PF455 HETEROGENEITY IN EPIGENETICALLY REGULATED LIN28A‐LET‐7A‐5P‐ROS AXIS LEADS TO HETEROGENEITY IN MEGAKARYOCYTE MATURATION

Abstract

Background:We have developed a robust ex vivo platelet like particle (PLP) generation system using self‐renewing megakaryocyte (MK) cell lines, imMKCLs, which was established by introducing three doxycycline (DOX)‐inducible transgenes, c‐MYC, BMI1, and BCL‐XL, into human induced pluripotent stem cell (iPSC)‐derived hematopoietic progenitor cells (HPCs) for extensive proliferation (Nakamura et al. Cell Stem Cell 2014). This system is able to simply switch cell stages, since imMKCLs proliferate in the presence of DOX, and when DOX is removed, imMKCLs enlarge, form proplatelets and release PLPs. Recently we reported turbulence could dramatically improve the efficiency of PLP production from imMKCLs (Ito et al. Cell 2018) and could manufacture adequate numbers of PLPs for each transfusion to patients. However, although imMKCLs display homogenous appearance at their proliferation stage, only a minor population eventually produces platelets at a level comparable to MKs in vivo.Aims:Elucidating the mechanism of the heterogeneity of imMKCL maturation and identifying a subpopulation of imMKCLs with high capacity to produce PLPs upon maturation.Methods:We applied miRNA switch technology, which could be used for detecting cell population subsets based on endogenous miRNA activity (Miki et al. Cell Stem Cell 2015), to imMKCLs to identify a crucial miRNA in the their maturation and further elucidated the mechanism behind.Results:We noticed that Let‐7a‐5p shows a distinguishable pattern with about 10% of the cells in the lower Let‐7a‐5p activity group (Group L), while other large majority in the higher activity group (Group H) already at the DOX present proliferation stage. Group L population revealed to produce significantly more PLPs than Group H population after DOX depletion. We then identified LIN28A, not LIN28B, was the upstream regulator of Let‐7a‐5p and its expression level was about 10‐fold higher in Group L than Group H. We also examined HPCs differentiated from ES cells and confirmed there was heterogeneity in LIN28A expression.Next we explored how the expression of LIN28A is regulated in imMKCLs. The CpG islands in LIN28A promoter region were significantly methylated in Group H cells and administration of pan histone deacetylase inhibitor, trichostatin A, upregulated the expression of LIN28A, suggesting LIN28A was regulated by the methylation of its promoter region in imMKCLs.Finally, we investigated the downstream target of LIN28A‐Let‐7a‐5p in MK maturation and PLPs production. LIN28A is involved in cell metabolisms and ROS generation (Shyh‐Chang et al. Cell 2013), which is also reported to be important for MK maturation (Motohashi et al. Blood 2010). In imMKCLs, Group L cells were found to produce more ROS than Group H cells using Flow Cytometry (MFI average 7926 vs 5334). By contrast, the expression of anti‐oxidant genes NFE2L2 and NQO1, which prevent ROS accumulation, was downregulated in Group L according to RT‐PCR.Summary/Conclusion:We clarified that the heterogeneity of imMKCL maturation is caused by the heterogeneity in LIN28A‐Let‐7a‐5p expression, which is already seemingly determined in HPC stage. Epigenetic regulation of LIN28A affects the expression of Let‐7a‐5p and the further downstream ROS, and eventually PLP generation in imMKCLs. By manipulating this pathway, we could resolve the heterogeneity of imMKCL maturation and thereby dramatically improve PLPs production ability leading to realize the commercialization of iPSC‐derived platelet products.