Production and nonclinical evaluation of an autologous iPSC-derived platelet product for the iPLAT1 clinical trial

Abstract

Donor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, ∼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)-based production system for an induced pluripotent stem cell-derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB.

Graphical abstract

Figure 1.

Flowchartof MCB establishmentfrom the patient’s peripheral blood. From the patient’s peripheral blood, mononuclear cells were purified and subjected to reprogramming to iPSCs using SNL cells as feeder cells (SNL-iPSC) and then transferred on Matrigel (M-iPSC). Using the revised "PSC-sac" method, M-iPSCs were differentiated into hematopoietic progenitor cells. 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 seed cell banks (SCB) and then as an MCB in liquid nitrogen.

Figure 2.

Production process of iPSC-PLTs from the imMKCL MCB. (A) Dox-ON condition for imMKCL proliferation: On day 1, the MCB cryopreserved in vials was thawed and cultured in a 6-well culture plate at 37°C and 5% CO₂. On days 4, 8, 11, 14, 17, and 20, the cells were transferred and cultured in a 90-mm culture dish, 250-mL culture flask, 2 500-mL culture flasks (both with 100 revolutions per minute [rpm] shaking), or 2-L, 10-L, and 50-L WAVE bags (each with rocking motion). On day 23, the imMKCLs were pelleted and washed twice using 2 ACP215 centrifugation systems and resuspended in Dox-OFF medium. (B) Dox-OFF condition for platelet production: the processed imMKCL solution was applied to 4 vessels of 10 L-scale VerMES bioreactors with 8 L medium solution in each and cultured with vertical stirring motion at 37°C for 6 days. (Ci) On day 29, the culture solution was added with 1/5 volume of ACD-A solution and concentrated to about 1/10 volume using a hollow fiber membrane filter. (Cii) imMKCLs and iPSC-PLTs were separated by continuous centrifugation using an ACP215 at 2500 rpm. With extruding solution, the platelet supernatant fraction volume became 4 L. (Ciii) The iPSC-PLT suspension was concentrated and extruded out to 700 mL using a hollow fiber membrane filter and then filtered through a leukocyte removal filter to remove residual megakaryocytes. (Civ) The iPSC-PLT suspension filtrate was centrifugated in an ACP215 at 200 mL per minute and 6000 rpm. Then, the pellets in the centrifuge pod were resuspended in 250 mL bicarbonate Ringer's solution (BRS) with 10% anticoagulant citrate dextrose solution (ACD)-A and 2.5% human serum albumin (HSA). From the 250 mL solution, 50 mL was drawn for evaluation.

Figure 3.

In vitro characteristics of iPSC-PLTs. (A) Representative flow cytometry scatter plots of cell surface A antigen and B antigen expression on JRC-PLTs of types A, B, AB, and O and on iPSC-PLTs. (B) Flow cytometry analysis of JRC-PLTs and iPSC-PLTs of batch 11, 13, 14, 15, 16, and 17. Mean fluorescence intensities (MFI) of CD41, CD42b, CD61, CD36, CD49b, and HLA-A/B/C are shown. For JRC-PLTs, the mean and standard deviation of MFI from 32 individuals are shown. (C) HPA genotypes of patient PBMCs and M35-1 imMKCL, determined using WAKFlow HPA typing reagents. (D) Flow cytometry histogram of iPSC-PLTs and HPA-1a/1a JRC-PLTs using HPA-1a-specific anti-CD61 (clone SZ21) antibody. (E) Scheme of the MR-MAIPA assay to identify HPA-1a antigen expression. (F) Readout ratio of anti–HPA-1a serum to negative control serum for iPSC-PLTs and HPA-1a/1a JRC-PLTs by MR-MAIPA, as in panel C. SZ21 antibody blocks the binding of anti–HPA-1a serum, resulting in values below the cutoff level for JRC-PLTs as well, thereby assuring specificity of the serum. (G) Sizes and percentages of the large IPF corresponding to iPSC-PLTs (batch 18) and blood donor–derived JRC-PLTs at different storage days. Representative data of 3 batches are shown. (H) Representative transmission electron micrograph images of iPSC-PLTs (batch 17) and JRC-PLTs. Scale bar: 1 μm.

Figure 3.

In vitro characteristics of iPSC-PLTs. (A) Representative flow cytometry scatter plots of cell surface A antigen and B antigen expression on JRC-PLTs of types A, B, AB, and O and on iPSC-PLTs. (B) Flow cytometry analysis of JRC-PLTs and iPSC-PLTs of batch 11, 13, 14, 15, 16, and 17. Mean fluorescence intensities (MFI) of CD41, CD42b, CD61, CD36, CD49b, and HLA-A/B/C are shown. For JRC-PLTs, the mean and standard deviation of MFI from 32 individuals are shown. (C) HPA genotypes of patient PBMCs and M35-1 imMKCL, determined using WAKFlow HPA typing reagents. (D) Flow cytometry histogram of iPSC-PLTs and HPA-1a/1a JRC-PLTs using HPA-1a-specific anti-CD61 (clone SZ21) antibody. (E) Scheme of the MR-MAIPA assay to identify HPA-1a antigen expression. (F) Readout ratio of anti–HPA-1a serum to negative control serum for iPSC-PLTs and HPA-1a/1a JRC-PLTs by MR-MAIPA, as in panel C. SZ21 antibody blocks the binding of anti–HPA-1a serum, resulting in values below the cutoff level for JRC-PLTs as well, thereby assuring specificity of the serum. (G) Sizes and percentages of the large IPF corresponding to iPSC-PLTs (batch 18) and blood donor–derived JRC-PLTs at different storage days. Representative data of 3 batches are shown. (H) Representative transmission electron micrograph images of iPSC-PLTs (batch 17) and JRC-PLTs. Scale bar: 1 μm.

Figure 4.

In vitro functional assessment of iPSC-PLTs shows the comparability with blood donor-derived platelets. (A) Representative flow cytometry images of P-selectin expression and PAC-1 binding of iPSC-PLTs (batch 09 and 17) with or without 100 μM ADP and 40 μM TRAP-6. (B) Flow cytometry data as in panel A for 4 batches of iPSC-PLT samples before processing, after filtration, and after the second ACP215 centrifugation for washing and after irradiation. (C) Aggregation assay. iPSC-PLTs (batch 07 and 09) were stimulated with 50 μM ADP or 5, 10, and 20 μg/mL collagen. (D) Autologous iPSC-PLTs of batch 16 in a blood bag were stored in a platelet preservation shaker. At the manufacturing date and after storage for 1, 2, 4, 6, and 8 days, the samples were tested for platelet count, CD42b expression, annexin V binding and in vitro function, and PAC-1 binding and P-selectin expression with or without ADP + TRAP-6 stimulation.

Figure 5.

In vivo functional assessment of iPSC-PLTs. (A) In vivo circulation in thrombocytopenic rabbit models for 3 batches of iPSC-PLTs as measured by the percentage of human and rabbit platelets in peripheral blood using flow cytometry. (B) In vivo hemostasis in thrombocytopenic rabbit models for iPSC-PLTs, as measured by the bleeding time of the ear incision before and after the transfusion of human iPSC-PLTs (batch 09). The maximum time was set to 600 seconds. (C) Circulation of iPSC-PLTs in NOG mice by IVIS imaging: imMKCLs or iPSC-PLTs expressing Venus-Akaluc were injected into NOG mice. Phosphate-buffered saline was injected for the control group. After 2, 6, 48, 72, and 168 hours, the mice were subjected to IVIS imaging.

Figure 6.

Nonclinical safety assessment of iPSC-PLTs. (A) Profiles of novel drugs in the test batches. All substances were a less-than-lifetime limit of 120 μg per day for administration of up to 1 month. (B) A table summarizing in vivo general toxicity tests. For the single-dose study in NOG mice, the mice were observed for general condition, weight, and feeding amount, and underwent blood tests. On days 14 and 28, half of the mice in each group were sacrificed for autopsy for macroscopic observation of the organs, organ weight, and histopathology. For single and repeated administration tests on rats, the rats were observed for general condition, weight, and feeding amount and underwent blood, urine, and ophthalmological tests for 2 weeks after the last dose. They were then sacrificed for autopsy for macroscopic observation of the organs, organ weight, and histopathology. Formulation buffer and saline of the same volume were used as controls. For repeated tests, samples were administered twice a week 4 times. (C) Hematoxylin and eosin–stained histology sections of day 14 NOG mice injected IV with a 0.1 mL suspension of 2 × 10⁸ platelets or formulation buffer of the same volume. (D) In vitro tumorigenicity test. (i) imMKCLs, iPSCs, or HL60 cells were cultured with or without the same nonirradiated cell type in 90-mm culture dishes. The number of grown colonies per dish was counted and averaged. (ii) An iPSC-PLT product of 2 mL and nonirradiated imMKCLs or HL60 cells were cultured in different combinations in suspension culture condition in 90-mm culture dishes. Similarly, an iPSC-PLT product of 2 mL and nonirradiated HeLa cells were cultured in different combinations in adherent culture condition in 90-mm culture dishes. The number of grown colonies per dish was counted and averaged.