Fig. 3: iMPCs regulate endothelial cell function.

A Growth quantification in ECs exposed to 2-fold concentrated conditioned medium from SMCs, MSCs, and iMPCs (n = 6, 8; ***P < 0.001). B Scratch assay comparing EC migration in conditioned medium from iMPCs and basal medium at 24 h (Left; Scale bar: 200 µm), with a quantified migration percentage of gap closure normalized to the basal medium control (Right; n = 4; ***P < 0.001). C Tube formation assay on Matrigel using conditioned medium, with a representative image (Scale bars: 200 µm) and quantification of total tube length (Right; n = 9; **P < 0.01, ***P < 0.001). D Schematic representation and fluorescent images showing the coculture of ECs and iMPCs (P1) within a microfluidic on-a-chip model. GFP-labeled iMPCs and DsRed-labeled ECs were embedded in a fibrin gel, and the formation of vascular structures was observed after 2 days. (Scale bars: 500 µm). E Immunofluorescent staining of the vascular network formed within the microfluidic chip. ECs are marked by CD31 and VE-Cadherin (red), and iMPCs by α-SMA and SM22 (green). Nuclei are counterstained with DAPI (blue). The inset shows a magnified view of an endothelial lumen surrounded by mural cells (yellow arrowheads). (Scale bars: 100 µm). F Subcutaneous implantation of ECs with or without mural cells into nude mice, with explanted grafts visually assessed at day 7 (Scale bar: 4 mm). G H&E staining identifying perfused blood vessels in implants at day 7 (yellow arrowheads) (Scale bars: 50 µm). H Microvessel density analysis per mm² area (n = 7; *P < 0.05). I IHC showing human-specific ECs (h-CD31 + ) and human perivascular cells (h-Vimentin + ) (Scale bar: 50 µm; inset 10 µm). J iMPC Tracing: GFP and α-SMA staining to track GFP-labeled iMPCs within the perivascular niche in vivo. (Scale bars: 50 µm; insets 10 µm). K Quantification of the percentage of human vessels with human mural cell coverage, comparing ECs implanted with SMCs, MSCs, and iMPCs (n = 4). All experiments in this Figure used nascent iMPCs right after differentiation (96 h). All data are mean ± s.e.m. n are biological replicates (A–C, H, K). Statistics are one-way ANOVA with Bonferroni’s post-test analysis (A–C, H, K). D, F were 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).