A Novel Megakaryocyte-Based Screening System Identifies Compounds That Promote Megakaryocyte Maturation and Platelet Production in Vitro

Platelets are anucleate cytoplasmic fragments derived from megakaryocytes (MKs) that play crucial roles in blood clotting system and innate immune response. The clinical needs of platelet transfusion are extensive and increasing, while current donor-derived platelet supply confronts challenges including limited donors, short shelf life of platelet products, the risk of bacterial and viral contamination and transfusion refractoriness. The construction of a non-donor-dependent system for efficient platelet generation to provide a consistent supply of platelets could address these concerns. Therefore, in vitro differentiation of human pluripotent stem cells (PSCs) have been studied to serve as a potential source of MKs and platelets to meet the demands of clinical transfusion. The immortalized megakaryocyte cell lines (imMKCLs) were previously established as cryopreservable master cells that can be robustly expanded and then produce platelets upon maturation, but the yield of platelets was far too short from that postulated in vivo (Nakamura, 2014). To overcome this issue, we sought to establish a reporter system that can monitor imMKCL maturation in vitro in high throughput manner to identify compounds that improve their maturation and platelet release.

Herein we generated TUBB1-VENUS reporter imMKCLs with a VENUS transgene integrated into the β1 tubulin (TUBB1) locus using CRISPR/Cas9 system. The expression of the cytoskeleton molecule TUBB1 has been identified to be restricted in MK/platelet lineage, and it is indispensable for platelet formation and function. The maturation of MKs accompanies the increased expression of TUBB1 along with crucial transcription factors including GATA1, FOG1 and NF-E2.

With this reporter line, the maturation of imMKCL is indicated by the fluorescence intensity of VENUS. Using this reporter imMKCL system for drug screening, we identified several compounds from a commercial drug library on facilitating platelet production efficiency. Interestingly, despite diverse categories (i.e. a Wnt inhibitor and a FLT3 inhibitor) of compounds were included, most drugs consistently induced significant suppression of the mRNA expression of CYP1B1, a typical downstream target of aryl-hydrocarbon receptor (AhR) pathway. Indeed, such molecules revealed an antagonizing action to aryl-hydrocarbon receptor (AhR) by pharmacological competitive assay using FICZ, an agonist to AhR. However, overexpression or knockdown of CYP1B1 did not directly affect platelet biogenesis in the presence of candidate drugs. These findings suggested that major MK maturation signaling is mediated by inhibition of AhR but independently of CYP1B1, a conventional AhR component.

The imMKCL reporter system identified several drugs that could contribute to ex vivo production of platelets through inhibition of AHR signaling. The system could also be useful as an experimental tool for studying MK maturation machinery.