Objective: We compared the capability of human umbilical vein endothelial cells (HUVECs), induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs), and human dermal blood endothelial cells (HDBECs) to effectively vascularize engineered human skin constructs (HSCs) in vitro and on immunodeficient mice. Approach: We quantified the angiogenesis within HSCs both in vitro and in vivo through computational analyses of immunofluorescent (IF) staining. We assayed with real-time quantitative PCR (RT-qPCR) the expression of key endothelial, dermal, and epidermal genes in 2D culture and HSCs. Epidermal integrity and proliferation were also evaluated through haematoxylin and eosin staining, and IF staining. Results: IF confirmed iEC commitment to endothelial phenotype. RT-qPCR showed HUVECs and iECs immaturity compared with HDBECs. In vitro, the vascular network extension was comparable for HDBECs and HUVECs despite differences in vascular diameter, whereas iECs formed unorganized rudimentary tubular structures. In vivo, all ECs produced discrete vascular networks of varying dimensions. HUVECs and HDBECs maintained a higher proliferation of basal keratinocytes. HDBECs had the best impact on extracellular matrix expression, and epidermal proliferation and differentiation. Innovation: To our knowledge, this study represents the first direct and quantitative comparison of HDBECs, HUVECs, and iECs angiogenic performance in HSCs. Conclusions: Our data indicate that HUVECs and iECs can be an alternative cell source to HDBEC to promote the short-term viability of prevascularized engineered grafts. Nevertheless, HDBECs maintain their capillary identity and outperform other EC types in promoting the maturation of the dermis and epidermis. These intrinsic characteristics of HDBECs may influence the long-term function of skin grafts.
Keywords: endothelial cell heterogeneity; engineered skin; iEC; skin graft; skin-specific vasculature; vascularized skin.