3D bioprinted human vascular organoid sheets promote functional ischemic repair and exhibit adaptive in vivo remodeling

Abstract

Ischemic vascular diseases remain a major clinical challenge, creating a need for engineered vascular tissues that can establish functional vascular networks and promote durable tissue repair. Conventional vascular organoids offer limited control over cellular composition and spatial organization, while the fate and adaptive remodeling of graft-derived human vascular cells after transplantation remain poorly understood. Human vascular organoid sheets (hVOS) were constructed by extrusion-based three-dimensional (3D) bioprinting of human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) and smooth muscle cells (SMCs) at a defined ratio within a gelatin methacryloyl (GelMA)-based bioink under chemically defined conditions. Vascular organization and cellular states were characterized using functional assays, immunofluorescence imaging, and single-cell RNA sequencing (scRNA-seq). Therapeutic efficacy and graft remodeling were evaluated in a murine hindlimb ischemia model using laser speckle perfusion imaging, histological analysis, intravital two-photon imaging, and scRNA-seq of recovered graft-derived human cells. Co-bioprinting ECs and SMCs accelerated vascular network formation and generated stable, interconnected vascular structures that underwent progressive maturation during culture. scRNA-seq identified diverse vascular and stromal populations and revealed transcriptional programs associated with vascular maturation, mechanotransduction, and hypoxic adaptation. Following transplantation, hVOS significantly improved blood perfusion, increased limb salvage, and promoted ischemic tissue repair. Intravital imaging detected circulating dextran within GFP-labeled hVOS-derived vascular structures at days 14 and 28, demonstrating perfusion of graft-derived vascular structures by the host circulation. Post-transplantation scRNA-seq revealed substantial adaptive remodeling of graft-derived ECs toward venous-biased and inflammatory states, accompanied by activation of NF-κB- and stress-associated programs. Transplanted SMCs and fibroblasts also exhibited coordinated transcriptional changes associated with wound healing and extracellular matrix remodeling. hVOS provide a reproducible and design-flexible 3D-bioprinted vascular tissue platform that enables controlled multicellular organization and formation of prevascularized constructs while supporting vascular integration and ischemic tissue repair in vivo. Single-cell analyses further reveal substantial adaptive remodeling of graft-derived vascular and stromal cells following transplantation. These findings support hVOS as a versatile platform for vascular regenerative medicine and for investigating the in vivo behavior of engineered human vascular tissues. Graphical abstract illustrating the generation and therapeutic application of human vascular organoid sheets (hVOS). hPSC-derived endothelial cells (hPSC-ECs) and smooth muscle cells (hPSC-SMCs) are precisely organized by 3D bioprinting to generate reproducible hVOS with enhanced vascular maturation driven by cellular, hypoxic, and biomechanical cues. Following transplantation into ischemic tissue, hVOS integrate with the host vasculature and undergo dynamic endothelial remodeling, enhancing perfusion and promoting tissue repair. Reproducible and Customizable 3D Bioprinted hVOS: hPSC-derived ECs and SMCs self-assembled into stable vascular networks within 3D-bioprinted hVOS, enabling controlled graft fabrication. Enhanced Vascular Maturation: hVOS promoted vascular maturation through coordinated cellular interactions, mechanical support, and hypoxic signaling. Therapeutic Efficacy in Hindlimb Ischemia: Transplantation of 3D-bioprinted hVOS improved blood perfusion and promoted tissue regeneration in a hindlimb ischemia model. In Vivo Reprogrammed of EC Function: Single-cell RNA sequencing revealed that the in vivo microenvironment reshaped EC functions within transplanted hVOS, contributing to vascular repair.