Modelling Hirschsprung disease with human pluripotent stem cell derived colonic organoids

Abstract

The recent breakthrough in stem cell research allows disease modeling in vitro, which is particularly useful for studying the genetic basis of congenital diseases. We have previously used Hirschsprung (HSCR)-specific nduced pluripotent stem cells (iPSC) to generate enteric neural crest cells (ENCCs) and unveil the biological impacts of genetic lesions in HSCR pathogenesis. In this study, we established human iPSC-based colonic organoids (HCO) to further illustrate how the gut environment and mesenchymal signals may influence the ENCC development and disease development. Our organoid model comprises two major components: ENCCs and gut endodermal cells. These two populations of cells were generated from hiPSCs, by sequentially activating the ACTIVIN and WNT pathways to promote the formation of definitive endoderm and the hindgut spheroids, respectively. The hindgut spheroids were then caudalized further by treatment of BMP2. The ENCCs, on the other hand, were generated using the dual SMAD inhibition differentiation method and enriched by FACS with HNK1 and p75NTR antibodies. hiPSC-ENCCs were then co-cultured with the hindgut spheroids in a three-dimensional matrigel culture, in which ENCCs received patterning signals from the gut endoderm and developed together with the endodermal cells. By thirty days, crypt-like structure was observed in the HCOs in which a distinct layer of gut epithelium (VILLIN+, CDH1+) expressing colonic specific marker (SATB2+), endocrine (CHGA+) and Goblet cells (MUC2+) were found. Intriguingly, ENCCs could be able to selfpattern and aligned with the epithelium and started differentiating into neurons (TUJ1+). After transplanting into the kidney capsule of immunodeficient mice, HCOs underwent morphogenesis and formed mature tissues with defined crypts and colonic epithelium, while ENCCs gave rise to nerve cells residing proximity to the submucosal and myenteric layers of smooth muscle fibers. Upon electrical-field stimulation, the engrafted tissues exhibited a sustained wave of contractions, which can be inhibited by the blockade of neuronal activity with tetrodotoxin. This model is currently being used to generate “HSCR colon” for a better understanding HSCR pathogenesis.