Derivation of pancreatic islet-like organoids from human gastric stem cells

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PROJECT SUMMARY Islet transplantation offers a potential cure for Type 1 diabetes (T1D). Wide adoption of this promising therapy requires abundant islet supplies and effective immune protection. My laboratory and others showed that it was feasible to derive insulin-secreting cells from gastrointestinal (GI) tissues. However, it has not been possible to mass-produce islet-like organoids from human GI tissues for detailed assessment of their translational potential. In preliminary studies, we established methods to culture human gastric stem cells (hGSCs) from biopsy or autopsy samples that can be expanded to billions. We developed a scalable 2-step method to produce thousands of GINS (Gastric Insulin Secreting) organoids by transient activation of NGN3 and stable expression of PDX1 and MAFA (collectively referred to as NPM factors). GINS organoids acquired glucose-stimulated-insulin-secretion (GSIS) within 10 days, and upon transplantation, rapidly reversed diabetes in mice and maintained normoglycemia for over 3 months, with no tumor formation. Human GINS organoids thus have favorable attributes as a potential cell product for T1D treatment. GINS organoids contain 25% of cells that closely resemble pancreatic β-cells but a paucity of GCG+ and SST+ cells. Human islets have 50-75% β-cells, 25-35% α-cells, and 5% δ-cells. Both α- and δ- cells exert paracrine effects on β-cell section. In this project, we aim to develop new clonal hGSC lines and novel nanoparticle-based mRNA transduction method suitable for mass production of organoids that closely mimic human islets in cell composition and function. These studies are based on preliminary data indicating that hGSC clonal lines are markedly different, with some predominantly producing β-like or α-/δ-like cells. Their differentiated progenies can thus be combined to yield islet- like organoids. We will further study the clonal lines for chromatin features and PDX1/MAFA genomic binding to gain mechanistic insight in GINS formation. Together, these studies constitute a major step in advancing the long-term goal of developing GINS organoids for T1D treatment.