Introduction: Platelet transfusion is a standard treatment for thrombocytopenia but is limited by donor shortages, contamination risks, and transfusion refractoriness. Ex vivo production of platelets from induced pluripotent stem cells (iPSCs) represents a promising alternative. We previously established immortalized megakaryocyte progenitor cell lines (imMKCLs), enabling clinical-scale production of iPSC-derived platelets (iPSC-PLTs) in a turbulent flow-based bioreactor (Ito et al. Cell, 2018), leading to the first-in-human clinical study of autologous iPSC-PLTs transfusion (Sugimoto et al. Blood, 2022). However, cellular heterogeneity in imMKCLs continues to hinder consistent and efficient iPSC-PLT production. To address this, we applied microRNA switch technology and unexpectedly identified an immune-biased subpopulation in imMCKLs characterized by reduced let-7 activity. This subset exhibited activated immune-related signaling and exerted a dominant negative effect, arresting proliferation and impairing iPSC-PLT yield. Further study revealed RAS-like proto-oncogene B (RALB) as a key modulator of immune phenotype and iPSC-PLT productivity in imMKCLs (Chen et al. Nature Communications, 2024). In this study, we investigate the upstream epigenetic and transcriptional regulation of the let-7–RALB axis to better understand and improve iPSC-PLT production.

Methods: We employed DNA methylation (bisulfite) analysis, transcription factor motif analysis, and siRNA-mediated gene knockdown to identify and validate upstream regulatory factors affecting let-7-RALB axis and final iPSC-PLT production.

Results: According to bulk RNA-seq and bisulfite sequencing analysis, we identified Lin-28 homolog A (LIN28A) as a negative regulator of let-7a-5p in imMKCLs, with its expression governed by DNA methylation. Notably, differential methylation patterns distinguished imMKCL subpopulations with high versus low let-7a-5p activities. Lentiviral-mediated LIN28A overexpression activated immune-related pathways and suppressed iPSC-PLT production. To uncover upstream regulators of LIN28A, we performed transcription factor motif analysis and siRNA-mediated knockdown, which identified signal transducer and activator of transcription 1 (STAT1) as a key regulator. STAT1 knockdown downregulated immune-related signaling, decreased he expression of CDKN2A and secretion of interleukin-8 (IL-8), and significantly enhanced iPSC-PLT yields. Notably, pharmacologic inhibition of STAT1 phosphorylation using flavopiridol or fludarabine boosted iPSC-PLT generation, further supporting its regulatory role.

Conclusion: Our findings define a novel STAT1-LIN28A-let-7a-5p-RALB axis that links immune signaling to platelet production in imMKCLs. This pathway represents a potential target for optimizing iPSC-PLT yields and quality through tuning immune signaling. These insights have critical implications for improving and standardizing the industrial-scale manufacturing of iPSC-PLTs for clinical application.