A calcium-driven transcriptional switch in mesenchymal stromal cells shapes postnatal intestinal development

Postnatal intestinal development depends on coordinated communication between epithelial and stromal cells, yet the transcriptional dynamics in intestinal mesenchymal stromal cells (iMSCs) during preweaning stages remain undefined in piglets. We investigated whether early postnatal iMSCs undergo significant transcriptional reprogramming that shifts their stromal cues to support epithelial transitioning from proliferation to a differentiation state. Jejunal iMSCs were isolated from mixed-sex piglets at postnatal Day 0, 7, and 21 (n = 4 biological replicates/age group) for enteroid coculture assays, RNA sequencing, and validation studies. Coculture experiments demonstrated that Day 0 iMSCs promoted the formation of large, cystic spheroidal enteroids (suggesting a high proliferative niche), coinciding with peak RSPO3 expression. In contrast, Day 7 iMSCs supported a more budded enteroid morphology (higher pro-differentiation niche), corresponding with elevated BMP4 expression (p-value ≤ 0.05). Consistent with the functional data, KEGG and GSEA analyses revealed major transcriptional reprogramming in Day 7 iMSCs compared with Day 0, with enrichment of calcium signaling and SMAD-pathway activation, including upregulation of calcium-responsive channels (TRPA1, CACNA1I, and P2RX7) (q-value ≤ 0.05). To test whether calcium influx acts as an upstream driver of this transcriptional switch during early postnatal intestinal development in piglets, Day 7 iMSCs were treated with the calcium channel inhibitor SKF-96365 (SKF, 5 µM, 2 h). Following the treatment, SKF significantly downregulated BMP4 and upregulated RSPO3 and WNT5A in Day 7 iMSCs, reversing the stromal niche expression patterns observed between Day 0 and Day 7 iMSCs (q-value ≤ 0.05). These results highlight the critical role of calcium signaling in promoting emerging pro-differentiation profiling of iMSCs during the early postnatal transition window. Together, the findings support a new model linking calcium-dependent signaling as a potential upstream regulator of the “transcriptional switch” in stromal cell reprogramming to promote epithelial maturation, suggesting a novel approach for future studies aimed at improving early neonatal gut maturation and resilience. Maryland Stem Cell Research Fund (GR101404) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.