Organization of Stem Cell-Derived Vascular Progenitors into Vascular Networks

Objectives: Translation of bladder regenerative technologies is limited by lack of angiogenesis in the engineered tissues. De novo angiogenesis requires coordination between endothelial cells (ECs) and stromal cells consisting of smooth muscle cells (SMCs) and fibroblasts. We hypothesize that the coordination necessary to form functional arteries requires communication between progenitor ECs (pECs) and progenitor SMCs (pSMCs), which may not occur between terminally differentiated cells. In this pilot study, we sought to test whether co-culture of pECs and pSMCs may reveal this type of coordination.

Methods: Human pluripotent stem cells (hPSC) were used to derive both vascular progenitor cell types. pECs were derived using a branching lineage-directed approach with stimulation to guide hPSCs into primitive streak, lateral mesoderm, and subsequent differentiation into pECs, which were virally transfected to express GFP. pSMCs were derived using a vascular differentiation protocol. We first performed optimization experiments to determine ideal coating substrates and growth media for 2D and 3D co-culture. For 2D co-culture, we tested combinations of collagen I, collagen IV and fibronectin as coating substrates. We cultured equal numbers of pECs and pSMCs in EC medium, SMC medium, and different ratios of both media combined. 2D co-culture experiments were then carried out for 9 days, during which cell-cell interaction was observed by time-lapse imaging. For 3D co-culture, we stained pSMCs with a commercial cell-tracking dye and then seeded equal numbers of pECs and pSMCs in hydrogel containing collagen I and IV. The 3D co-cultures in hydrogel were cultured in 1:1 ratio of EC and SMC media for 17 days. Cell location within the hydrogels was visualized using fluorescence and confocal microscopes.

Results: Co-culture of pECs and pSMCs on combinations of different coating substrates revealed that neither cell type survived well on fibronectin, but both survived equally well (9 days) in culture on collagen IV alone and on different combinations of collagen I and IV. Growth media testing with different ratios of EC and SMC media determined that both cell types survived well in 1:1 ratio of the two media. Time-lapse imaging of 2D co-culture on collagen IV alone and combinations of collagen I and IV with 1:1 media mixture revealed organized clusters of pECs circled by pSMCs by day 3 of culture, and pSMCs did not infiltrate the pEC clusters. Fluorescence and confocal imaging of 3D co-culture in hydrogel showed vascular-like networks of pECs with adjacent pSMCs by day 17 of culture.

Conclusions: Organization of pECs into clusters and vascular-like networks surrounded by pSMCs suggests coordination between progenitor cells. Future studies using terminally differentiated ECs and SMCs as controls will help differentiate the role of progenitor cells in guiding angiogenesis in engineered tissues.