Single-cell RNA Sequencing for Profiling Ganglionic and Aganglionic Colonic Segments from Patients with Hirschsprung...

Area of Science:

• Gastroenterology
• Developmental Biology
• Immunology

Background:

• Hirschsprung disease (HSCR) is a congenital intestinal motility disorder defined by aganglionosis in the distal bowel.
• Single-cell RNA sequencing (scRNA-seq) offers higher resolution than bulk RNA sequencing for studying cellular heterogeneity.

Purpose of the Study:

• To establish a standardized scRNA-seq workflow for characterizing the transcriptional landscape of HSCR affected tissues.
• To compare the cellular and molecular differences between ganglionic and aganglionic segments in HSCR patients.

Main Methods:

• Colon tissues from HSCR patients were processed for scRNA-seq after identifying ganglionic and aganglionic regions via H&E staining.
• Single-cell suspensions were generated, libraries constructed, sequenced, and data quality-filtered, normalized, and dimensionally reduced.
• Cells were clustered and annotated using marker genes, followed by comparative analyses of cell-type composition and differential gene expression.

Main Results:

• The study successfully generated scRNA-seq data from HSCR patient colon tissues.
• Significant differences in cell-type composition and immune microenvironment were observed between ganglionic and aganglionic segments.
• Key immune cell subsets exhibited differentially expressed genes, highlighting distinct immune responses.

Conclusions:

• The established scRNA-seq workflow provides a robust method for dissecting HSCR pathophysiology at the single-cell level.
• Distinct immune microenvironments exist between affected and unaffected bowel segments in HSCR.
• These findings offer novel molecular insights into the pathogenesis of Hirschsprung disease.