Fig. 4: Maturation of iMPCs upon interaction with ECs.

A Co-Culture and Transcriptomics: Diagram showing iMPCs and ECs in co-culture for bulk RNA sequencing. The lower panel indicates up-regulated and down-regulated gene counts in co-cultured iMPCs (co-iMPCs) versus mono-cultured iMPCs. B Transcriptomic Correlation: Pearson’s correlation plot delineating transcriptional profiles among SMCs, co-iMPCs, MSCs, iMPCs, iSMCs, and iPSCs. C Principal Component Analysis: Transcriptional comparison of co-iMPCs with primary SMCs and MSCs relative to iMPCs and iPSCs (n = 3). D Gene Expression Heatmap: Differential gene expression patterns in co-iMPCs compared to iMPCs. E Gene Ontology Enrichment: Up-regulated genes in co-iMPCs associated with mature mural cell functions. F, G Marker Gene Expression: RT-qPCR analysis of (F) SMC and (G) pericyte markers, showing enhanced expression in co-iMPCs versus mono-cultured iMPCs (n = 3, 4, 6, 7, 9; *P < 0.05, **P < 0.01, ***P < 0.001). All PCR data is normalized to GAPDH. H, I Immunofluorescence Characterization of co-iMPCs: co-iMPCs were sorted as CD31- cells from the co-culture. H Sorted cells stained for α-SMA (green) with 3G5 (pericyte marker, red) and nuclei counterstained with DAPI (blue), demonstrating the presence of both SMCs (α-SMA + -/3G5-) and pericytes (α-SMA-/3G5 + ) (Scale bar: 100 µm). I Sorted cells stained for α-SMA (green) (green), MYH11 (red), and DAPI (blue). The co-localization of MYH11 and α-SMA is indicative of cells with a more mature SMC phenotype (yellow arrowheads). (Scale bar: 50 µm). All experiments in this Figure used nascent iMPCs right after differentiation (96 h). All data are mean ± s.e.m. n are biological replicates (F, G). Statistics are one-way ANOVA with Bonferroni’s post-test analysis (F, G). A was partially created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).