J. L. Mueller1, R. Stavely1, R. A. Guyer1, S. Bhave1, R. Hotta1, A. M. Goldstein1 1Massachusetts General Hospital, Pediatric Surgery, Boston, MA, USA
Introduction: Development of the enteric nervous system (ENS) requires coordinated interactions between neural crest-derived cells and the extracellular matrix (ECM) through which they migrate. Stem cell therapy offers a potential to reestablish the ENS in the aganglionic colon of Hirschsprung disease, however the aganglionic gut environment may not be permissive for transplanted ENS stem cells. We therefore sought to utilize single-cell RNA sequencing to understand the transcriptional changes in the ECM within the aganglionic colon of Hirschsprung mice.
Methods: The muscular layers of the distal 2 cm of aganglionic colon from two 10-14 day old PLP1GFP; Ednrb-/- mice (KO Hirschsprung mice with GFP labeled glia) and the corresponding ganglionic segment from wild-type (WT) controls were used. 10X Genetics v3.1 kits were utilized to prepare scRNA-seq libraries. Sequencing was performed at the Harvard University Bauer Core Facility and data was analyzed with the Seurat R package. After cell filtration, datasets were integrated using the “FindIntegrationAnchors” command, principal component analysis was performed, neighbors were identified, UMAP projection was performed, and clusters were identified. Differential gene expression (DGE) analysis was performed comparing the KO aganglionic colon and WT ganglionic colon.
Results: We identified 23 clusters which were labeled according to their unique transcriptome (Figure 1). The distal aganglionic KO colon was missing neurons and intraganglionic glial cells. DGE analysis revealed a wide array of ECM alterations in the aganglionic colon, particularly amongst glial cells, macrophages, smooth muscle cells (SMCs), and fibroblasts. Glial cells express many ECM genes, suggesting that they secrete their own ECM to modulate their interactions with their environment. The aganglionic colonic transcriptome is pro-inflammatory, where macrophages express numerous migratory ECM genes consistent with their response to inflammation. KO fibroblasts overexpress Col6a5, encoding an inhibitory ECM protein against neural crest migration, and underexpress various pro-migratory ECM genes, including Adamdec1 and Itgb4, while the KO SMCs underexpress other potentially permissive genes, including Thsd4 and Npnt.
Conclusion: This is the first study to evaluate the transcriptome of a Hirschsprung model at the single cell level. We identify numerous transcriptional ECM changes in the aganglionic distal colon which create a non-permissive environment for stem cell therapy. We also observe that glial cells have high ECM gene expression, which is critically important in the context of stem cell therapy, as glial cells are a component of our transplanted cells and are therefore modifiable.
