A β1-tubulin–based megakaryocyte maturation reporter system identifies novel drugs that promote platelet production

Abstract

During maturation, megakaryocytes (MKs) express β1-tubulin (TUBB1) and rearrange their microtubule components to enlarge, form proplatelets, and eventually release platelets. The development of a platform to identify in vitro conditions that would efficiently promote MK development could potentially enable large-scale platelet production. Here, we show that an immortalized MK cell line (imMKCL) genetically modified to express the β1-tubulin-Venus reporter provides a practical system to efficiently monitor the in vitro production of platelet-like particles (PLPs). The Venus transgene was inserted downstream of the locus in imMKCLs using CRISPR/Cas9, and the expression was visualized by Venus fluorescence intensity. This imMKCL reporter line was then used for high-throughput drug screening. We identified several compounds that significantly improved the efficiency of PLP production in vitro under feeder-free conditions and showed a significant tendency to recover platelets in vivo in a mouse thrombocytopenia model induced by anti-GPIbα antibody administration. Interestingly, most of these compounds, including a WNT signaling pathway inhibitor, Wnt-C59, antagonized the aryl hydrocarbon receptor (AhR) to increase PLP production, confirming the crucial role of AhR inhibition in MK maturation. Consistently, small interfering RNA treatment against AhR increased the Venus intensity and PLP production. TCS 359, an FLT3 inhibitor, significantly increased PLP production independently of FLT3 or AhR. This study highlights the usefulness of the β1-tubulin reporter MK line as a useful tool to study the mechanisms underlying thrombopoiesis and to identify novel inducers of ex vivo platelet production.

Good Manufacturing Practice‐Grade of Megakaryocytes Produced by a Novel Ex Vivo Culturing Platform

“…Previous studies demonstrated that SR1 promoted MK differentiation and maturation in addition to its effect on HSC expansion. 22,23 On the other hand, little is known about the effect of VPA and C433 on megakaryopoiesis. We then focused on the assessment of VPA and C433 on differentiating SMC-expanded CD34 + cells toward the megakaryocytic lineage.…”

Megakaryocytic Expansion in Gilteritinib-Treated Acute Myeloid Leukemia Patients Is Associated With AXL Inhibition

“…Lin − Sca1 + kit + Flt3 + HSCs are highly proliferative and show lymphoid or myeloid differentiation potential, but lack E-Mk potential. Treatment with Flt3 inhibitor also promotes significantly increased platelet-like particle production from MK cell line (5). To address the association of Flt3 and Mk development, we reviewed bone marrow biopsy from patient with and without Flt3 mutation and found that patients with Flt3 mutants showed statistically higher numbers of megakaryocytes in post-therapy marrows.…”

“…During hematopoiesis, megakaryocytes (Mk) develop from hematopoietic stem cells (HSCs) under tight control of Wnt/b-catenin signaling (3,4). Seo and colleagues demonstrated that Wnt signaling inhibitor, Wnt-C59, and Flt3 inhibitor, TCS 359, significantly increased platelet-like particle production from Mk supporting the crucial role of Wnt signaling pathway and Flt3 in Mk development and maturation (5).…”

Generation and manipulation of human iPSC-derived platelets

“…Through an assessment of relevant substances or drugs, we found that the combination of a ROCK inhibitor and an AhR antagonist enables platelet generation without feeder cells for imMKCL cultivation [ 73 ]. For further high-throughput screening of drugs that enhance megakaryocyte maturation, we developed an imMKCL with a fluorescent reporter that correlates with the expression of a maturation-specific protein [ 106 ].…”