intestinal motility in HSCR human tissue has not been shown. What this study adds We describe, for the first time, the detailed characterization of an ENS progenitor population derived from human pluripotent stem cell lines using our efficient protocol. Further, we demonstrate the ability of ENS progenitors to differentiate into enteric neurons in vitro and mediate functional rescue following transplantation into explants of human Hirschsprung disease tissue. How this study might affect research, practice or policy These results clearly show the potential of hPSC-derived ENS progenitors in stem cell therapy of Hirschsprung disease for progression towards clinical trials. This study highlights the significant advantages of using human surgical discard tissue for testing the efficacy of stem cell therapies. The described ex vivo model can be used to test different therapeutic approaches prior to clinical trials.