Production of iPSC-PLTs from imMKCL MCB and their functional properties. (A) Somatic cells (peripheral blood mononuclear cells) from an aplastic anemia patient with alloPTR owing to anti–HPA-1a, were reprogrammed using episomal vectors. iPSCs were differentiated to hematopoietic progenitor cells using the revised pluripotent stem cell (PSC)-sac method. Then, under megakaryocyte differentiating condition, c-MYC, BMI-1, and BCL-XL were sequentially transduced by lentiviral vectors to establish imMKCLs. A single imMKCL clone was selected based on expandability and platelet production and stocked as a master cell bank (MCB) in liquid nitrogen. From the MCB, iPSC-PLTs were produced under good manufacturing practice (GMP)–based conditions: an MCB vial was thawed, cultured with a thrombopoietin mimetic (TA-316), stem cell factor and doxycycline, and expanded for 23 days. After expansion, the cells were transferred to medium without doxycycline but with a Rho-associated kinase inhibitor (Y-39983), an aryl hydrocarbon receptor antagonist (GNF-316), and an ADAM17 inhibitor (KP-457). The 6-day maturation culture was done in 4 vessels of 8 L-scale VerMES, which are turbulent flow bioreactors. iPSC-PLTs released into the media were concentrated, separated, and washed using hollow fibers and an ACP215 centrifugation system with low and high settings and depleted for nucleated cells using leukocyte depleting filters. Approximately 10 × 10¹¹ iPSC-PLTs were resuspended in 200 mL bicarbonate linger solution with 20% acid citrate dextrose and 2.5% human serum albumin and packaged into a blood bag. The final package included ∼10⁷ imMKCLs but was gamma-ray irradiated with 25 Gy to eliminate tumorigenicity. (B) Sizes and percentages of the large immature platelet fraction (IPF) corresponding with iPSC-PLTs (batch 18) and blood donor–derived Japanese Red Cross product platelets (JRC-PLTs) at different storage days. Representative data of 3 batches are shown. (C) Representative transmission electron micrograph images of iPSC-PLTs (batch 17) and JRC-PLTs. Scale bars, 1 μm. (D) In vitro functional assay of iPSC-PLTs: (i) representative flow cytometric analysis data for iPSC-PLT samples before processing, after filtration, and after a second ACP215 centrifugation for washing and after irradiation and (ii) aggregation assay. iPSC-PLTs were stimulated with 50 μM adenosine diphosphate (ADP) or 5, 10, and 20 μg/mL collagen. (E) In vivo circulation in thrombocytopenic rabbit models for 6 batches of iPSC-PLTs (3 batches per rabbit, 2 rabbits) by measuring the ratio of human and rabbit platelets in peripheral blood using flow cytometry. (F) In vivo hemostasis in thrombocytopenic rabbit models for iPSC-PLTs by measuring the bleeding time of the ear incision before and after the transfusion of human iPSC-PLTs (batch 09). The maximum measuring time was 600 seconds. (G) imMKCLs or iPSC-PLTs introduced with Akaluc expression vectors were injected into NOD/Shi-scid,IL-2RγKO (NOG) mice. Phosphate-buffered saline (PBS) was injected for the control group. Results of in vivo imaging after 2, 6, and 48 hours are shown. CCI, corrected count increment; max, maximum; min, minimum.