Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

Hemostatic function of cold-stored platelets in a thrombocytopenic rabbit bleeding model.

BACKGROUND: Transfusion of cold-stored platelet concentrates (CS-PCs) appears effective in massively bleeding patients. However, few studies have evaluated their in vivo hemostatic function in severe thrombocytopenia. STUDY DESIGN AND METHODS: The in vivo function of plasma-depleted human PCs was evaluated in rabbits with a blocked reticuloendothelial system and busulfan-induced thrombocytopenia. On day 1, a human apheresis PC was processed in a platelet additive solution (PAS-PC) and split evenly for cold or room temperature storage (RTS). On days 3, 6, or 9, RTS- or CS-PAS-PCs were transfused (4.0 × 109 platelets/kg) after plasma depletion into two to four rabbits that developed adequate thrombocytopenia (<25 × 109 /L). Ear bleeding time was measured by two incisions in small veins. The hemostatic rate was defined as the percentage of rabbits achieving bleeding cessation within 600 s at either incision. The experiment was repeated using five different PCs on each storage day. RESULTS: The mean pre-transfusion rabbit platelet count was 8.6 ± 5.2 × 109 /L. The hemostatic rates with RTS- and CS-PAS-PCs were both 100% on day 3, 93 ± 15% and 73 ± 15% on day 6 (p = .07), and 65 ± 36% and 73 ± 37% on day 9 (p = .27), respectively, with no statistical differences. Total platelet counts were significantly lower after CS-PAS-PC than RTS-PAS-PC transfusion on all days (e.g., 58.7 ± 5.7 vs. 42.4 ± 14.7 × 109 /L, p = .0007, day 9), and did not reach 50 × 109 /L in several experiments. Platelet count increments correlated significantly with hemostatic efficacy for CS-PAS-PC transfusion only. DISCUSSION: CS-PAS-PCs might achieve similar hemostasis as RTS-PAS-PCs in thrombocytopenic patients with mild bleeding. Hemostatic efficacy could be improved by transfusing more CS-PAS-PCs.

Adoptive cell therapy using tumor-infiltrating lymphocytes for melanoma refractory to immune-checkpoint inhibitors.

To evaluate the feasibility of adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) in Japanese patients with melanoma who failed immune-checkpoint inhibitor therapy, an open-label, single-arm, pilot study was conducted. We investigated the immunological and genetic factors of the pretreatment tumor and expanded TILs that may be associated with the clinical response. The treatment protocol comprised preparation of TIL culture, lympho-depleting non-myeloablative preconditioning with cyclophosphamide and fludarabine, TIL infusion, and intravenous administration of low-dose IL-2. Three patients of clinical subtypes mucosal, superficial spreading, and acral melanoma underwent TIL-ACT. Most severe adverse events, including fever and leukopenia, were manageable with the supportive regimen specified in the protocol, suggesting that the TIL-ACT regimen is suitable for Japanese patients with melanoma. One patient showed a short-term partial response, one relatively long-stable disease, and one experienced disease progression. Whole-exome and transcriptional sequencing of isolated tumor cells and immunohistochemical analyses before TIL-ACT revealed various immunostimulatory factors, including a high tumor mutation burden and immune cell-recruiting chemokines, as well as various immunosuppressive factors including TGF-ß, VEGF, Wnt/ß-catenin, and MAPK signaling and epithelial-to-mesenchymal transition, which might influence the efficacy of TIL-ACT. Our results imply mechanisms for the antitumor effect of and resistance to TIL-ACT. Further studies of immune-resistant mechanisms of TIL-ACT are warranted. This study is registered with the UMIN Clinical Trial Registry (UMIN 000011431).

Refined methods to evaluate the in vivo hemostatic function and viability of transfused human platelets in rabbit models.

BACKGROUND: To bridge the gap between in vitro function and clinical efficacy of platelet (PLT) transfusion products, reliable in vivo PLT functional assays for hemostasis and survival in animal models are required. However, there are no standardized methods for assessing the in vivo quality of transfused human PLTs. STUDY DESIGN AND METHODS: Plasma-depleted human PLT concentrates (PCs; Day 3, Day 5, Day 7, Day 10, and damaged) were transfused into busulfan-induced rabbits with thrombocytopenia with prolonged bleeding times 1 day after treatment with ethyl palmitate (EP) to block their reticuloendothelial systems. The hemostatic effect of PC transfusion was evaluated by the ear fine vein bleeding time. For the in vivo survival assay, splenectomized EP-treated rabbits were transfused with human PCs, and viability of the human PLTs in the rabbits was determined by flow cytometry using human PLT-specific antibodies and Trucount tubes. RESULTS: The hemostatic effect of PCs was slightly reduced with increasing storage periods for early time points, but more dramatically reduced for later time points. PLT survival was similar after 3 and 7 days of storage, but PLTs stored for 10 days showed significantly poorer survival than those stored only 3 days. CONCLUSION: Our new and improved protocol for in vivo assessment of transfused PLTs is sufficiently sensitive to detect subtle changes in hemostatic function and viability of human PLTs transfused into rabbit models. This protocol could contribute to preclinical in vivo functional assessment and clinical quality assurance of emerging novel PLT products such as cultured cell-derived human PLTs.

Calcium-dependent activation and autolysis of Arabidopsis metacaspase 2d.

Metacaspases (MCPs) are members of a new family of cysteine proteases found in plants, fungi, and protozoa that are structurally related to metazoan caspases. Recent studies showed that plant MCPs are arginine/lysine-specific cysteine proteases with caspase-like processing activities in vitro and in vivo, and some of the plant type II MCPs exhibit Ca(2+) dependence for their endopeptidase activity in vitro. However, the mechanisms and biological relevance of Ca(2+) dependence and self-processing of plant MCPs remains unclear. Here we show that recombinant AtMCP2d, the most abundantly expressed member of Arabidopsis type II MCPs at the transcriptional level, exhibits a strict Ca(2+) dependence for its catalytic activation that is apparently mediated by intramolecular self-cleavage mechanism. However, rapid inactivation of AtMCP2d activity concomitant with Ca(2+)-induced self-processing at multiple internal sites was observed. Because active AtMCP2d can cleave its inactive form, intermolecular cleavage (autolysis) of AtMCP2d could also occur under our assay conditions. Ca(2+)-induced self-processing of recombinant AtMCP2d was found to correlate with the sequential appearance of at least six intermediates, including self-cleaved forms, during the proenzyme purification process. Six of these peptides were characterized, and the cleavage sites were mapped through N-terminal protein sequencing. Mutation analysis of AtMCP2d revealed that cleavage after Lys-225, which is a highly conserved residue among the six Arabidopsis type II MCPs, is critical for the catalytic activation by Ca(2+), and we demonstrate that this residue is essential for AtMCP2d activation of H(2)O(2)-induced cell death in yeast. Together, our results provide clues to understand the mode of regulation for this class of proteases.

Arabidopsis metacaspase 2d is a positive mediator of cell death induced during biotic and abiotic stresses.

Cysteine proteases such as caspases play important roles in programmed cell death (PCD) of metazoans. Plant metacaspases (MCPs), a family of cysteine proteases structurally related to caspases, have been hypothesized to be ancestors of metazoan caspases, despite their different substrate specificity. Arabidopsis thaliana contains six type II MCP genes (AtMCP2a-f). Whether and how these individual members are involved in controlling PCD in plants remains largely unknown. Here we investigated the function and regulation of AtMCP2d, the predominant and constitutively expressed member of type II MCPs, in stress-inducible PCD. Two AtMCP2d mutants (mcp2d-1 and mcp2d-3) exhibited reduced sensitivity to PCD-inducing mycotoxin fumonisin B1 as well as oxidative stress inducers, whereas AtMCP2d over-expressors were more sensitive to these agents, and exhibited accelerated cell-death progression. We found that AtMCP2d exclusively localizes to the cytosol, and its accumulation and self-processing patterns were age-dependent in leaves. Importantly, active proteolytic processing of AtMCP2d proteins dependent on its catalytic activity was observed in mature leaves during mycotoxin-induced cell death. We also found that mcp2d-1 leaves exhibited reduced cell death in response to Pseudomonas syringae carrying avirulent gene avrRpt2, and that self-processing of AtMCP2d was also detected in wild-type leaves in response to this pathogen. Furthermore, increases in processed AtMCP2d proteins were found to correlate with conditional cell-death induction in two lesion-mimic mutants (cpr22 and ssi4) that exhibit spontaneous cell-death phenotypes. Taken together, our data strongly suggest that AtMCP2d plays a positive regulatory role in biotic and abiotic stress-induced PCD.

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.

SHD1 is a novel cytokine-inducible, negative feedback regulator of STAT5-dependent transcription.

STAT5 is a critical mediator of a variety of cytokine signaling whose transcriptional activity is regulated by associating with various proteins. During a search for STAT5-interacting proteins, we identified SHD1, a mammalian homologue of yeast gene Sac3, as a potential interacter. SHD1 was localized in the nucleus, and induced by cytokines that activate STAT5, such as erythropoietin, interleukin-2 (IL-2), or IL-3. SHD1 interacted specifically with STAT5A and STAT5B, and interestingly, it specifically repressed STAT5-dependent transcription in vitro without affecting the stability or phosphorylation of STAT5 protein. Gene disruption study revealed that T, B, or bone marrow cells from mice lacking SHD1 were hyperresponsive to T-cell-receptor engagement, or stimulation with various STAT5-activating cytokines. These results suggest that SHD1 is a novel cytokine-inducible negative feedback regulator of STAT5.

Genome-wide transposon tagging reveals location-dependent effects on transcription and chromatin organization in Arabidopsis.

The interphase nucleus exists as a highly dynamic system, the physical properties of which have functional importance in gene regulation. Not only can gene expression be influenced by the local sequence context, but also by the architecture of the nucleus in three-dimensions (3D), and by the interactions between these levels via chromatin modifications. A challenging task is to resolve the complex interplay between sequence- and genome structure-based control mechanisms. Here, we created a collection of 277 Arabidopsis lines that allow the visual tracking of individual loci in living plants while comparing gene expression potential at these locations, via an identical reporter cassette. Our studies revealed regional gene silencing near a heterochromatin island, via DNA methylation, that is correlated with mobility constraint and nucleolar association. We also found an example of nucleolar association that does not correlate with gene suppression, suggesting that distinct mechanisms exist that can mediate interactions between chromatin and the nucleolus. These studies demonstrate the utility of this novel resource in unifying structural and functional studies towards a more comprehensive model of how global chromatin organization may coordinate gene expression over large scales.

Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3.

BACKGROUND: Integrin receptors, composed of transmembrane alpha and beta subunits, are essential for the development and functioning of multicellular animals. Agonist stimulation leads cells to regulate integrin affinity ("activation"), thus controlling cell adhesion and migration, controlling extracellular-matrix assembly, and contributing to angiogenesis, tumor cell metastasis, inflammation, the immune response, and hemostasis. A final step in integrin activation is the binding of talin, a cytoskeletal protein, to integrin beta cytoplasmic domains. Many different signaling molecules that regulate integrin affinity have been described, but a pathway that connects agonist stimulation to talin binding and activation has not been mapped. RESULTS: We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet alphaIIbbeta3. Phorbol myristate acetate (PMA) activated alphaIIbbeta3 only after the increased expression of both recombinant protein kinase Calpha (PKCalpha) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas activated Rap1A(G12V) bypassed the requirement for PKC, establishing that Rap1 is downstream of PKC. Talin binding to integrins mediates Rap1-induced activation because Rap1A(G12V) failed to activate alphaIIbbeta3 in cells expressing integrin binding-defective talin (W359A). Rap1 activated integrins by forming an integrin-associated complex containing talin in combination with the Rap effector, RIAM. Furthermore, siRNA-mediated knockdown of RIAM blocked integrin activation. CONCLUSIONS: We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.

Generation of megakaryocytes and platelets from human subcutaneous adipose tissues.

Recent advances in regenerative medicine have created a broad spectrum of stem cell research. Among them, tissue stem cell regulations are important issues to clarify the molecular mechanism of differentiation. Adipose tissues have been shown to contain abundant preadipocytes, which are multipotent to differentiate into cells including adipocytes, chondrocytes, and osteoblasts. In this study, we have first shown that megakaryocytes and platelets can be generated from adipocyte precursor cells. Human adipocyte precursor cells were cultured in conditioned media for 12 days to differentiate adipocytes, followed by 12 days of culture in media containing thrombopoietin. The ultrastructures of adipocyte precursor cell- and bone marrow CD34-positive cell-derived megakaryocytes and platelets were similar. In addition, adipocyte precursor cell-derived platelets exhibited surface expression of P-selectin and bound fibrinogen upon stimulation with platelet agonists, suggesting that these platelets were functional. This is the first demonstration that human subcutaneous adipocyte precursor cells can generate megakaryocyte and functional platelets in an in vitro culture system.

Bax Inhibitor-1, a conserved cell death suppressor, is a key molecular switch downstream from a variety of biotic and abiotic stress signals in plants.

In Nature plants are constantly challenged by a variety of environmental stresses that could lead to disruptions in cellular homeostasis. Programmed cell death (PCD) is a fundamental cellular process that is often associated with defense responses to pathogens, during development and in response to abiotic stresses in fungi, animals and plants. Although there are many characteristics shared between different types of PCD events, it remains unknown whether a common mechanism drives various types of PCD in eukaryotes. One candidate regulator for such a mechanism is Bax Inhibitor-1 (BI-1), an evolutionary conserved, endoplasmic reticulum (ER)-resident protein that represents an ancient cell death regulator that potentially regulates PCD in all eukaryotes. Recent findings strongly suggested that BI-1 plays an important role in the conserved ER stress response pathway to modulate cell death induction in response to multiple types of cell death signals. As ER stress signaling pathways has been suggested to play important roles not only in the control of ER homeostasis but also in other biological processes such as the response to pathogens and abiotic stress in plants, BI-1 might function to control the convergence point that modulates the level of the "pro-survival and pro-death" signals under multiple stress conditions.

Moonlighting vacuolar protease: multiple jobs for a busy protein.

In this Genomics Era with a wealth of annotated sequence data, it is easy to pigeonhole a protein into a particular function. However, Noa Matarasso et al. recently found a vacuolar protease that can also function as a transcription factor. This work illustrates that a protein can serve multiple roles in a cell, raising intriguing questions as to the extent that genomic information can be deciphered de novo.

Analyzing the mechanism of Rap1 activation in platelets: Rap1 activation is related to the release reaction mediated through the collagen receptor GPVI.

The abundant Rap1 in platelets becomes activated when these cells are stimulated by various agonists, but its function has remained unknown. In view of this, we developed an assay to quantitatively measure activated Rap1 and used it to determine relationships between Rap1 activation and several platelet functions: integrin alpha2beta1 activation, tyrosine phosphorylation, and the release reaction. We looked at how these processes are affected by the protein kinase C inhibitor BIMI, tyrosine kinase inhibitor PP2, PI 3-kinase inhibitor wortmannin, and ADP scavenger apyrase. In CRP (collagen related peptide)-activated platelets, all the inhibitors severely inhibited Rap1 activation, but had little effect on integrin alpha2beta1 activation, indicating that the integrin activation mechanism is different from the Rap1 activation mechanism, at least in GPVI-dependent activation. With p85alpha-null mouse platelets, we demonstrated that Rap1 activation involves PI 3-kinase p85alpha-dependent tyrosine phosphorylation. All the inhibitors similarly decreased Rap1 activation and the serotonin release reaction, and the inhibition of Rap1 activation was not due to the lack of released ADP. Our results indicate that platelet Rap1 activation is closely related to the release reaction and not to integrin alpha2beta1 activation in GPVI-mediated platelet activation.

Flagellin from an incompatible strain of Acidovorax avenae mediates H2O2 generation accompanying hypersensitive cell death and expression of PAL, Cht-1, and PBZ1, but not of Lox in rice.

Acidovorax avenae causes a brown stripe disease in monocot plants. We recently reported that a rice-incompatible strain of A. avenae caused hypersensitive cell death in rice and that the flagellin of the incompatible strain was involved in this response. The incompatible strain induced the rapid generation of H2O2 accompanying hypersensitive cell death and the expression of defense genes such as PAL, Cht-1, PBZ1, and LOX, whereas the compatible strain did not. The purified incompatible flagellin also induced the expression of PAL, Cht-1, and PBZ1, but LOX expression was not induced by the incompatible flagellin. PAL and LOX enzymatic activities were increased by inoculation with the incompatible strain, whereas only PAL activity was increased by the incompatible flagellin. Interestingly, the flagellin-deficient incompatible strain lost the ability to generate H2O2 and induce hypersensitive cell death, but PAL, Cht-1, and PBZ1 expression still were induced by inoculation with the deficient strain, suggesting that induction of these genes is regulated not only by flagellin but also by some other signal. Thus, the incompatible flagellin of A. avenae is a specific elicitor in rice, but it is not the only factor capable of inducing the rice defense system.

A novel polymorphism, 70Leu/Phe, disrupts a consensus Leu residue within the leucine-rich repeat sequence of platelet glycoprotein Ibalpha.

Platelet glycoprotein (GP) Ib/IX/V complex mediates high-shear dependent platelet activation through an interaction with the von Willebrand factor (vWF). All four subunits of the complex have a structural motif, the leucine-rich repeat (LRR) sequence, with leucines in conserved positions. Here we report a new polymorphism, Leu/Phe at residue 70 of GPIbalpha, which disrupts the consensus sequence of the LRR in the vWF binding domain. Genotype frequencies among 142 healthy Japanese subjects were 92.3%, 7.7%, and 0.0%, for the 70Leu/Leu, 70Leu/Phe, and 75Phe/Phe genotypes, respectively. Ristocetin-induced or shear-induced platelet aggregation was not significantly different between the 70Leu/Leu and 70Leu/Phe genotypes. In in vitro studies, a recombinant GPIbalpha fragment with 70Phe (L70F) as compared to that with 70Leu (WT) had low reactivity to anti-GPIbalpha monoclonal antibodies, GUR20-5 and Hip1, both of which recognize conformation-specific epitopes within the 45-kDa domain. Ristocetin-induced 125I-vWF binding to L70F, however, did not differ from that to WT.