Endothelial Cells Differentiated from Human Induced Pluripotent Stem Cells Form Aligned Network Structures in Engineered Neural Tissue

Background/Objectives: Endothelial cells play a key role in peripheral nerve regeneration, forming aligned vasculature which bridges the gap in the injured nerve tissue and guides the regrowing tissue. This work aimed to mimic key features of this aligned vasculature by differentiating endothelial cells from human induced pluripotent stem cells (hiPSCs) and incorporating them into engineered neural tissue (EngNT). Methods: hiPSCs were differentiated into endothelial cells with the temporal addition of growth factors and biomolecules. These hiPSC-derived endothelial cells (hiPSC-ECs) were incorporated into EngNT fabricated from collagen hydrogels using the gel aspiration-ejection (GAE) technique and maintained in vitro to allow endothelial network formation. Results: At the mRNA and protein level, pluripotency marker expression decreased and endothelial cell marker expression increased over the course of hiPSC differentiation to endothelial cells. The derived endothelial cells expressed CD31, CD144, ENG, VEGFR2, and VWF, and formed network structures in the matrix tubulogenesis assay. hiPSC-ECs incorporated into EngNT were viable and aligned. They formed highly aligned tube-like structures containing lumens after four days in culture and the EngNT constructs supported neurite growth in vitro when co-cultured with rat dorsal root ganglion (DRG) neurons. Conclusions: This work rapidly generated engineered nerve tissue containing highly aligned endothelial tube-like structures, resembling key features of the early nerve regeneration bridge. Therefore, this 3D engineered tissue provides a platform to study the effects of endothelial cell structures in nerve repair treatment and translational development.