(F1060) A PROTOCOL FOR THE GENERATION OF FUNCTIONAL PANCREATIC β-CELL BY COMBINING SCFA SUPPLEMENTATION AND METABOLIC REGULATION THROUGH METHIONINE DEPRIVAL
Graduate Student Institute of Science Tokyo, Tokyo, Japan
Abstract: Transplantation of stem cell-derived β-cells (SC-β) presents a promising approach for treating diabetes, relying on high differentiation efficiency and robust functional maturity. Our previous research demonstrated that short-term deprival of reducing methionine and zinc before differentiation enhances pancreatic differentiation efficiency (Cell Reports, 2022; Star Protocols, 2023). Based on these findings, we developed an improved protocol that combines methionine modulation with supplementation of short-chain fatty acids (SCFAs), metabolites of intestinal microbiota that circulate systemically, to further enhance glucose-stimulated insulin secretion (GSIS) in SC-β cells. In this study, we evaluated the effects of SCFAs—specifically, acetic acid, propionic acid, and butyric acid—on SC-β differentiation during key stages, including endoderm formation (Stage 1), pancreatic progenitor differentiation (Stage 4), and endocrine maturation (Stage 6). We found that the addition of butyrate during Stage 1 inhibited endoderm differentiation and led to cell death. Moreover, its addition at Stage 4 allowed the cells to differentiate into β-cells, leading to a loss of glucose responsiveness. Notably, butyrate supplementation during Stage 6 substantially enhanced GSIS without compromising differentiation efficiency. In contrast, acetic and propionic acids did not demonstrate any beneficial effects on GSIS at Stage 6. RNA sequencing analysis revealed that butyrate addition during Stage 6 altered the expression of key genes associated with β-cell function and maturation. These findings highlight the synergistic potential of methionine modulation and SCFA supplementation in promoting the functional maturation of SC-β cells, providing a solid foundation for advancing stem cell-based therapies for diabetes.
