410.9: Expansion and differentiation of human pancreas-derived islet cell precursors into functional islet cell organoids

Background: Emerging technologies in human embryonic stem cell (hES) and inducible pluripotent stem cell (iPSC) differentiation protocols designed to mimic stages of islet cell development have led to promising sources of stem-cell derived β-like cells for potential islet cell transplantation. We recently isolated adult human islet cell precursors (hICPs) from COBE bag remnants obtained during islet cell isolation procedures. We hypothesized that hICPs could be expanded and differentiated into functional islet-like organoids for use in islet cell transplantation. Methods: Adult hICP gene marker expression was compared to stages of hES-derived β- and α-like cells single-cell transcriptome profiles. Proliferation of hICPs was stimulated by cytokines, stressed islet exosomes, and GSK3β/DYRK inhibitors. Small molecule ISX9 was used to stimulate calcineurin (CN)/NFAT signaling and islet cell differentiation. High throughput QPCR arrays measured cell replication and survival genes. QPCR was used to validate and measure expression of cell cycle genes and islet cell differentiation transcription factors. Flow cytometry was used to identify RGS16+ and Nestin+ hICPs and determine β:α-cell ratios in differentiated islet cell organoids. ELISA was used to measure release of insulin and glucagon from islet cell organoids upon high and low glucose stimulation, respectively. Results: Adult hICPs were determined to be equivalent to stages 5-7 of hES-derived pancreatic endocrine precursors by reference to islet progenitor markers RGS16 and Nestin. Treatment of hICPs with exosomes purified from stressed human islet cells induced cell cycle gene expression and expansion of islet cell organoids. Gene profiling analyses of hICPs treated with ISX9, harmine, and GNF4877 promoted NFAT signaling and induction of several cell cycle and anti-apoptotic genes. Stimulated CN/NFAT signaling directly upregulated transcription factors identified to drive efficient islet cell differentiation. Hh/SMO inhibition promoted β-cell differentiation and Hh agonists induced genes to favor α-like cells. Differentiated islet cell organoid products released 16.8 ± 1.7 pg/IEQ/30 min insulin in response to high glucose and 40.5 ±8.5 pg/IEQ/30 min glucagon in response to low glucose which were comparable to those of human islets. Conclusion: These results demonstrate feasibility to selectively target CN/NFAT, AKT/GSK3β, and Hh/SMO signaling components in adult hICPs to induce expansion and differentiation into islet cell organoids with functional β- and α-like phenotypes. Further standardization of protocols to generate functional islet organoids from adult hICPs could provide widespread utility for islet cell replacement strategies for islet transplantation.