Ex vivo platelet production from iPS cells offers a promising solution to overcome the limitations of donor-derived platelet transfusions, including short shelf life, supply shortages, and alloimmune complications. While researchers in the Eto Laboratory have previously developed immortalized megakaryocyte progenitor cell lines (imMKCLs) capable of producing functional iPS cell-derived platelets (iPSC-PLTs) at a clinical scale, cellular heterogeneity and senescence within imMKCLs remain major obstacles to consistent and high-yield manufacturing.

In this study, the researchers identified LIN28A as a key upstream regulator of the let-7-RALB axis. Overexpression of LIN28A suppressed let-7a-5p, increased RALB expression, and activated immune-related signaling, leading to reduced proliferation and impaired platelet production. DNA methylation analysis revealed that LIN28A expression is epigenetically regulated via differential methylation of CpG islands, with hypomethylation correlating with higher LIN28A levels in cells with low let-7 activity.

The team performed motif enrichment analysis and siRNA-mediated knockdown experiments to identify transcriptional regulators of LIN28A, with STAT1 emerging as a critical upstream factor. Knockdown of STAT1 reduced LIN28A expression, restored let-7a-5p levels, and significantly enhanced iPSC-PLT production under both static and turbulent flow conditions. Importantly, platelet functionality remained intact, and markers of cellular senescence, including CDKN2A and IL-8, were suppressed.

Further experiments demonstrated that pharmacological inhibition of STAT1 phosphorylation using fludarabine and flavopiridol also improved platelet yield, particularly under turbulent flow. While flavopiridol aligned with the expected molecular changes--upregulating let-7a-5p and downregulating immune genes--fludarabine appeared to act via an alternative mechanism. These findings suggest that STAT1 activity, especially its phosphorylation state, plays a pivotal role in regulating the LIN28A-let-7-RALB axis and the senescence phenotype of imMKCLs.

This study establishes STAT1 as a central regulator of platelet biogenesis from iPS cells and highlights its role in modulating immune signaling and cellular aging. By targeting STAT1, either genetically or pharmacologically, the researchers offer a new strategy to optimize ex vivo platelet production and improve the quality and consistency of iPS cell-derived platelet products. These insights pave the way for more efficient and scalable manufacturing systems, with potential applications in transfusion medicine and regenerative therapies.

• doi: 10.1182/bloodadvances.2024015557