Abstract
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<0.01). Subsequently, c-MYC, BMI1, and BCL-XL genes were turned off for inducing platelet yield. After another 4-days of culture, matured megakaryocytes and platelets were collected and analyzed by flow cytometer. Total number of CD41+CD42b+ platelets with MK-001 was increased >2-fold compared with that of rhTPO (p<0.001), whereas 1000ng/mL of Eltrombopag had little effect on platelet production. imMKCLs cultured with MK-001 contained a lot of large multinucleated cells and showed high levels of DNA content as well as those cultured with rhTPO. Intracellular phosphorylation analysis (BD Phosflow assay) revealed that MK-001 activated phosphorylation of components of three major TPO signaling pathways, JAK/STAT, MAPK, and PI3K/AKT within imMKCLs. PAC-1 bindings to platelets produced with MK-001, specific to an activated aIIbb3 integrin by adenosine 5-diphosphate (ADP), thrombin, or phorbol 12-myristate 13-acetate (PMA), were the same levels as those produced with rhTPO. These results indicated MK-001 promotes the production of functional platelets from imMKCLs more efficiently than rhTPO or Eltrombopag.
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.
Incomplete restoration of Mpl expression in the mpl−/− mouse produces partial correction of the stem cell–repopulating defect and paradoxical thrombocytosis
