Introduction and Objective: Despite recent improvements in generating functional human insulin-producing β cells, stem cell-derived beta-like cells (sBC) still develop and function less efficiently than bona fide β cells from cadaveric islets. Therefore, we sought to identify key factors affecting efficient sBC generation during differentiation, with a focus on reducing time for sBC generation while improving efficiency and function.Methods: We tested multiple endocrine induction and maturation parameters compared to previous methodology controls during differentiation, evaluating phenotypic and function via proteomic analyses and functional assessment in vitro and in vivo.Results: We demonstrate that currently employed endocrine induction cocktails provide opposing signals for cell specification resulting in incomplete sBC induction despite efficient endocrine commitment (Fig. 1A-B). We show that continuous TGFβ inhibition in the presence of thyroid hormone T3 increases sBC yield and function, partly by inducing a beta-like phenotype in non-β endocrine cells (Fig.1C-E). Additional experiments revealed a novel combination of small molecules, including HDAC class II inhibition, that further enhances functional sBC generation with significantly increased in vivo performance (Fig. 1F-K).Conclusion: These data provide a detailed framework for the improved generation of functional human β cells for cell therapy and biological research.
A.T. Kratz: None. H. Russ: Other – Advisory panel and consultant, stock holder; Current; Sernova, Corp. Other – Scientific founder, Consultant, Advisory panel, stock holder; Current; Tolerance Bio. Consultant; Current; Guidepoint consulting, Axxon Advisors.
The work in the Russ lab is or was supported by R01DK120444, R01DK132387 and R01NS122911, a new investigator award from NIDDK/HIRN RRID: SCR_014393; UC24 DK104162, and SRA grants 2-SRA-2023-1313-S-B and 3-SRA-2023-1367-S-B by Breakthrough T1D (BT1D).ATK is supported by the NIH NIDDK T32 Training Program in Type 1 Diabetes and Biomedical Engineering T32 DK10876.
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