A let-7 microRNA-RALB axis links the immune properties of iPSC-derived megakaryocytes with platelet producibility.

We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are dependent on donors and therefore variable in supply. However, heterogeneity characterized by thrombopoiesis-biased or immune-biased megakaryocytes (MKs) continues to pose a bottleneck against the standardization of iPSC-PLT manufacturing. To address this problem, here we employ microRNA (miRNA) switch biotechnology to distinguish subpopulations of imMKCLs, the MK cell lines producing iPSC-PLTs. Upon miRNA switch-based screening, we find imMKCLs with lower let-7 activity exhibit an immune-skewed transcriptional signature. Notably, the low activity of let-7a-5p results in the upregulation of RAS like proto-oncogene B (RALB) expression, which is crucial for the lineage determination of immune-biased imMKCL subpopulations and leads to the activation of interferon-dependent signaling. The dysregulation of immune properties/subpopulations, along with the secretion of inflammatory cytokines, contributes to a decline in the quality of the whole imMKCL population.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells.

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such "hu-NK mice" rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

Ex vivo manufacturing of platelets: beyond the first-in-human clinical trial using autologous iPSC-platelets.

Platelet transfusion is a common clinical approach to providing platelets to patients suffering from thrombocytopenia or other ailments that require an additional platelet source. However, a stable supply of platelet products is challenged by aging societies, pandemics, and other factors. Many groups have made extensive efforts toward the in vitro generation of platelets for clinical application. We established immortalized megakaryocyte progenitor cell lines (imMKCLs) from human induced pluripotent stem cells (iPSCs) and achieved clinical-scale manufacturing of iPSC-derived platelets (iPSC-PLTs) from them by identifying turbulent flow as a key physical condition. We later completed the iPLAT1 study, the first-in-human clinical trial using autologous iPSC-PLTs. This review summarizes current findings on the ex vivo generation of iPSC-PLTs that led to the iPLAT1 study and beyond. We also discuss new insights regarding the heterogeneity of megakaryocytes and the implications for the ex vivo generation of iPSC-PLTs.

[Clinical applications of iPS cell-derived platelets].

The COVID-19 pandemic has cast a shadow over transfusion medicine based on the blood donation system. However, managing alloimmune platelet transfusion refractoriness (allo-PTR) has already been difficult. As a first step toward resolving this issue using induced pluripotent stem cell-derived platelet products (iPSC-PLTs), a clinical trial of autologous products (iPLAT1) was conducted in a patient with allo-PTR caused by anti-HPA-1a antibodies who had no compatible donor, and safety was confirmed. To produce iPSC-PLTs, a master cell bank (MCB) of expandable megakaryocyte lines (imMKCLs) is established from iPSCs. From this MCB, iPSC-PLTs are manufactured using a newly developed turbulent-type bioreactor and various compounds. Their quality, safety, and efficacy are confirmed by extensive preclinical studies. Based on the findings of the iPLAT1 study, a clinical trial of allo-transfusion of HLA homozygous iPSC-PLTs is currently ongoing and HLA class I-deficient O-type universal iPSC-PLTs are also being developed. iPSC-PLTs are expected to solve various problems, including allo-PTR in platelet transfusion, and greatly contribute to the advancement of transfusion medicine.

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

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.

Silencing of p53 and CDKN1A establishes sustainable immortalized megakaryocyte progenitor cells from human iPSCs.

Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

Fit-For-All iPSC-Derived Cell Therapies and Their Evaluation in Humanized Mice With NK Cell Immunity.

Human induced pluripotent stem cells (iPSCs) can be limitlessly expanded and differentiated into almost all cell types. Moreover, they are amenable to gene manipulation and, because they are established from somatic cells, can be established from essentially any person. Based on these characteristics, iPSCs have been extensively studied as cell sources for tissue grafts, blood transfusions and cancer immunotherapies, and related clinical trials have started. From an immune-matching perspective, autologous iPSCs are perfectly compatible in principle, but also require a prolonged time for reaching the final products, have high cost, and person-to-person variation hindering their common use. Therefore, certified iPSCs with reduced immunogenicity are expected to become off-the-shelf sources, such as those made from human leukocyte antigen (HLA)-homozygous individuals or genetically modified for HLA depletion. Preclinical tests using immunodeficient mice reconstituted with a human immune system (HIS) serve as an important tool to assess the human alloresponse against iPSC-derived cells. Especially, HIS mice reconstituted with not only human T cells but also human natural killer (NK) cells are considered crucial. NK cells attack so-called "missing self" cells that do not express self HLA class I, which include HLA-homozygous cells that express only one allele type and HLA-depleted cells. However, conventional HIS mice lack enough reconstituted human NK cells for these tests. Several measures have been developed to overcome this issue including the administration of cytokines that enhance NK cell expansion, such as IL-2 and IL-15, the administration of vectors that express those cytokines, and genetic manipulation to express the cytokines or to enhance the reconstitution of human myeloid cells that express IL15R-alpha. Using such HIS mice with enhanced human NK cell reconstitution, alloresponses against HLA-homozygous and HLA-depleted cells have been studied. However, most studies used HLA-downregulated tumor cells as the target cells and tested in vitro after purifying human cells from HIS mice. In this review, we give an overview of the current state of iPSCs in cell therapies, strategies to lessen their immunogenic potential, and then expound on the development of HIS mice with reconstituted NK cells, followed by their utilization in evaluating future universal HLA-engineered iPSC-derived cells.

Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin ß1-ILK-ß-catenin-JUN axis.

Recombinant matrices have enabled feeder cell-free maintenance cultures of human pluripotent stem cells (hPSCs), with laminin 511-E8 fragment (LM511-E8) being widely used. However, we herein report that hPSCs maintained on LM511-E8 resist differentiating to multipotent hematopoietic progenitor cells (HPCs), unlike hPSCs maintained on LM421-E8 or LM121-E8. The latter two LM-E8s bound weakly to hPSCs compared with LM511-E8 and activated the canonical Wnt/ß-catenin signaling pathway. Moreover, the extracellular LM-E8-dependent preferential hematopoiesis was associated with a higher expression of integrin ß1 (ITGB1) and downstream integrin-linked protein kinase (ILK), ß-catenin and phosphorylated JUN. Accordingly, the lower coating concentration of LM511-E8 or addition of a Wnt/ß-catenin signaling activator, CHIR99021, facilitated higher HPC yield. In contrast, the inhibition of ILK, Wnt or JNK by inhibitors or mRNA knockdown suppressed the HPC yield. These findings suggest that extracellular laminin scaffolds modulate the hematopoietic differentiation potential of hPSCs by activating the ITGB1-ILK-ß-catenin-JUN axis at the undifferentiated stage. Finally, the combination of low-concentrated LM511-E8 and a revised hPSC-sac method, which adds bFGF, SB431542 and heparin to the conventional method, enabled a higher yield of HPCs and higher rate for definitive hematopoiesis, suggesting a useful protocol for obtaining differentiated hematopoietic cells from hPSCs in general.

Dual fungal infection with Lomentospora prolificans and Aspergillus fumigatus: A pathogenetic diagnosis elucidated after two decades by sequential examinations.

A 44-year-old male Japanese was admitted for further post-remission treatments for acute myeloid leukemia. He developed a right orbital abscess. An isolate of Lomentospora prolificans was obtained from the lesion, and orbital biopsy also revealed the presence of Aspergillus fumigatus. This fatal case involved a concurrent dual fungal infection.

Development of platelet replacement therapy using human induced pluripotent stem cells.

In the body, platelets mainly work as a hemostatic agent, and the lack of platelets can cause serious bleeding. Induced pluripotent stem (iPS) cells potentially allow for a stable supply of platelets that are independent of donors and eliminate the risk of infection. However, a major challenge in iPS cell-based systems is producing the number of platelets required for a single transfusion (more than 200 billion in Japan). Thus, development in large-scale culturing technology is required. In previous studies, we generated a self-renewable, immortalized megakaryocyte cell line by transfecting iPS cell-derived hematopoietic progenitor cells with c-MYC, BMI1, and BCL-XL genes. Optimization of the culture conditions, including the discovery of a novel fluid-physical factor, turbulence, in the production of platelets in vivo, and the development of bioreactors that apply turbulence have enabled us to generate platelets of clinical quality and quantity. We have further generated platelets deleted of HLA class I expression by using genetic modification technology for patients suffering from alloimmune transfusion refractoriness, since these patients are underserved by current blood donation systems. In this review, we highlight current research and our recent work on iPS cell-derived platelet induction.

Generation and manipulation of human iPSC-derived platelets.

The discovery of iPSCs has led to the ex vivo production of differentiated cells for regenerative medicine. In the case of transfusion products, the derivation of platelets from iPSCs is expected to complement our current blood-donor supplied transfusion system through donor-independent production with complete pathogen-free assurance. This derivation can also overcome alloimmune platelet transfusion refractoriness by resulting in autologous, HLA-homologous or HLA-deficient products. Several developments were necessary to produce a massive number of platelets required for a single transfusion. First, expandable megakaryocytes were established from iPSCs through transgene expression. Second, a turbulent-type bioreactor with improved platelet yield and quality was developed. Third, novel drugs that enabled efficient feeder cell-free conditions were developed. Fourth, the platelet-containing suspension was purified and resuspended in an appropriate buffer. Finally, the platelet product needed to be assured for competency and safety including non-tumorigenicity through in vitro and in vivo preclinical tests. Based on these advancements, a clinical trial has started. The generation of human iPSC-derived platelets could evolve transfusion medicine to the next stage and assure a ubiquitous, safe supply of platelet products. Further, considering the feasibility of gene manipulations in iPSCs, other platelet products may bring forth novel therapeutic measures.

Oncolytic herpes simplex virus type 1 (HSV-1) in combination with lenalidomide for plasma cell neoplasms.

Oncolytic viruses exert an anti-tumour effect through two mechanisms: direct oncolytic and indirect immune-mediated mechanisms. Although oncolytic herpes simplex virus type 1 (HSV-1) has been approved for melanoma treatment and is being examined for its applicability to a broad spectrum of malignancies, it is not known whether it has an anti-myeloma effect. In the present study, we show that the third-generation oncolytic HSV-1, T-01, had a direct oncolytic effect on five of six human myeloma cell lines in vitro. The anti-tumour effect was enhanced in the presence of peripheral blood mononuclear cells (PBMCs) from healthy individuals and, to a lesser extent, from patients with myeloma. The enhancing effect of PBMCs was abrogated by blocking type I interferons (IFNs) or by depleting plasmacytoid dendritic cells (pDCs) or natural killer (NK) cells, suggesting that pDC-derived type I IFNs and NK cells dominated the anti-tumour effect. Furthermore, the combination of T-01 and lenalidomide exhibited enhanced cytotoxicity, and the triple combination of T-01, lenalidomide and IFN-α had a maximal effect. These data indicate that oncolytic HSV-1 represents a viable therapy for plasma cell neoplasms through direct oncolysis and immune activation governed by pDCs and NK cells. Lenalidomide is likely to augment the anti-myeloma effect of HSV-1.

Oncolytic Virus Therapy with HSV-1 for Hematological Malignancies.

Oncolytic herpes simplex virus type 1 (HSV-1) has been investigated to expand its application to various malignancies. Because hematopoietic cells are resistant to HSV-1, its application to hematological malignancies has been rare. Here, we show that the third generation oncolytic HSV-1, T-01, infected and killed 18 of 26 human cell lines and 8 of 15 primary cells derived from various lineages of hematological malignancies. T-01 replicated at low levels in the cell lines. Viral entry and the oncolytic effect were positively correlated with the expression level of nectin-1 and to a lesser extent 3-O-sulfated heparan sulfate, receptors for glycoprotein D of HSV-1, on tumor cells. Transfection of nectin-1 into nectin-1-negative tumor cells made them susceptible to T-01. The oncolytic effects did not appear to correlate with the expression or phosphorylation of antiviral molecules in the cyclic GMP-AMP (cGAS)-stimulator of interferon genes (STING) and PKR-eIF2α pathways. In an immunocompetent mouse model, intratumoral injection of T-01 into lymphoma induced regression of injected, as well as non-injected, contralateral tumors accompanied by abundant infiltration of antigen-specific CD8+ T cells. These data suggest that intratumoral injection of oncolytic HSV-1 may be applicable to systemic hematological malignancies. Nectin-1 expression may be the most useful biomarker for optimal efficacy.

Ex vivo generation of platelet products from human iPS cells.

Platelet products are used in treatments for thrombocytopenia caused by hematopoietic diseases, chemotherapy, massive hemorrhages, extracorporeal circulation, and others. Their manufacturing depends on volunteers who donate blood. However, it is becoming increasingly necessary to reinforce this blood donation system with other blood sources due to the increase in demand and shortage of supply accompanying aging societies. In addition, blood-borne infections and alloimmune platelet transfusion refractoriness are not completely resolved. Since human induced pluripotent stem cell (iPSC)-platelet products can be supplied independently from the donor, it is expected to complement current platelet products. One big hurdle with iPSC-based systems is the production of 10 units, which is equivalent to 200 billion platelets. To overcome this issue, we established immortalized megakaryocyte cell lines (imMKCLs) by introducing three transgenes, c-MYC, BMI1, and BCL-XL, sequentially into hematopoietic and megakaryocytic progenitor stage cells derived from iPSCs. The three transgenes are regulated in a Tet-ON manner, enabling the addition and depletion of doxycycline to expand and maturate the imMKCLs, respectively. In addition, we succeeded in discovering drug combinations that enable feeder-free culture conditions in the imMKCL cultivation. Furthermore, we discovered the importance of turbulence in thrombopoiesis through live bone marrow imaging and developed a bioreactor based on the concept of turbulent flow. Eventually, through the identification of two key fluid physic parameters, turbulent energy and shear stress, we succeeded in scaling up the bioreactor to qualitatively and quantitatively achieve clinically applicable levels. Interestingly, three soluble factors released from imMKCLs in the turbulent flow condition, macrophage migration inhibitory factor (MIF), insulin growth factor binding protein 2 (IGFBP2), and nardilysin (NRDC), enhanced platelet production. Based on these developments, we initiated the first-in-human clinical trial of iPSC-derived platelets to a patient with alloimmune platelet transfusion refractoriness (allo-PTR) using an autologous product. In this review, we detail current research in this field and our study about the ex vivo production of iPSC-derived platelets.

iPSC-Derived Platelets Depleted of HLA Class I Are Inert to Anti-HLA Class I and Natural Killer Cell Immunity.

The ex vivo production of platelets depleted of human leukocyte antigen class I (HLA-I) could serve as a universal measure to overcome platelet transfusion refractoriness caused by HLA-I incompatibility. Here, we developed human induced pluripotent cell-derived HLA-I-deficient platelets (HLA-KO iPLATs) in a clinically applicable imMKCL system by genetic manipulation and assessed their immunogenic properties including natural killer (NK) cells, which reject HLA-I downregulated cells. HLA-KO iPLATs were deficient for all HLA-I but did not elicit a cytotoxic response by NK cells in vitro and showed circulation equal to wild-type iPLATs upon transfusion in our newly established Hu-NK-MSTRG mice reconstituted with human NK cells. Additionally, HLA-KO iPLATs successfully circulated in an alloimmune platelet transfusion refractoriness model of Hu-NK-MISTRG mice. Mechanistically, the lack of NK cell-activating ligands on platelets may be responsible for evading the NK cell response. This study revealed the unique non-immunogenic property of platelets and provides a proof of concept for the clinical application of HLA-KO iPLATs.

[Reconstitution of the hematopoietic system and clinical applications of iPS cells].

Human iPS cells are somatic cells reprogrammed to the pluripotent state. Because of their pluripotent nature, iPS cells are now commonly used to model several developmental processes including hematopoiesis in vitro. The in vitro models can be used to study the mechanisms regulating not only normal hematopoiesis but also hematological diseases ranging from monogenic congenital disorders to genetically multifactorial malignancies. Those disease models can also be used to investigate novel treatments through procedures including high throughput drug screening. The possible clinical applications of iPS cell-derived hematopoietic cells include immunotherapy with T lymphocytes, NK cells and macrophages, and transfusion therapy with platelets and red blood cells. Platelets have now been produced from iPS cells in quantities sufficient for clinical use. By developing expandable immortalized megakaryocyte cell lines (imMKCLs), several novel drugs and turbulence-incorporated bioreactors, efficient and scalable generation of platelets was achieved. This review summarizes the current status of iPS cell research in hematopoiesis with details on iPS cell-derived platelets.

Anti-inflammatory modulation of human myeloid-derived dendritic cell subsets by lenalidomide.

Although immunomodulatory drugs (IMiDs) were originally developed as anti-inflammatory drugs, they are effective for multiple myeloma. In order to gain further insights into the immunomodulatory mechanisms of IMiDs for the treatment of inflammatory disorders and myeloma, we investigated the influence of a representative IMiD, lenalidomide, on human primary dendritic cell (DC) subsets: myeloid-derived CD1c+ DCs, CD141+ DCs, and plasmacytoid DCs. Lenalidomide did not affect the viability or expression of costimulatory molecules, but it potently suppressed the production of the key inflammatory cytokines IL-12 and IL-23, and enhanced the production of the anti-inflammatory cytokine IL-10 by CD1c+ DCs. Lenalidomide also suppressed the production of IFN-α by CD141+ DCs but not that by plasmacytoid DCs. Lenalidomide likely targets pathways downstream of the nuclear translocation of the transcription factors nuclear factor κB (NF-κB) and IFN regulatory 5 (IRF5) in CD1c+ DCs. Consistent with the direct immunomodulatory effects on DCs, lenalidomide decreased the capacity of CD1c+ DCs to induce differentiation of naïve CD4+ T cells into effector cells producing immune activating and myeloma-promoting cytokines. This study demonstrated that lenalidomide has anti-inflammatory effects via the modulation of cytokine production by human myeloid-derived DCs. Such effects on DCs may allow for beneficial immunomodulation aiding in the treatment of inflammatory disorders and multiple myeloma.