Heterogeneity in cell-cycle dynamics of synthetic mRNA-induced β-cell proliferation

Adult pancreatic β-cells are mostly locked in quiescence, limiting large-scale analysis of their cell cycle. To overcome this limitation, we used synthetic in vitro transcribed mRNAs encoding Cyclin D1 and CDK4 to induce proliferation in up to 70% of primary rat β-cells. Flow cytometry-based analysis of cell-cycle markers revealed substantial heterogeneity in β-cell cell-cycle progression, identifying five distinct groups of proliferating β-cells based on G1 entry timing. Total cell-cycle length ranged from approximately 26 to 34 hours and was primarily determined by variability in G1 duration (13–19 hours). In the fastest-dividing β-cells, G1, S, and G2M lasted approximately 13, 6, and 7 hours, respectively, whereas later-entering β-cells exhibited progressively longer G1 phase. Additionally, we identified a small β-cell subpopulation that failed to complete division and may have exited the cell cycle. These findings reveal cell-cycle entry and G1 progression as a major source of heterogeneity in β-cell proliferation and provide quantitative benchmark for developing strategies to enhance controlled β-cell regeneration in diabetes.