(549a) Flow Induced Vascularization in a Human Pluripotent Stem Cell (hPSC)-Based Islet-like Organoid

Islets of Langerhans are vital in the regulation of blood glucose levels primarily through the production of glucagon by the alpha cells and insulin by the beta cells. When insulin synthesis is hindered, cells aren’t able to take up glucose causing a diabetic state to develop. This can occur from autoimmune beta cell death (type 1 diabetes) or insulin resistance of cells (type 2 diabetes), and both types normally require exogenous insulin replacement. A long-term solution for treatment is islet transplantation, but due to the lack of sufficient donors, this method can’t be used on a large scale. An alternative method would utilize human pluripotent stem cells (hPSCs) to create islet like organoids that can be used for implantation. Organoids are in vitro synthesized tissue that replicates the function and structure of in vivo organs. Primary human islets are composed of endocrine cells, stromal cells, and a dense vascular network. The aim of this project is to reproduce these aspects of the islet to more accurately form islet-like organoids that match of the structure and function of islets, with the hypothesis that the inclusion of the vessel network would aid in function of the endocrine cells through the replication of the system that normally exists in vivo.

Forming the organoid utilized a controlled heterotypic aggregation platform that our lab has developed based on a hydrogel platform that efficiently forms spheroids from the hPSC-derived endocrine cells and endothelial cells. This platform utilizes microwells formed from hydrogels that allows bulk aggregation in a standard 12 well tissue culture plate. With the formation of these spheroids, we were able to achieve intra-organoid vascularization with the inclusion of a stromal cells and adipose microvascular fragments under static culture conditions. The resulting gene expression of maturing pancreatic beta cell markers (NKX, PDX1, and Insulin), an islet specific endothelial gene (API), and an endothelial diaphragm fenestration indicator (PLVAP) increased compared to homotypic aggregates of iPSC-derived islets and the initial microvascular fragments. Additionally, glucose responsiveness in the formed triculture organoids were significantly higher compared to the hPSC-islet monoculture aggregates, with the monoculture doubling in insulin secretion to 0.32 micro-insulin units (uIU) per aggregate with a 13mM increase in glucose and the triculture insulin secretion tripling to 0.58uIU per aggregate under the same conditions. Furthermore, we have introduced the organoids after initial aggregation into a microfluidic device that would allow for shear flow through the organoid to drive angiogenesis. We have developed an alginate encapsulation method in the device that sustained the organoid spherical structure while allowing for shear flow across the organoid, which improved network development. The formed vascularized organoids are very capable as a regenerative therapy for diabetes and for disease modeling in a microphysiological system.