In vitro priming of human mesenchymal stromal/stem cells into 3D capillary-like structures : Implication of the Src/JAK/STAT3 signaling pathway in vasculogenic mimicry

Background: Mesenchymal stromal/stem cells (MSC) can enhance the neovascularization processes required for wound healing and are widely used as an autologous source of progenitor cells in stem cell-based therapies. However, incomplete MSC differentiation towards a vascular endothelial cell phenotype questions their involvement in an alternative process to angiogenesis, namely vasculogenic mimicry (VM), and the signal transducing events that regulate their in vitro priming into 3D capillary-like structures. Methods: Human MSC were primed on top of Cultrex matrix in order to recapitulate an in vitro 3D phenotype of VM. Total RNA was extracted, and differential gene expression assessed through RNA-Seq analysis and RT-qPCR. Transient gene silencing was achieved using specific siRNA. AG490, Tofacitinib, and PP2 pharmacological effects on VM 3D structures were analyzed using the Wimasis software. Results: In vitro VM occurred within 4 hours and was prevented by the JAK/STAT3 inhibitors AG490 and Tofacitinib, as well as by the Src inhibitor PP2. RNA-Seq highlighted STAT3 as a signaling hub contributing to VM when transcripts from 3D cultures were compared to those from 2D monolayers. Concomitant increases in IL6, IL1b, CSF1, CSF2, STAT3, FOXC2, RPSA, FN1, and SNAI1 transcript levels suggest the acquisition of a combined angiogenic, inflammatory and epithelial-to-mesenchymal transition phenotype in 3D cultures. Increases in STAT3, FOXC2, RPSA, Fibronectin, and Snail protein expression were confirmed in 3D cultures. STAT3 and RPSA gene silencing abrogated in vitro VM. Conclusions: In vitro VM priming of MSC into 3D structures requires Src/JAK/STAT3 signaling. This molecular evidence supports a clinically feasible MSC-mediated pseudo-vasculature process that could temporarily sustain grafts through VM until the host vasculature is able to infiltrate and remodel injured tissues.