Abstract: The gastric muscularis mucosa (MM) is a thin layer of smooth muscle within the gastrointestinal wall. However, the development of human gastric MM remains poorly understood. Previously, we investigated gastric MM development using gastric organoids (GOs) derived from human induced pluripotent stem cells (hiPSCs). We reported that long-term culture led to the formation of GOs accompanied by MM, and that epithelial-derived Sonic Hedgehog (SHH) and TGFβ signaling, as well as mechanical factors, were involved in MM development. To further explore this process, we conducted a time-course analysis of spatial transcriptomics using the Xenium in situ platform on hiPSC-derived GOs. Spatial transcriptomic analysis is particularly suitable for studying specimens containing diverse histological structures, such as tissues and organoids. UMAP analysis of this dataset revealed that epithelial and smooth muscle clusters were positioned on opposite sides relative to the hiPSC cluster. Additionally, RNA expression profiling of individual clusters has the potential to identify MM precursor cells. Based on this analysis, differentially expressed genes (DEGs) in the gastric epithelial cluster were significantly associated with gene ontologies related to gland development and epithelial morphogenesis. In contrast, DEGs in the mature smooth muscle cluster were significantly enriched in gene ontology terms related to the collagen-containing extracellular matrix. These findings suggest that the subepithelial mechanical environment contributes to MM formation. UMAP analysis, incorporating GOs treated with SHH and TGFβ inhibitors, revealed that while a candidate MM precursor cluster was maintained, mature smooth muscle cells were markedly reduced, indicating that these inhibitors prevent smooth muscle maturation. Furthermore, pseudotime analysis based on single-cell RNA expression demonstrated that the gastric epithelial cluster preceded smooth muscle development. This suggests that epithelial maturation in GOs precedes MM formation, which is consistent with our previous report demonstrating that epithelial-derived factors contribute to MM development.
Funding Source: JSPS KAKENHI Grant Number 23K15041
