Naoshi Sugimoto and Koji Eto
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

Preface

This year marks the 200^th anniversary of the first successful blood transfusions by Dr. James Blundell, saving the lives of four women with severe postpartum bleeding. Today, regenerative medicine using iPSC-derived cells is entering clinical application. In 2020, we completed the iPLAT1 study (jRCTa050190117), the world’s first clinical trial of iPSC-derived platelet product (iPSC-PLTs). Building on the success of that study, in which we manufactured “autologous” iPSC-PLTs from the subject’s own iPS cells, we are now developing a universal product for the next clinical trial. Specifically, we are working towards HLA-knockout (KO) iPSC-PLTs with improved circulation capacity and more efficient manufacturing procedures to prepare allogeneic formulations. Currently, our group is the only one in the world to have achieved the clinical application of ex vivo-manufactured platelet products. Here, we will outline the current status of our work toward a future clinical trial using universal HLA-KO iPSC-PLTs.

Manufacturing Autologous iPSC-PLTs for the iPLAT Study

Megakaryocytes differentiated from hematopoietic stem cells undergo endomitosis (nuclear division) during maturation, becoming polyploid with approximately 64N chromosomes and reaching a size of 50–100 μm. The expanded cytoplasm forms DMS (demarcation membrane system), leading to platelet production through a fragmentation mechanism or platelet release from the tip of proplatelets—cytoplasmic protrusions extending into bone marrow sinusoids. The number of platelets produced per megakaryocyte is calculated to be between 800 and several thousand. While this platelet production has traditionally been considered to occur primarily in the bone marrow, multiple recent studies have observed a high proportion of platelet production in the spleen and lungs. The in vitro production of platelets essentially equated to solving two key challenges: how to generate a sufficient number of megakaryocytes (present at only about 0.01% in bone marrow) and how to establish a maturation environment that promotes the release of high-quality platelets.

We achieved the generation of large quantities of megakaryocytes by leveraging imMKCLs—expandable megakaryocyte progenitor cell lines. imMKCLs are established by introducing expression vectors for the c-MYC, BCL-XL, and BMI1 genes during megakaryocyte differentiation from hematopoietic progenitor cells derived from iPS cells (1). Expression of these three transgenes is under the control of the Tet-ON system, which induces their expression in response to doxycycline added to the culture media, leading to the robust proliferation of imMKCLs by conferring proliferative, anti-apoptotic, and anti-senescence properties (Dox-ON). Upon depletion of doxycycline, imMKCLs spontaneously mature and release platelets (Dox-OFF). Furthermore, the development of novel drugs enabling platelet production under liquid culture conditions distinct from the in vivo environment, along with the development of a “turbulent flow” bioreactor, facilitates clinical-scale iPSC-PLT manufacturing (2).

In the iPLAT1 trial, the imMKCL used was established from iPS cells derived from the subject’s peripheral blood mononuclear cells. An imMKCL clone with excellent proliferation and platelet production capacity was cryopreserved as the master cell bank (MCB) (3, 4). For iPSC-PLT production, 2 × 10⁶ MCB-imMKCL cells were first cultured in 3 mL medium under the Dox-ON condition. After 23 days, they were amplified to approximately 3 × 10¹⁰ cells in a 20 L culture. The medium contained doxycycline, SCF (stem cell factor), and the proprietary thrombopoietin receptor agonist TA-316. Cells were then washed and transferred to Dox-OFF culture using four 8 L VerMES bioreactors (3, 4). This platelet-producing medium contained a ROCK inhibitor and an AhR antagonist to promote maturation and an ADAM17 inhibitor to prevent GPIbα cleavage. Based on in vivo observations, we previously found that turbulent flow is a crucial physical factor contributing to platelet generation from megakaryocytes. To recapitulate similar fluid dynamics, we developed a new culture device, VerMES, with vertical motion, capable of generating turbulence with optimal shear stress and turbulent energy (2).

Six days after Dox-OFF culture, iPSC-PLTs were packaged via megakaryocyte separation and washing/replacement/concentration using a hollow fiber membrane filter and a continuous centrifugation device. The final product contained approximately 1 × 10¹⁰ iPSC-PLTs in 200 mL of bicarbonate Ringer’s solution/20% ACD/2.5% human serum albumin. Subsequently, to eliminate the tumorigenicity of residual imMKCL, irradiation with 25 Gy of radiation, as is typically done with conventional platelet preparations, was performed (3, 4).

The MCB passed viral safety testing and identity verification, manufacturing additives were confirmed safe based on residual concentration and toxicity/mutagenicity tests, and the final product, iPSC-PLTs, cleared general toxicity testing. In tumorigenicity studies with imMKCLs and iPSC-PLTs, no proliferative cells were detected after 25 Gy irradiation (4). Although iPSC-PLTs were approximately 1.5 times larger than donated platelets (3.5–4 μm), electron microscopy revealed equivalent intracellular structures, and in vitro testing demonstrated comparable quality and function. Tests in a rabbit model demonstrated competent circulation and hemostatic ability. Distribution tests using fluorescently labeled iPSC-PLTs conducted after completion of the iPLAT1 trial also confirmed systemic circulation (3, 4). Furthermore, the selection of manufacturing raw materials and development of manufacturing methods in accordance with GCTP (Good Gene, Cellular, and Tissue-based Products Manufacturing Practice), as well as the establishment of quality and safety test items, were determined following consultations with the regulatory authority, PMDA (4).

Achievement of Primary Endpoints and Lessons Learned from the iPLAT1 Trial

The iPLAT1 trial, designed as a dose-escalation Phase 1 trial, was approved by Kyoto University and the Ministry of Health, Labour and Welfare, and ultimately, designated as compliant with the Act on the Safety Assurance of Regenerative Medical Treatment in October 2018. It was subsequently registered to the Japan Registry of Clinical Trials as jRCTa050190117 (3).

The study subject was a multiparous woman with aplastic anemia complicated by alloimmune platelet transfusion refractoriness due to anti-HPA-1a antibodies (3). Individuals with the same HPA-1b/1b phenotype compatible for transfusion to this subject constituted less than 0.002% of the Japanese population and were absent among registered donors of the Japanese Red Cross Society. Fortunately, the aplastic anemia itself improved with cyclosporine monotherapy, making it ideal from a safety perspective. Eventually, the iPLAT1 trial was conducted in a stable disease state in which cyclosporine treatment was terminated.

The initial dose of 0.5 Japan units (platelet count 0.1 × 10¹¹; 1/20th the volume of a standard platelet transfusion) was transfused in May 2019, followed by 1.5 units in August, and 5 units in January of the following year. The primary endpoint was safety, based on the occurrence of adverse events. The secondary endpoint was efficacy, measured by the CCI (corrected platelet increment) values at 1 hour and 24 hours post-transfusion (3).

An external safety and efficacy review committee confirmed that there were no issues with dose escalation after the first and second doses. After a one-year observation period following the final dose, the primary endpoint of safety was finally determined to have been successfully achieved. However, the secondary endpoint CCI did not show a clear post-transfusion increase either at 1 hour or 24 hours (3).

So why did the circulation of iPSC-PLTs not show up in the measurements? At the maximum dose of 5 units, an increase in peripheral blood platelet count should have been measurable. In this regard, we arrived at multiple possibilities. First, reflecting the good control of the underlying disease, the platelet count was already high before transfusion (90,000/μL), making it difficult to detect small changes (3). Nevertheless, flow cytometry analysis detected the presence of large platelets in blood samples one hour post-transfusion, and their fraction gradually decreased, suggesting that large iPSC-PLTs were circulating (3). In other words, conventional hematology analyzers may have failed to capture the larger iPSC-PLTs. Alternatively, the peak circulation of iPSC-PLTs might have occurred 2-6 hours post-transfusion—later than conventional platelet transfusions—and the measurement points may not have captured the characteristic temporal changes (3, 4), if circulation dynamics mirrored those observed in animal studies. Collectively, these findings suggest that while iPSC-PLTs were indeed circulating, they were simply not being accurately measured.

We also questioned whether there could be an issue with the circulatory function of the iPSC-PLTs. For example, since the platelets administered in the iPLAT1 trial were autologous preparations, the likelihood of immune rejection was considered extremely low. However, the possibility of antigen modification due to the influence of additives cannot be ruled out.

Following iPSC-PLT transfusion, a slight increase in D-dimer was observed (3), and thus, the possibility that iPSC-PLTs formed thrombi cannot be ruled out. However, no clinical findings suggestive of thrombosis or embolism were noted, nor were thrombi detected by non-invasive lower extremity venous ultrasound. Furthermore, D-dimer levels subsequently decreased spontaneously. However, consistent with the D-dimer course, a transient increase in white blood cell count was also observed (3). This observation drew attention due to its potential association with thromboinflammation and the contribution of immune megakaryocytes, both identified in severe COVID-19. Recent single-cell RNA sequencing analyses have reported that megakaryocytes in vivo include subsets supporting bone marrow niche maintenance and immune or inflammatory responses. Similarly, since we identified the presence of an immune subset within imMKCLs (5), the possibility that this subset may have induced thromboinflammation warrants further investigation.

Post-iPLAT1 Trial Research and Development

Following the iPLAT1 trial, we set out to perform reverse translational research aimed at fully elucidating the potential deficit in the circulatory function. This effort includes analyzing the presence or absence of anti-iPSC-PLT antibodies and the immune megakaryocyte-like property of imMKCL MCB, as well as verifying low-level platelet activation and desialylation during the manufacturing process. Activated platelets and desialylated platelets promote thrombosis through coagulation factor assembly associated with phosphatidylserine expression on the cell surface and lead to uptake by hepatocytes due to galactose exposure. Furthermore, we are developing measurement methods suitable for iPSC-PLTs and creating humanized mice that replicate the circulation of iPSC-PLTs in the human body.

To advance iPSC-PLTs into a commercially viable formulation, manufacturing processes must also be refined. Currently, imMKCL yields approximately 100 iPSC-PLTs per megakaryocyte cell, which is considered a benchmark for success. However, this falls short of the estimated thousands of platelets produced per megakaryocyte in the body. In this regard, we are considering the suppression or exclusion of immune megakaryocyte-like cells as well as focusing on the allocation of healthy mitochondria to iPSC-PLTs as key factors for improvement. Meanwhile, given that manufacturing based on GCTP allows for the maximum prevention of pathogenic microorganism contamination, there is potential to significantly exceed the current 6-day shelf life of donated platelets if quality can be maintained.

In terms of universal application, we are currently developing HLA class I-deficient iPSC-PLTs toward clinical trials. HLA class I possesses an enormous repertoire, and its incompatibility accounts for the majority of cases of alloimmune platelet transfusion refractoriness, which complicates approximately 5-15% of platelet transfusion patients. HLA-deleted products not only enable safe use in patients with alloimmune platelet transfusion refractoriness but also inherently become a single product, facilitating cost reduction through mass production. They are also well-suited as a foundational platform for specific products like HPA-modified products, functionally engineered products, and platelet-rich plasma (PRP) therapy products. Furthermore, they could serve as an ideal cell base for antibody detection tests during platelet transfusion refractoriness.

In summary, we are developing universal iPSC-PLTs using a manufacturing method that incorporates research and development focused on improving quality by enhancing circulatory capacity and manufacturing efficiency. Our goal is to create a formulation that is truly implementable at the required scales to revolutionize blood transfusion therapy for all patients in the world for the first time in two centuries.

References

1.         Nakamura S, Takayama N, Hirata S, Seo H, Endo H, Ochi K, et al. Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells. Cell Stem Cell. 2014;14(4):535-48.

2.         Ito Y, Nakamura S, Sugimoto N, Shigemori T, Kato Y, Ohno M, et al. Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production. Cell. 2018;174(3):636-48.e18.

3.         Sugimoto N, Kanda J, Nakamura S, Kitano T, Hishizawa M, Kondo T, et al. iPLAT1: the first-in-human clinical trial of iPSC-derived platelets as a phase 1 autologous transfusion study. Blood. 2022;140(22):2398-402.

4.         Sugimoto N, Nakamura S, Shimizu S, Shigemasa A, Kanda J, Matsuyama N, et al. Production and nonclinical evaluation of an autologous iPSC-derived platelet product for the iPLAT1 clinical trial. Blood Adv. 2022;6(23):6056-69.

5.         Chen SJ, Hashimoto K, Fujio K, Hayashi K, Paul SK, Yuzuriha A, et al. A let-7 microRNA-RALB axis links the immune properties of iPSC-derived megakaryocytes with platelet producibility. Nat Commun. 2024;15(1):2588.