Age-dependent functional and transcriptional differences in intestinal mesenchymal stromal cells during early postnatal development in piglets.

Intestinal mesenchymal stromal cells (iMSCs) regulate postnatal epithelial development, yet their temporal dynamics remain poorly defined in early postnatal piglets. Here, we investigated functional and transcriptional changes in jejunal iMSCs isolated from 0-, 7-, and 21-Day-old piglets. In an enteroid-iMSC coculture system, Day 0 iMSCs supported predominantly cystic enteroids with the highest surface area and proliferation index, whereas Day 7 iMSCs increased multi-budded enteroid morphology with intermediate proliferation, showing age-dependent differences in iMSC-mediated epithelial growth modulation. Relative real-time qPCR revealed that these functional differences aligned with niche-associated expression patterns in iMSCs. Specifically, RSPO3 level was highest at Day 0, BMP4 and PDGFRα expression peaked at Day 7, and WNT and BMP antagonists (SFRP1 and GREM1, respectively) increased in Day 21 iMSC samples. Bulk RNA sequencing further revealed shifts in iMSC transcriptomes across developmental stages, with Day 7 iMSCs exhibiting a transitional transcriptional profile enriched for genes associated with cytoskeletal reorganization and cell fate regulation compared with Day 0 iMSCs. Notably, pathway enrichment and gene set analyses identified multiple calcium channel genes and related pathways in Day 7 iMSCs compared with Day 0 iMSCs. Short-term pharmacological inhibition of calcium channels in Day 7 iMSCs was associated with rapid changes in niche factor levels, including decreased BMP4 and increased RSPO3 and WNT expressions. Together, these findings suggest age-related functional and transcriptional differences in iMSCs across early postnatal intestinal development. Our results highlight that calcium channel-related processes may be associated with changes observed in the Day 7 iMSC profile, providing a framework for future studies on iMSC-epithelial interactions during early intestinal development in piglets.