Ex vivo Engineering of Platelets
Authors: Eto Koji 
Published: November 2019

Platelet transfusion is necessary for patients in thrombocytopenic states. Due to short shelf life of platelet product, the shortage of blood donors in the younger population as the consequence of aging societies in developed countries and platelet transfusion refractoriness (PTR) caused by alloimmune response, the risk of platelet product shortage has been a concern. Therefore, many attempts to substitute donor-dependent platelet product have been proposed. Induced pluripotent stem cell (iPSC) derived-platelet like particle product (which we refer as iPS-platelets) is aimed to provide measures against alloimmune PTR as well as to complement the current blood donor-dependent system.To generate the huge number of platelets required for transfusion into thrombocytopenia patients (200-300 billion per transfusion) ex vivo, megakaryopoiesis and subsequent platelet biogenesis process from megakaryocytes needed to be both substantially elucidated in mechanical systems. For the former, our key measure is to expand the immortalized megakaryocyte cell lines (imMKCLs) derived from iPSCs, whereby c-MYC, BMI-1, and Bcl-XL are overexpressed under doxycycline-inducible system to regulate proliferation of megakaryocyte progenitors (Cell Stem Cell, 2014). For the latter, in vivovisualization of mouse bone marrow revealed the presence of turbulence adjacent to megakaryocytes actively releasing platelets, which prompted us to utilize a unique turbulent flow-incorporated bioreactor to produce iPS-platelets. Furthermore, we succeeded in identifying turbulent energy and shear stress as essential physical parameters and further determined the optimal values, thereby to enable efficient and intact ex vivoplatelet manufacture, as exemplified by lowered Annexin V binding values in iPS-platelets, comparable with donor-human platelets. Simultaneously, we developed a culture medium cocktail including a thrombopoietin (TPO) mimetic small compound, an ADAM10/17 inhibitor for the maintenance of GPIb-alpha expression on platelets, an arylhydrocarbon receptor inhibitor, and a ROCK inhibitor for feeder cell-independent megakaryocyte maturation (Stem Cells Transl Med, 2017; Blood Adv, 2017; Blood Adv, 2018). By scaling up of the bioreactor, 100 billion iPS-platelets were produced in 8 L scale. In vitro and in vivo evaluation of iPS-platelets showed the functionality comparable with blood donor-derived platelets (Cell, 2018), including the newly developed thrombocytopenia rabbit model (Transfusion, 2017). Meanwhile, an iPS cell-derived human leukocyte antigen (HLA) class-I-null imMKCL as a universal HLA platelet source is potentially the best solution for alloimmune PTR. However, the immunogenic properties are still unclear, especially regarding natural killer (NK) cells that attack HLA-downregulated cells. Therefore, we evaluated the immunological reaction between HLA-null platelets and NK cells by using our newly developed alloimmune PTR in vivo model in which human NK cells are highly reconstituted in MISTRG mice (Suzuki and Sugimoto et al.,manuscript in revision). This pre-clinical study model should provide a proof-of concept for the clinical application of HLA-null iPS-platelets as a universal platelet product for future transfusion.
References Nakamura S, et al. Expandable Megakaryocyte Cell Lines Enable Clinically Applicable Generation of Platelets from Human Induced Pluripotent Stem Cells. Cell Stem Cell. 2014, 14, 535-548.Hirata S, et al. Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein IbαRetention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell-Derived Platelets. Stem Cells Transl Med. 2017, 6, 720-730.Aihara A, et al. Novel TPO receptor agonist TA-316 contributes to platelet biogenesis from human iPS cells. Blood Adv. 2017, 1, 468-476.Seo H, Chen SJ, et al. A β1-tubulin-based megakaryocyte maturation reporter system identifies novel drugs that promote platelet production. Blood Adv. 2018, 2, 2262-2272.Ito Y, Nakamura S, et al. Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production. Cell. 2018, 174, 636-648.Watanabe N, et al. Refined methods to evaluate the in vivo hemostatic function and viability of transfused human platelets in rabbit models. Transfusion. 2017, 57, 2035-2044.

Disclosures
Eto: Megakaryon Co. Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

OffLabel Disclosure:
Our published results that include some drugs, i.e., TA-316 (Blood Advances, 1(7):468-476, 2017), KP457 (Stem Cells Transl Med, 6(3):720-730, 2017), and SR1 (Cell, 174(3):636-648, 2018)