Introduction and Objective: Human pluripotent stem cell-derived pancreatic β-like cells (hPSC-β cells) represent a promising cell source for diabetes therapy. However, their clinical translation is hampered by time- and cost-intensive differentiation protocols. Establishing a scalable expansion system for hPSC-derived pancreatic endoderm (progenitor) cells (hPSC-PEs) would enable accessible regenerative therapies. Methods: To identify the mechanisms limiting their expansion, we performed single-nucleus multi-omics analysis, machine learning approach, and chemical compound screening for hPSC-PEs. Results: The expansion of hPSC-PEs is restricted by cellular senescence and epithelial-mesenchymal transition (EMT) driven by a mechanotransduction-dependent canonical TGF-β pathway. We identified GRHL2 as a critical gatekeeper at the bifurcation between senescence and EMT, and PRDM16 as a direct transcriptional activator of the pancreatic progenitor determinants PDX1 and NKX6-1. By preventing senescence and EMT using a chemically defined cocktail, we achieved long-term expansion of hPSC-PEs for over one year under feeder-free conditions while preserving their capacity to differentiate into hPSC-β cells. Conclusion: These findings elucidate fundamental mechanisms constraining progenitor cell expansion and establish a robust expansion system, thereby paving the way for scalable transplantation therapies. Disclosure Y. Hatano: None. R. Ito: None. A. Kimura: None. K. Aihara: None. H. Tsujimoto: Employee; Current; Rege Nephro Co., Ltd. R. Hirayama: Employee; Current; RegeNephro. K. Osafune: Advisory Panel; Current; iPS Portal, Inc. Stock/Shareholder; Current; iPS Portal, Inc. Board Member; Current; RegeNephro Co., Ltd. Stock/Shareholder; Current; RegeNephro Co., Ltd. Advisory Panel; Current; RegeNephro Co., Ltd.
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