Thursday, May 2, 2024
5/2/2024
SUMMARY Human pancreatic islets are the key regulator of whole-body lipid and glucose homeostasis. The dysfunction of hormone secretion from islets, and/or loss of ß cell mass is an important part of the pathogenesis of diabetes, often requiring insulin or islet replacement. Human induced pluripotent stem cells (hiPSCs) provide a potential alternative source to cadaveric human pancreatic islets, but the generated islets from hiPSCs in vitro commonly shows functional heterogeneity (batch-to-batch difference) and limited scalability. In addition, even with autologous delivery strategies, hiPSC derived islets (hiPSC-islets) still require life-long immune suppression because of a hyper-active immune reaction especially in type 1 diabetes (T1D) patients. Therefore, universal human PSCs that evade immune detection constitute a major goal of translational research in T1D research. Our research proposal focuses on investigating methods for the functional selection, mass production, and immune protection of hiPSC-islets. The rationale is that Identifying such a pathway is necessary for large- scale islet cell therapy for diabetes. Our preliminary results revealed that 1) the mineral absorption FXYD2 pathway is a novel hallmark of functional heterogeneity of hiPSC-islets. 2) Newly developed giant islet (GiSLETs) technology provides better scalability than traditional hiPSC-islets. 3) PD-L1 provides immune evasive function in hiPSC-islets. We hypothesize that FXYD2 enriched GiSLETs with PD-L1 expression provides long-term viability and functionality in immune competent diabetic condition. This hypothesis will be tested with the following specific aims. Aim 1: Identification of FXYD2 as functional stem cell derived ß cell marker. Using a newly developed three-dimensional protocol to mimic human islet organogenesis, we will test the role of FXYD2 in islet functionality, a core mineral absorption pathway using genetic and physiological approaches. Aim 2: Explore the in vivo efficacy of FXYD2 enriched GiSLETs. We hypothesized that FXYD2 can be selection marker of functional GiSLETs. We will isolate FXYD2 enriched GiSLETs (fGiSLETs) by FXYD2 promoter driven reporter system and will explore the efficacy for glycemic control of fGiSLETs in T1D model mice. Aim 3: Explore the long-term efficacy of immune evasive GiSLETs in allogenic and autoimmune reaction. To investigate whether overexpression of PD-L1 with HLA-A&B deletion will limit the immune response of fGiSLETs upon transplantation and prolong survival in human patients, we will use humanized CD34+ mice (CD34+-NGS-SGM3, Jackson Laboratory) as an in vivo graft rejection model. The significance of the proposed study is in its focus on the mechanisms involved in human islet maturation, tissue engineering of practical scaling of functional insulin producing cells by GiSLETs. The success of this study will enable us to create more robust, reproducible, and functional multi-cellular islet-organoids from hiPSCs. This relies on our innovative concepts of functional selection, mass production and immune protection for cell therapy in diabetes.
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