Physical and Biochemical Determinants of Endocrine Fate in 3D Suspension Culture Based Stem Cell-Derived β-Cell Differentiation.

Efficient and reproducible generation of functional stem cell-derived β cells (sBC) remains a major challenge for basic research and cell replacement therapy, partly due to incomplete understanding of endocrine induction during differentiation and challenges with clinical scale-up. Here, we dissect the individual contributions of commonly used endocrine differentiation factors on pancreatic progenitor maintenance, endocrine commitment, and hormone subset generation in a scalable 3D differentiation system. We demonstrated that starting pluripotent stem cell cluster size is a critical determinant for downstream sBC generation. We also verify that a commonly employed combination of endocrine induction molecules efficiently drives endocrine lineage commitment but yields limited β-cell generation. Detailed analysis of the effects of individual endocrine induction molecules revealed distinct effects: EGF or KGF preserved NKX6.1+ progenitors without induction of endocrine differentiation; Notch or BMP inhibition robustly induced endocrine marker expression but concurrently reduced NKX6.1 expression, resulting in predominant generation of glucagon-expressing cells; retinoic acid, thyroid hormone (T3), or TGFβ inhibition maintained high NKX6.1 levels while also promoting efficient insulin+ endocrine differentiation. These findings indicate NKX6.1 protein maintenance as a key determinant of human β-cell generation and show that endocrine differentiation factors exert divergent effects on lineage progression.