(549b) Pluripotent Stem Cell-Derived Organoids to Model Fetal Pancreatic Development and Maturation

Type 1 diabetes mellitus is characterized by the autoimmune destruction of insulin-producing β-cells within the pancreatic islets and it affects millions of people around the globe. Human pluripotent stem cells (hPSCs) are an intriguing option to replace the lost β-cells in patients and are currently being used in clinical studies that employ encapsulation to protect the cells from the immune system. Approaches for β-cell replacement therapies follow either hPSC differentiation in vitro to mature, functional β-cells, followed by in vivo transplantation, or hPSC differentiation in vitro towards pancreatic progenitors, which are transplanted for in vivo maturation. However, complete maturity of the hPSC-derived β-cells in vitro has been difficult to achieve. Therefore, we hypothesize that monolayer induction of pancreatic progenitors from hPSCs, followed by organoid maturation will enhance both maturity and the number of derived β-cells. Organoids provide the three-dimensional environment and signaling that progenitor cells experience during development. Thus, we suggest that this cultivation model could be used to study the signals and pathways involved in β-cell differentiation and maturation. Utilizing culture conditions such as low oxygen (5%), which better mimics physiological oxygen levels available during progenitor development, we have derived a pancreatic progenitor population from hPSCs that expresses high levels of pancreatic and duodenal homeobox 1 (Pdx1) genes, and demonstrate increases in pancreatic progenitor specific markers neurogenin 3 (Ngn3) and NK2 homeobox 2 (Nkx2.2) gene expression over 11 days of monolayer culture, signifying endocrine commitment of pancreatic progenitors. We are further characterizing protein expression in this population with immunostaining, western blot, and functional glucose tolerance tests. We also hypothesize that creating organoids in the form of hollow cysts will provide the best platform to model pancreatic development. This structure most closely recapitulates the structures formed during pancreatic development and has not been very well explored as a pancreatic organoid model. During development, the pancreas forms a hollow branch-like structure where endocrine cells migrate from the epithelial layer and aggregate to form the fetal islets. Therefore, we are devising methods, which include the addition of growth factor-reduced matrigel, soluble factors, and other cell types; to form cyst-like organoids from our D11 cell population with the goal of mimicking this branched structure. Preliminary organoid models, embedded in matrigel, developed both cyst-like and solid structures in culture, which we believe develop from differing cell types within our D11 population; however, the organoids have shown elevated expression of non-pancreatic lineage markers. Yet, these organoids could serve as a promising system to model pancreatic development once conditions and day of culture have been optimized. On the other hand, organoids formed in suspension culture have shown elevated levels of Nkx6.1 and insulin, both expressed by the pancreatic β-cells. Our future work will be to understand how modeling fetal states can enhance organoid maturation and evaluate the function of these organoids with in vitro and in vivo models of diabetes