DCEF-directed 3D angiogenic sprouting using human stem cell-derived vascular organoids

Simran Shergill

Angiogenesis is crucial for wound healing as it ensures optimal tissue perfusion during the proliferation phase. In chronic wounds, the lack of a healthy blood supply caused by impaired angiogenesis prohibits effective healing. Sprouting angiogenesis, a process of new capillary vessel growth from pre-existing vessels, is modulated by several microenvironmental stimuli, including growth factors and mechanical forces. Thus far, most studies centered on triggering sprouting angiogenesis for treatment of chronic wounds have focused on applying chemical gradients of growth factors to endothelial cells, but this approach has many limitations. Here, we propose using direct current (DC) electric field (EF) stimulation to induce and direct sprouting angiogenesis of endothelial cells. DCEF, a less well studied endogenous signal, has been proposed to have the ability to elicit angiogenic cellular responses that facilitate wound healing. Prior research indicates that EF can induce or influence cellular proliferation, differentiation, and migration of cells in vitro. Thus, we utilized this non-conventional approach to induce angiogenesis in vitro. First, we used VeraVec endothelial cells to determine the electrical responses in a 2D electrotaxis assay with DCEFs of physiological strengths, emulating an in vivo condition. VeraVec cells showed robust directional migration in a voltage-dependent manner. We observed significant bias in migration direction toward the anode but did not see any changes in cell migration speed. Next, we developed 3D vascular aggregates using human stem cells and exposed them to DCEF. To quantify the angiogenic effects of the DCEF exposure, after 2 days of 3D culture in the presence of growth factors, we measured and compared the numbers, lengths, and angles of sprouting vessels following tip cells towards both the anode and cathode. Within the 3D assay, we demonstrated collective 3D migration of the stem cell aggregates, as well as directional vessel sprouting towards the anode. Vessel growth not only was directed toward the anode, but cathodal growth was prohibited. We also noted that vessel density was higher on the anodal side of the aggregates, confirming the sprouting bias toward the anode. Our findings support directed endothelial cell migration toward the anode in both 2D and 3D environments. The 3D assay provides a clinically relevant model which emulates in vivo conditions, demonstrating that angiogenesis can be guided and promoted using DC EF. We expect that this novel technique will have distinct advantages over currently available approaches to provide a complementary and powerful technique to control angiogenesis for wound healing due to its non-invasive nature.

DCEF-directed 3D angiogenic sprouting using human stem cell-derived vascular organoids

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