Engineering islet-like organoids from gastric stem cells for T1D cell replacement therapy

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PROJECT SUMMARY Islet transplantation offers a potential cure for Type 1 diabetes (T1D). Wide adoption of a cell therapy requires abundant islet supplies and effective immune protection without long-term systemic immune suppression. My laboratory and others showed that it was feasible to derive insulin-secreting cells from renewable and abundant gastrointestinal (GI) stem cells. 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 samples and expanded them to billions. The hGSCs were engineered for transient activation of NGN3 and stable expression of PDX1 and MAFA (collectively referred to as NPM factors), leading to formation of thousands of GINS (Gastric Insulin Secreting) organoids. 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 with a scalable derivation method. GINS organoids contain 25-30% of cells that closely resemble pancreatic Beta-cells. In this project, we will evaluate the hypothesis that clonal hGSC lines yielding a higher percentage of Beta-like cells can be readily identified from donor tissues. We will develop a standard derivation protocol using genetic knockin of NPM and clonal selection with the aim to consistently produce highly functional GINS grafts from donors. GINS cells lack key autoantigens and may be naturally less immunogenic than islet Beta-cells. We will confer further autoimmune protection by constitutive expression of two potent immune regulators PD-L1 an CD47. Normal and immune-evasive organoids will be evaluated in vitro and in vivo with a panel of antigen-specific cytotoxic CD8 T cells, the main effector of Beta-cell demise in T1D. Together, these studies will create a technology for reliable production of autologous GINS grafts with strong autoimmune protection, suitable for long-term glycemic control without immune isolation or suppression.