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1292 Exposure of hiPSC-Derived Mature Megakaryocytes to Vincristine Boosts Platelet Biogenesis in Vitro

Program: Oral and Poster Abstracts
Session: 401. Blood Transfusion: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Bleeding and Clotting, Biological therapies, platelet disorders, Diseases, thrombocytopenias, Therapies, Transfusion
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Yasuo Harada, PhD1,2*, Emiri Nakamura1*, Trevor Bingham3*, Leonard I. Zon, MD4, George Q. Daley, MD, PhD5,6,7, Andrew Frelinger, PhD8, Koji Eto, MD, PhD1,9 and Thorsten M. Schlaeger, PhD10*

1Centre of iPS Cell Research and Application, Kyoto University, Kyoto, Japan
2Otsuka Pharmaceutical, Co., Ltd., Osaka, Japan
3Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA
4Harvard Stem Cell Institute, Cambridge, MA
5Department of Hematology/Oncology, Harvard Medical School/Boston Children's Hospital, Boston, MA
6Harvard University, Cambridge, MA
7Harvard Medical School/Boston Children's Hospital, Boston, MA
8Boston Children's Hospital, Boston, MA
9Department of Regenerative Medicine, Chiba University, Chiba, Japan
10Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA

Millions of healthy donor platelet (PLT) units are needed each year for therapeutic transfusions of patients with thrombocytopenia and bleeding disorders, but shortages are common. The pandemic worsened donor shortages in the US and Japan. To ensure the predictability of supply, and to mitigate clinical complications associated with conventional donor PLTs, we have developed human induced pluripotent stem cell (hiPSC)-derived megakaryocyte (MK) cell lines, imMKCLs, as a source of in vitro PLT production (iPSC-PLTs)(Nakamura et al., Cell Stem Cell, 2014; Sone et al., Stem Cell Rep, 2021), and manufactured platelets are now in clinical trials using a previously published protocol (Sugimoto et al., Blood, 2022; Sugimoto et al., Blood Adv, 2022). To optimize and improve the in vitro manufacturing protocol of iPSC-PLTs from imMKCLs, we conducted a comprehensive imaging-based compound screening which identified microtubule (MT) destabilizing agents, including the vinca alkaloid vincristine (VCR), that appear to boost proplatelet formation. Combined use of VCR at 10 μM and turbulent hydrodynamic culture conditions (Ito et al., Cell, 2018) allowed production of 23.0 PLTs per imMKCL, 2.5 fold higher than without VCR. This successful result was observed only when VCR was administered at day 3 of 6-days of the maturation phase, when imMKCLs have become polyploid, while exposure at earlier timepoints induced apoptosis. However, PLTs produced in this manner showed poor agonist-induced surface expression of P-selectin or the PAC1 epitope. Therefore, we next examined dose-dependent action of VCR on both yield of iPSC-PLTs and their function. At nanomolar concentrations (10 nM), VCR still exhibited significant PLT biogenesis-boosting activity, but without apparent impairment of PLT in vitro functionality. Interestingly, this low concentration of VCR still resulted in reduced marginal MT band staining in imMKCLs, suggesting a potential role of weakened membrane structure/status in enhanced iPSC-PLT generation. Currently, we are attempting to address if PLTs made from low-dose VCR-exposed imMKCLs have reduced marginal MT bands and whether they restore hemostasis when transfused into immunodeficient thrombocytopenic mice. Our study unveils the potential of VCR as a potent enhancer of PLT biogenesis in the imMKCL system, particularly when combined with optimized turbulent flow production systems. Our results provide a means to make production of iPSC-PLTs more cost-efficient and suggest a mechanism for the previously unexplained observation that vinca alkaloids raise PLT counts in vivo.

Disclosures: Harada: Otsuka Pharmaceuticals Co., Ltd.: Current Employment. Daley: Megakaryon Inc.: Research Funding. Eto: Otsuka Pharmaceuticals Co., Ltd.: Research Funding; Megakaryon Inc.: Research Funding. Schlaeger: Megakaryon Inc.: Research Funding.

OffLabel Disclosure: Vincristine (Oncovin) - also known as Leurocristine is an anti-cancer drug used in chemotherapy.

*signifies non-member of ASH