A Novel Chemical Approach to Expand Platelets Using Immortalized Megakaryocyte Progenitor Cells Derived from Human Induced Pluripotent Stem Cells

Blood platelets can be obtained only by blood donation and reveal short-shelf life by the reason that they must be maintained with plasma at 20-24 degrees with shaking. These factors lead to shortage of donor platelets for clinical use. To overcome this issue, we developed a clinically applicable strategy for the derivation of functional platelets from human pluripotent stem cells (PSCs). We previously reported in vitro culture methods for producing functional platelets from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) (Takayama et al. Blood 2008, J Exp Med 2010). We also have established immortalized megakaryocyte progenitor cell lines (imMKCLs) with long-term expansion capability from hiPSC-derived hematopoietic progenitors with three defined factors, c-MYC, BMI1 and, BCL-XL (Nakamura et al. Cell Stem Cell, 2014). Although imMKCLs can be promising source of functional platelets for transfusion, further inventive efforts are needed to expand imMKCLs more efficiently towards clinical application. Thrombopoietin (TPO) is a cytokine initially identified as the primary regulator of megakaryocyte differentiation and platelet production. TPO is also an essential supplement for expanding platelets from imMKCLs. Recently, several nonpeptidyl small-molecule compounds have been developed to activate the TPO receptor, c-MPL and promote platelet production such as SB-497115 (Eltrombopag), an orally available drug for thrombocytopenia. Chemically synthesized c-MPL agonists reveal the advantage in terms of biological safety, low-immunogenicity or, low-cost manufacturing as compared to peptide-based ligands for platelet production. To obtain a c-MPL agonist that expands imMKCLs more efficiently and cost effectively than recombinant human TPO (rhTPO), we firstly screened small-molecule c-MPL agonists by evaluating its effects on platelet production from hiPSCs. Consequently, we identified “MK-001”, as the most potent compound that increases platelet productivity, as evidenced by the effects of MK-001 on the proliferation, differentiation, cell signaling, and platelet production from imMKCLs. We also studied the functionality of imMKCL-derived platelets. imMKCLs were cultured for 15 days with passage every 3 or 4 days with rhTPO in the presence of either rhTPO, Eltrombopag or MK-001 employing the same method as previously described (Nakamura et al. Cell Stem Cell, 2014). Total cell number was measured by Trypan Blue staining and automated cell counter. On day11, the number of total cells cultured with 200ng/mL of MK-001 was increased >1.5-fold compared with that of 50ng/mL of rhTPO (p 2-fold compared with that of rhTPO (p In conclusion, the c-MPL agonist MK-001 could be applicable as an indispensable tool for expansion of platelets from hiPSCs with a combination of imMKCL system. Disclosures Nakauchi: Nissan Chemical Industries: Research Funding. Eto: Nissan Chemical Industries: Research Funding.