Human iPSC-derived mesodermal progenitor cells preserve their vasculogenesis potential after extrusion and form hierarchically organized blood vessels

Abstract

Post-fabrication formation of a proper vasculature remains an unresolved challenge in bioprinting. Established strategies focus on the supply of the fabricated structure with nutrients and oxygen and either rely on the mere formation of a channel system using fugitive inks, or additionally use mature endothelial cells and/or peri-endothelial cells such as smooth muscle cells for the formation of blood vessels in vitro. Functional vessels, however, exhibit a hierarchical organization and multilayered wall structure that is important for their function. Human induced pluripotent stem cell-derived mesodermal progenitor cells (hiMPCs) have been shown to possess the capacity to form blood vessels in vitro, but have so far not been assessed for their applicability in bioprinting processes. Here, we demonstrate that hiMPCs, after formulation into an alginate/collagen type 1 bioink and subsequent extrusion, retain their ability to give rise to the formation of complex vessels that display a hierarchical network in a process that mimicks the embryonic steps of vessel formation by vasculogenesis. Histological evaluations at different time points of extrusion revealed initial formation of spheres, followed by lumen formation and further structural maturation as evidenced by building a multilayered vessel wall and a vascular network. These findings are supported by immunostainings for endothelial and peri-endothelial cell markers as well as electron microscopic analyses at the ultrastructural level. Moreover, capillary-like vessel structures deposited a basement membrane-like matrix structure at the basal side between the vessel wall and the alginate-collagen matrix. These results evidence the applicability and great potential of hiMPCs for the bioprinting of vascular structures mimicking the basic morphogenetic steps of de novo vessel formation during embryogenesis.

Fabrication and Morphological Control of Nonspherical Alginate Hydrogel Particles

“…The optical microscopy images show the morphologies in terms of width, length, and spread area of the Y1 and HT-22 cells on the surfaces of pure CA and hybrid A 1 :G 0.5 hydrogel particles (Figure b). We observed that the cells clustered and formed 3D cell spheres on the CA hydrogel particle surfaces (Cultures 1 and 2) due to insufficient cell–matrix interactions, similar to previously reported works. ,, The size of the cell spheres increased with increasing culture days. As a biomaterial, alginate hydrogel is mechanically tough but not adhesive enough to provide cell attachment. ,, Cells cultured on the surfaces of hybrid A 1 :G 0.5 hydrogel particles exhibited spreading (Culture 3) because of the cell-adhesive property of Gel (Gel possesses the RGD sequence of collagen). ,− Immediately after the culture, both the cells on the hybrid A 1 :G 0.5 hydrogel particle surfaces showed spherical morphology (as also observed on CA hydrogel particle surfaces).…”

“…We observed that the cells clustered and formed 3D cell spheres on the CA hydrogel particle surfaces (Cultures 1 and 2) due to insufficient cell−matrix interactions, similar to previously reported works. 20,67,68 The size of the cell spheres increased with increasing culture days. As a biomaterial, alginate hydrogel is mechanically tough but not adhesive enough to provide cell attachment.…”

“…As a biomaterial, alginate hydrogel is mechanically tough but not adhesive enough to provide cell attachment. 20,67,68 Cells cultured on the surfaces of hybrid A 1 :G 0.5 hydrogel particles exhibited spreading (Culture 3) because of the cell-adhesive property of Gel (Gel possesses the RGD sequence of collagen). 20,69−71 Immediately after the culture, both the cells on the hybrid A 1 :G 0.5 hydrogel particle surfaces showed spherical morphology (as also observed on CA hydrogel particle surfaces).…”

Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling

“…Previous studies reported that the presence of mesodermal progenitor [ 66 – 68 ] or pericytes [ 54 , 55 ] is critical for vasculature formation in various organoids. In agreement with these findings, we found that hPSC-derived HscLCs were crucial to the development of functional microvasculature in mLOs.…”

Stratified tissue biofabrication by rotational internal flow layer engineering

“…This is due to a combination of low viscosity and extended thermal gelation time 13,16 , ultimately requiring a high level of self-support and temperature control when transitioning from a liquid to a gel 56,57 a challenge prevalent throughout biofabrication, with technologies often requiring specific bioink hydrogel formulations. Collagen itself is often combined with other hydrogels such as alginate 58 to enable bioprinting. The RIFLE system circumvents this problem for stratified tissue with the stable suspension of the liquid phase hydrogel in the desired microscale spatial constraint prior to application of the gelation reagent or process.…”