Human enteric nervous system progenitor transplantation improves 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 the 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 affect 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 a cell population exhibiting transcriptional and functional hallmarks of early ENS progenitors. Following transplantation, hPSC-derived ENS progenitors integrate, migrate and form neurons/glia within explanted human HSCR colon samples. Importantly, the transplanted HSCR tissue displayed significantly increased basal contractile activity and increased responses to electrical stimulation compared with control tissue.

Conclusion: Our findings demonstrate, for the first time, the potential of hPSC-derived ENS progenitors to repopulate and increase functional responses in human HSCR patient colonic tissue.

Keywords: ENTERIC NERVOUS SYSTEM; GASTROINTESTINAL SURGERY; HIRSCHSPRUNG'S DISEASE; STEM CELLS.

Figure 1

High yields of homogenous vagal NC can be robustly generated from hESCs and induced pluripotent stem cells. (A) Schematic representation of the treatment conditions used to generate vagal NC/enteric nervous system progenitors in vitro. (B) Flow cytometry-based quantification of SOX10 expressing cells in day (D) 6 cultures derived from the indicated human pluripotent stem cell lines. Error bars=SEM (n=3–7 independent differentiations). (C) qPCR expression analysis of SOX10 and key vagal HOX genes carried out in the same D6 cultures as those shown in B (error bars=SEM; n=3–7 independent differentiations). (D) Quantification of p75 and CD49d-positive cells in D6 cultures following immunostaining and flow cytometry (error bars=SEM; n=3). Each biological repeat is represented by a unique symbol. (E) UMAP visualisation of 17 928 cells and their distribution in three samples (D0, D4 and D6) corresponding to unbiasedly defined clusters. (F) UMAP plots showing expression of a set of selected marker genes. (G) Dot plot visualisation of the selected expressed marker genes for each cluster. (H) Selective re-clustering of D4 and D6 samples. (I) RNA velocity analysis of D4 and D6 samples. (J) Cytotrace and pseudotime analysis of D4 and D6 samples. ANC, anterior NC; hESCs, human embryonic stem cells; NC, neural crest.

Figure 2

Human embryonic stem cell (hESC)-derived vagal neural crest (NC)/enteric nervous system (ENS) progenitors can be differentiated to enteric-like neurons and glia in vitro. (A–L) Representative images of day 21 neurons immunolabeled with indicated ENS markers following in vitro differentiation of hESC-derived (H9 background) vagal NC cells. (M–O) Analysis and quantification of hESC-derived (H9 background) neuronal Ca²⁺ response to depolarisation with high K⁺ (M), activation with DMPP (N) and electrical stimulation (O). Left: overview of the regions of interest (ROIs) employed for analysis. Right: individual line traces of the responding cells, the change in fluorescence (F/F0) is plotted over time (s). The traces are randomly chosen for illustration. The application of high K⁺/DMPP/electrical stimulation is represented by the grey bar in the graph. Scale bars: 100 µm.

Figure 3

ZsGreen-labelled ENS progenitors survive transplantation into Hirschsprung disease (HSCR) surgical discard tissue and spread through endogenous tissue. (A–C) Schematic representation of ex vivo transplantation procedure conducted on patient-derived HSCR colonic tissue. (D–F) Representative stereoscopic images of ZsGreen⁺ donor ENS progenitor cells following ex vivo transplantation into patient-derived HSCR tissue at (D) day 7 (D7), (E) D14 and (F) D21. (G–I) Representative images showing delineation of ZsGreen⁺ donor cell coverage (from D to F) achieved using a custom-designed FIJI Macro. (J) Summary data showing quantification of donor cell coverage at D7, D14 and D21; n=4 transplanted segments. (K) Representative image showing superimposed outlines of the extent of migration at D7 (yellow outline), D14 (cyan outline) and D21 (magenta outline) from panel D to F. Arrows show direction of spread from the presumptive transplant site. (L) High magnification imaging revealed donor cells migrating from the site of transplantation in all directions (arrows) and ZsGreen⁺ cells that were observed closely associated with endogenous vasculature (arrowheads). Scale bars: D–F: 2 mm; L: 500 µm. CM, circular muscle; ENS, enteric nervous system; LM, longitudinal muscle; MYP, myenteric plexus; SMP, submuscosal plexus.

Figure 4

Enteric nervous system (ENS) progenitors transplanted into Hirschsprung disease (HSCR) surgical discard tissue differentiate into neurons and glia. (A–Aii) Representative images, acquired by light sheet fluorescence microscopy, of cleared HSCR patient-derived tissue at D21 post-transplantation in three planes: XY (A), ZX (Ai) and ZY (Aii). ZsGreen⁺ ENS progenitors could be seen clustered around the initial transplant site and migrating from the transplant site in streams. (B–D) Representative confocal images of cleared transplanted tissue at D21 demonstrating the presence of ZsGreen⁺TUBB3⁺ cells within recipient HSCR patient-derived tissue segments (arrows). Insets show TUBB3⁺ transplanted cells captured at higher magnification, extending axon-like processes (arrowheads). (E–G) ZsGreen⁺S100β⁺ donor-derived cells were also readily detected. Scale bars: A–Aii: 1 mm; D, G: 100 µm.

Figure 5

Transplantation of enteric nervous system (ENS) progenitors into Hirschsprung disease (HSCR) surgical discard tissue increases both baseline contractility and response to electrical stimulation via neuronal activity. (A) Representative traces showing basal contractile activity in HSCR patient-derived tissue 21 days after sham (grey) or ENS progenitor-transplantation (green). (B–D) Summary data of baseline contractile frequency (B), amplitude (C) and cumulative area under the curve (AUC; D). (E) Representative contractility traces of sham (grey) or ENS progenitor transplanted (green) tissues in response to electrical field stimulation (EFS; red bar) in control conditions. (F, G) Summary data of amplitude (F) and AUC (G) in response to EFS in control conditions. (H) Representative contractility traces of sham (grey) or ENS progenitor-transplanted (green) tissues in response to electrical field stimulation (EFS; red bar) in the presence of 1 µM tetrodotoxin (TTX). (I) Summary data of AUC in response to EFS in TTX. To account for variability in tissue size, all functional analyses (peak contractile amplitude (g) and area under the curve (g.s) measurements) were normalised to wet tissue weight (g). *p<0.05. Error bars=SEM.

Figure 6

Cholinergic and nitrergic activity contribute to contractile responses within transplanted Hirschsprung disease tissue. (A–F) Representative contractility traces of sham (grey) or enteric nervous system (ENS) progenitor transplanted (green) tissues in response to electrical field stimulation (EFS; red bar) in control conditions (A, B), in the presence of atropine alone (C, D) and in the presence of atropine+L NAME (E, F). (G, H) Summary analysis of normalised area under the curve (AUC) response in sham (G) or ENS progenitor-transplanted (H) tissues. To account for variability in tissue size, area under the curve (g.s) measurements were normalised to wet tissue weight (g). *p<0.05. Error bars=SEM.

Figure 7

Enteric nervous system (ENS) progenitor transplantation increases the response of Hirschsprung disease (HSCR) surgical discard tissue samples to electrical stimulation. To account for patient variability (in terms of age, gender, disease severity and positioning of the sample along the oro-anal axis) samples were normalised by patient using feature scaling (Z=[X–min(X)]/[min(X)–max(X)]). (A–G) Slope analysis for each pair of tissue samples under examination by patient. (H) Summary data showing scaled area under the curve (AUC) analysis of ENS progenitor-transplanted samples compared with sham-transplanted. *p<0.05. Error bars=SEM.