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.

Revised "hPSC-Sac Method" for Simple and Efficient Differentiation of Human Pluripotent Stem Cells to Hematopoietic Progenitor Cells.

The human hematopoietic differentiation in vitro of human pluripotent stem cells (hPSCs) has provided new tools to elucidate the mechanisms of related genetic abnormalities, such as congenital diseases and acquired hematopoietic malignancies, and to discover new treatments. The differentiation can also be applied to developing a stable source of blood products for transfusion with minimal risk of several blood-borne infections. We previously proposed a method for hematopoietic progenitor cell (HPC) differentiation, the "hPSC-sac method", in which hPSCs are cocultured with C3H10T1/2 mouse stromal cells and mixed with a single cytokine, VEGF. The hPSC-sac method can differentiate hPSCs to multiple blood lineages. Here we describe improvements in the method by adding bFGF, TGFß inhibitor and heparin to the culture, which increases the yield of CD34+CD43+ HPCs 50-fold compared with the original protocol. This revised hPSC-sac method is expected to contribute to the development of disease models and regenerative medicine using hematopoietic lineage 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.

Tyrosyl-tRNA synthetase stimulates thrombopoietin-independent hematopoiesis accelerating recovery from thrombocytopenia.

New mechanisms behind blood cell formation continue to be uncovered, with therapeutic approaches for hematological diseases being of great interest. Here we report an enzyme in protein synthesis, known for cell-based activities beyond translation, is a factor inducing megakaryocyte-biased hematopoiesis, most likely under stress conditions. We show an activated form of tyrosyl-tRNA synthetase (YRSACT), prepared either by rationally designed mutagenesis or alternative splicing, induces expansion of a previously unrecognized high-ploidy Sca-1+ megakaryocyte population capable of accelerating platelet replenishment after depletion. Moreover, YRSACT targets monocytic cells to induce secretion of transacting cytokines that enhance megakaryocyte expansion stimulating the Toll-like receptor/MyD88 pathway. Platelet replenishment by YRSACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from induced pluripotent stem cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. We suggest megakaryocyte-biased hematopoiesis induced by YRSACT offers new approaches for treating thrombocytopenia, boosting yields from cell-culture production of platelet concentrates for transfusion, and bridging therapy for hematopoietic stem cell transplantation.

Clinical experience of bendamustine for adult Langerhans cell sarcoma.

A 40-year-old man was diagnosed with Langerhans cell histiocytosis (LCH) in October 2010. His LCH was refractory to conventional chemotherapy, and thus worsened to Langerhans cell sarcoma (LCS) in May 2011. Although we repeated combination chemotherapies, new infiltration of the liver and bone marrow, as well as primary lesions of the bone, lymph nodes, and skin, appeared. These intensive chemotherapies caused candida liver abscesses, invasive aspergillosis, disseminated varicella zoster virus infection and bacterial sepsis. We administered bendamustine for chemotherapy, which resulted in a partial response (PR) with no severe adverse events. Because of pancytopenia caused by secondary myelodysplastic syndrome, we stopped the bendamustine chemotherapy after two courses. PR was maintained for 4 months. We plan to perform allogeneic hematopoietic stem cell transplantation from a sibling donor after a conditioning regimen. Optimal therapy for adult LCH, which is a rare and treatment-resistant disease, has yet to be established. Bendamustine is a potential chemotherapeutic agent for standard treatment of LCS.

Discrepancy in EBV-DNA load between peripheral blood and cerebrospinal fluid in a patient with isolated CNS post-transplant lymphoproliferative disorder.

Post-transplant lymphoproliferative disorder (PTLD) is a fatal complication of allogeneic hematopoietic stem cell transplantation (HSCT) that is caused by reactivation of Epstein-Barr virus (EBV). A successful approach, monitoring EBV-DNA load in peripheral blood (PB) accompanied by preemptive rituximab therapy, has recently been reported. Here, we describe a 29-year-old woman who developed isolated central nervous system (CNS) PTLD. She received HSCT against acute myelogenous leukemia from a related human leukocyte antigen-haploidentical donor, following a conditioning regimen that included antithymocyte globulin. Tacrolimus and methylprednisolone were given as prophylaxis for graft-versus-host disease. On day +172, the patient's consciousness deteriorated. Magnetic resonance imaging showed six ring-enhanced lesions in the cerebral hemispheres. These tumors were diagnosed, via a craniotomy and tumorectomy, as PTLD. EBV-DNA load was elevated in the cerebrospinal fluid (CSF) but not detected in PB. She was treated with whole-brain irradiation and rituximab, and achieved partial remission of the tumors. This case serves as a reminder that vigilance is required regarding the development of isolated CNS PTLD; it is worth examining EBV-DNA replication in CSF for diagnosis even when the EBV-DNA load is negative in PB.

Successful treatment of autoimmune hemolytic anemia associated with multicentric Castleman disease by anti-interleukin-6 receptor antibody (tocilizumab) therapy.

We describe herein the successful treatment of severe autoimmune hemolytic anemia (AIHA) in a patient with multicentric Castleman disease (MCD) by humanized anti-interleukin-6 (IL-6) receptor antibody (tocilizumab) therapy. Inflammatory anemia is commonly reported; however, AIHA is a very rare complication of MCD. In 1996, a 45-year-old Japanese woman was referred to our hospital because of generalized lymphadenopathy, anemia and skin eruptions. Lymph node biopsy demonstrated MCD. She was treated with prednisolone (1 mg/kg/day), which improved the anemia and skin eruptions. In 2009, she suddenly developed Coombs-positive hemolytic anemia. The blood count was as follows: hemoglobin 4.7 g/dl, platelets 490 × 10(9)/l and white blood cell count 9.8 × 10(9)/l. Both direct and indirect Coombs' tests were strongly positive. She was treated with 8 mg/kg tocilizumab every 2 weeks. One month later, her hemoglobin levels rose dramatically to 10.9 g/dl and her haptoglobin level, hypergammaglobulinemia and clinical symptoms had also markedly improved. To the best of our knowledge, this is the first report of the efficacy of tocilizumab in AIHA associated with MCD. The well-established role of IL-6 in the pathogenesis of MCD may have been responsible for the improvement in the AIHA associated with MCD. Anti-IL-6 receptor antibody treatment could be an attractive therapeutic approach for AIHA associated with MCD.