Single-cell RNA sequencing in Hirschsprung's disease tissues reveals lack of neuronal differentiation in the aganglionic colon segment

Abstract

The enteric nervous system (ENS) is a complex network of neurons and glial cells. Hirschsprung's disease (HSCR) is a congenital condition characterized by the absence of ganglion cells in the distal colon, leading to functional bowel obstruction. In this study, we used single-cell RNA sequencing (scRNA-seq) and whole genome sequencing (WGS) to analyze healthy and aganglionic colon segments from HSCR patients. Using scRNA-seq, we identified 13 major cell types in patient samples and observed that neural progenitor cells were present in both healthy and aganglionic colon regions, while mature neurons were absent from aganglionic colon. In these progenitor cells, critical differentiation pathway genes displayed reduced expression in the aganglionic colon, suggesting a disruption in their transition to mature neuronal cell types. Furthermore, transcriptomic analysis revealed significant alterations in gene expression across several stromal cell types. These transcriptomic shifts, particularly in mast cells, support the hypothesis that altered gene expression in the microenvironment of neural progenitor cells contributes to impaired differentiation. Our findings support the hypothesis that neural precursors in HSCR are capable of migration, but they are defective in their differentiation to mature cell types. Our analysis provides insights into potential therapeutic targets to stimulate neurogenesis in the aganglionic colon.

Figure 1:. Experimental design and overview of scRNA-seq analysis of HSCR patient samples

(A) Representative UMAP embedding overlay showing the single cell transcriptome distribution of healthy and aganglionic colon samples. (B) Feature plots of representative marker gene expressions used for cell type identification. (C) Heatmap of top 10 most significantly upregulated genes of identified cell clusters in a representative HSCR patient. (D) Representative UMAP embedding of single cell transcriptomes from HSCR healthy and aganglionic colon samples with cell type annotations. (E) Calculated ratios of cell-types in the healthy and aganglionic colon samples (n=3, mean±SD, T-test).

Figure 2:. Identification of neural-progenitor cells in the aganglionic colon of HSCR patient

(A) UMAP plots showing expression of neural progenitor cell- (NGFR), mature neuron (UCHL1) and mature glial cell (S100B) marker gene expression in a representative patient sample. (B) UMAP plots showing expression of neural progenitor cell- (NGFR) and mature neuron (UCHL1) marker gene expression in a representative patient sample split by tissue origin. Barcharts show mean+SD of neural progenitor and mature neuron cell ratios of three HSCR patients. (C) Heatmap showing the expression of neural progenitor, mature neuron and mature glial cell markers and HSCR-associated genes in identified mature neurons and neural progenitor cells. (D) Fluorescent microscopic images of p75 (NGFR) and DAPI stained colon samples from HSCR patients show the presence of neural progenitor cells in the healthy and the aganglionic colon samples alike. (E) Dotplot of gene expression of key differentiation pathway receptor genes expressed on healthy and aganglionic colon segment resident neural progenitor cells (representative plot).

Figure 3:. Expression of key DEG genes in healthy and aganglionic colon specimens

(Wilcoxon rank sum test, min.diff.pct = 0.25, logfc.threshold = 0.25).

Figure 4:. Genomic analysis of HSCR patient samples

(A) Number of identified somatic variants and healthy- or aganglionic colon segments specific variants in three HSCR patients. (B) Representative allele frequency plot of identified colon specific variants showing healthy colon specific (blue) and aganglionic colon specific (red) variants. (C) CNV analysis on three HSCR patients using FACETS. Plots show copy number state (bottom), log-odds ratio (middle) and log-ratio (top) for healthy- (top row) and aganglionic (bottom row) samples.