C. R. Schlieve1, K. L. Fowler1, I. Hajjali1, X. Hou1, T. C. Grikscheit1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA
Introduction: Disruption of enteric nervous system (ENS) development and function leads to varying degrees of pathology, including Hirschsprung Disease (HD). Impaired migration of enteric neural crest cells (ENCC) within the gastrointestinal tract results in an inability to relax intestinal smooth muscle causing obstructive symptoms. Surgical resection of the aganglionic portion of the colon in HD decreases mortality, but does not completely resolve symptoms. Therefore, restoration of the ENS through cellular transplantation has become a promising area of research for the treatment of enteric neuropathies. We have previously generated ENCC from human pluripotent stem cells (hPSC) and demonstrated their ability to differentiate into diverse classes of neurons and glia. Additionally, injection of hPSC-derived ENCC into the muscular layer of the colon demonstrated extensive migration and rescued mortality in an EDNRB knockout mouse model that mimics HD. However, successful integration of hPSC-derived ENC into human HD colon has not been demonstrated. The purpose of our study was to explore hPSC-derived ENC supplementation to restore ENS components in tissue-engineered colon (TEC) from patients with HD.
Methods: Multicellular clusters of epithelial and mesenchymal cells, termed organoid units (OU), were derived from colon obtained from patients with HD. Human embryonic stem cell line H9 was exposed to LDN193189, SB431542, and CHIR99021 under defined conditions with the addition of Retinoic Acid to promote differentiation into ENCC. Unsorted ENCC were mixed with HD OU prior to implantation, seeded onto a biodegradable scaffold, wrapped in the omentum of adult NOD/SCID mice, and allowed to mature for 1 month. Implants were analyzed for development of ENS cell types through immunostaining of neurons (Tuj1) and glia (GFAP/s100b). Neurons were evaluated for expression of excitatory marker choline acetyltransferese (ChAT) and inhibitory marker neuronal nitric oxide synthase (nNOS).
Results: Human iPSC-derived ENCC supplementation restored components of the ENS in TEC generated from HD patients. Aganglionosis of HD donor samples was confirmed by intraoperative frozen section and H&E analysis. HD TEC with ENCC co-implantation resulted in intestinal constructs with villi, underlying smooth muscle and the presence of enteric neurons and glia. Excitatory (ChAT/Tuj1) and inhibitory (nNOS/Tuj1) neurons and glia (s100b) were identified within the submucosa and muscular layers of HD TEC supplemented with ENCC and were absent in HD TEC alone.
Conclusion: Our findings validate a novel approach to restoring human ENS cell types in HD colonic tissue through hPSC-derived ENC supplementation. This advancement is a necessary step toward establishing cellular therapies for future treatment of enteric neuropathies.