STAT1-mediated epigenetic regulation of LIN28A controls iPSC-derived platelet production through the let-7-RALB axis

Ex vivo platelet (PLT) production from induced pluripotent stem cells (iPSCs) represents a potential solution to address the limitations of donor-dependent PLT transfusion therapy. Although our established immortalized megakaryocyte progenitor cell lines (imMKCLs) from iPSCs enable large-scale production of functional iPSC-derived PLT products, cellular heterogeneity and senescence remain significant challenges for robust industrial-scale manufacturing. We recently identified RAS-like proto-oncogene B as a key regulator of immune properties and PLT productivity of imMKCLs, acting downstream of the let-7a-5p microRNA. This study aims to identify the upstream regulators of let-7a-5p in this context. Herein, we demonstrate that the expression of Lin-28 homolog A (LIN28A), which negatively regulates let-7a-5p, is controlled in imMKCLs through DNA methylation-dependent mechanisms. Analysis of the LIN28A locus revealed distinct methylation patterns between let-7high and let-7low populations within an intronic CpG island. Overexpression of LIN28A upregulated immune-related signaling and diminished PLT production from imMKCLs. We further examined for transcriptional regulators by motif enrichment analysis and small interfering RNA-mediated knockdown, identifying STAT1 as an upstream regulator of LIN28A. Knockdown of STAT1 led to the suppression of immune-related gene expression, resulting in increased PLT production. Inhibition of STAT1 phosphorylation with fludarabine and flavopiridol enhanced PLT generation, uncovering a novel role in PLT generation beyond their established functions in cell cycle arrest and apoptosis. In conclusion, our findings unveil the modulating roles of immune and senescence signaling in imMKCLs to optimize cell and culture conditions for large-scale PLT manufacturing.