X-irradiated umbilical cord blood cells retain their regenerative effect in experimental stroke.

Although regenerative therapy with stem cells is believed to be affected by their proliferation and differentiation potential, there is insufficient evidence regarding the molecular and cellular mechanisms underlying this regenerative effect. We recently found that gap junction-mediated cell-cell transfer of small metabolites occurred very rapidly after stem cell treatment in a mouse model of experimental stroke. This study aimed to investigate whether the tissue repair ability of umbilical cord blood cells is affected by X-irradiation at 15 Gy or more, which suppresses their proliferative ability. In this study, X-irradiated mononuclear (XR) cells were prepared from umbilical cord blood. Even though hematopoietic stem/progenitor cell activity was diminished in the XR cells, the regenerative activity was surprisingly conserved and promoted recovery from experimental stroke in mice. Thus, our study provides evidence regarding the possible therapeutic mechanism by which damaged cerebrovascular endothelial cells or perivascular astrocytes may be rescued by low-molecular-weight metabolites supplied by injected XR cells in 10 min as energy sources, resulting in improved blood flow and neurogenesis in the infarction area. Thus, XR cells may exert their tissue repair capabilities by triggering neo-neuro-angiogenesis, rather than via cell-autonomous effects.

Erratum: Acquisition of neural fate by combination of BMP blockade and chromatin modification.

[This corrects the article DOI: 10.1016/j.isci.2023.107887.].

Acquisition of neural fate by combination of BMP blockade and chromatin modification.

Neural induction is a process where naive cells are converted into committed cells with neural characteristics, and it occurs at the earliest step during embryogenesis. Although the signaling molecules and chromatin remodeling for neural induction have been identified, the mutual relationships between these molecules are yet to be fully understood. By taking advantage of the neural differentiation system of mouse embryonic stem (ES) cells, we discovered that the BMP signal regulates the expression of several polycomb repressor complex (PRC) component genes. We particularly focused on Polyhomeotic Homolog 1 (Phc1) and established Phc1-knockout (Phc1-KO) ES cells. We found that Phc1-KO failed to acquire the neural fate, and the cells remained in pluripotent or primitive non-neural states. Chromatin accessibility analysis suggests that Phc1 is essential for chromatin packing. Aberrant upregulation of the BMP signal was confirmed in the Phc1 homozygotic mutant embryos. Taken together, Phc1 is required for neural differentiation through epigenetic modification.

A novel quantitative method to evaluate the contribution of platelet products to white thrombus formation in reconstituted blood under flow conditions.

BACKGROUND AND OBJECTIVES: Currently, the quality of platelet (PLT) products is evaluated using a series of in vitro tests, which only analyse PLTs as an inspection material. However, it would be ideal to assess the physiological functions of PLTs under conditions similar to the sequential blood haemostatic process. In this study, we attempted to establish an in vitro system where the thrombogenicity of PLT products was evaluated in the presence of red blood cells (RBCs) and plasma using a microchamber under constant shear stress (600/s). MATERIALS AND METHODS: Blood samples were reconstituted by mixing PLT products, standard human plasma (SHP) and standard RBCs. Each component was serially diluted keeping the other two components fixed. The samples were applied onto a flow chamber system (Total Thrombus-formation Analysis System [T-TAS]), and white thrombus formation (WTF) was assessed under large arterial shear conditions. RESULTS: We observed a good correlation between the PLT numbers in the test samples and WTF. The WTF of samples containing ≦10% SHP was significantly lower than those containing ≧40% SHP, and no difference was observed in WTF among samples containing 40%-100% SHP. WTF significantly declined in the absence of RBCs, whereas no change in WTF was observed in the presence of RBCs, over haematocrit range of 12.5%-50%. CONCLUSION: The WTF assessed on the T-TAS using reconstituted blood may serve as a new physiological blood thrombus test to quantitatively determine the quality of PLT products.

Automated preparation of washed platelet concentrates through spinning-membrane filtration.

BACKGROUND: Washed platelet concentrates (WPC), prepared with an automated system cell processor (ACP), have recently been approved to be manufactured and marketed in Japan. From the perspective of risk management, it is preferable to secure alternative technologies for ACP. Here, we conducted a study to evaluate the quality of WPC prepared using an automated membrane filtration-based system, Lovo. STUDY DESIGN AND METHODS: Replaced PCs prepared from apheresis PCs were equally divided into control and test units, and subsequently washed using ACP and Lovo respectively. Work and operational efficiencies were evaluated by in vitro analyses, including total handling time, platelet recovery, and plasma protein removal rate. Product quality, including a set of biochemical and physiological indicators of platelets and supernatants, were assessed before and 3 days after washing. RESULTS: In vitro platelet recovery rates and plasma protein removal rates were >85% and >95%, respectively, in both groups. The pH values on day 0 were significantly high (6.97 vs. 6.86) due to low pCO2 in the test group, while no significant differences in glucose consumption and lactate production were observed between the two groups. The levels of hypotonic shock responses, aggregation response, platelet shape, CD62P expression, and sCD62P concentration were similar in both groups during the 3-day storage period. CONCLUSION: Platelet washing with Lovo provides platelet quality equivalent to, or better than, conventional washing with ACP. Thus, the new automated system, Lovo, can be considered as an alternative to ACP for WPC preparation.

Comparison of the programmed freezer method and deep freezer method in the manufacturing of frozen red blood cell products.

BACKGROUND AND OBJECTIVES: Frozen-thawed red blood cells (FTRCs) are useful blood components to patients with rare blood phenotypes. However, frozen red blood cells (FRCs) sometimes cause significant haemolysis after thawing due to the freeze/thaw process. In this study, we aimed to focus on the former process and reduce process-related haemolysis. MATERIALS AND METHODS: Five-day-old red blood cells (RBCs) (5D) or 9-week-old RBCs (9 W) were glycerolized, pooled and split into two aliquots. RBCs were frozen using either the programmed freezer (PF) method or the deep freezer (DF) method. After 4-8 weeks, the FRCs were thawed and washed. In vitro characteristics were compared between the PF and DF methods. Nine week were used as a starting material for FTRCs with the assumption that they can mimic disqualified FTRCs with respect to Hb recovery. RESULTS: The PF method resulted in a significantly higher Hb recovery rate than the DF method (5D: 85.9 ± 2.1 vs. 81.1% ± 3.5%, p < 0.001) (9 W: 56.8 ± 4.0 vs. 52.4% ± 3.5%, p < 0.001). Both 5D and 9W-derived FTRCs immediately after preparation prepared by the PF method were more resistible to haemolysis than those prepared by the DF method. On the other hand, there were no significant differences between PF and DF methods in Adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). CONCLUSION: The PF method was more suitable for RBC freezing than the DF method in terms of Hb recovery in FTRCs. Although it was only 4%-5%, the improvement in the Hb recovery rate will contribute to a more stable supply.

Passive immune basophil activation test for the identification of allergic episodes from various adverse events elicited by haematopoietic cell transplantation: A pilot study.

BACKGROUND AND OBJECTIVES: Haematopoietic cell transplantation (HCT) therapy tends to be associated with various complications including engraftment failure, regimen-related toxicities, and infectious diseases. In addition, HC infusion itself occasionally elicits adverse events (AEs), one of the most common AEs is an allergic reaction. As appropriate laboratory tests have not yet been established to distinguish allergy-mediated AEs from other complications, clinical responses for HCT-related AEs can only be nonspecific. In this pilot study, using passive immune basophil activation test (pi-BAT), we attempted to distinguish an HC infusion-induced allergic reaction from various HCT-related AEs. MATERIALS AND METHODS: Using pi-BAT, we examined 34 patients who underwent HCT, that is, 11 with AEs and 23 without AEs as controls. RESULTS: Two of the eleven AE cases were pi-BAT positive and, the rest of nine AE cases were negative, while all non-AE cases were negative. Both of the two positive cases showed erythema, tachycardia, plus cough. Because erythema is one of the representative symptom of allergy, those cases could be classified as allergic reaction cases or anaphylaxis cases if tachycardia and cough were concomitant symptoms of erythema. Among the nine AEs with pi-BAT negative result, four cases showed urticaria, four showed vomiting plus diarrhoea, and one showed cough. Urticaria case was strongly suspected of allergy, however, the AE cases were pi-BAT negative. CONCLUSION: The pi-BAT may be useful as an auxiliary diagnostic tool to confirm the possible involvement of HC infusion in HCT-related AEs and identify an immunologic mechanism for HCT-related hypersensitivity reactions.

Dual preparation of plasma and platelet concentrates in platelet additive solution from platelet concentrates in plasma using a novel filtration system.

BACKGROUND AND OBJECTIVES: Platelet concentrates suspended in a platelet additive solution (PAS-PC) are associated with a reduction in allergic response and are suitable for preparing pathogen-inactivated PC. We aimed to develop an efficient platform for the dual preparation of PAS-PC and platelet-poor plasma. MATERIALS AND METHODS: PAS-PC was prepared in six steps by using a hollow-fibre system based on cross-flow filtration: priming, loading PC, loading PAS, collection of filtered liquid (flow-through) and collection of platelets by washing with PAS followed by washing with air. In this study, the efficacy of platelet and plasma protein recovery and characteristics of recovered PAS-PC and flow-through plasma were analysed in detail. RESULTS: Recoveries of platelet in PAS-PC and plasma protein in the flow-through were 95.4% ± 3.7% and 61.6% ± 5.0%, respectively. The residual plasma protein in PAS-PC was 34.1% ± 2.8%. Although the expression level of CD62P, a platelet activation marker, in recovered platelets was approximately 1.2-fold of that in original platelets, swirling patterns were well retained, and aggregation in PAS-PC was not visible. Agonist-induced aggregabilities, platelet morphology and hypotonic shock recovery were conserved. The patterns of plasma protein and lipoprotein in the flow-through were comparable with those in the original PCs. The multimeric pattern analysis of VWF remained unaltered. CONCLUSION: We propose a highly efficient preparation system that enables the simultaneous production of PAS-PC and platelet-poor plasma. It also achieves a high recovery of functionally well-retained platelets with very low activation.

UV light-emitting diode (UV-LED) at 265 nm as a potential light source for disinfecting human platelet concentrates.

The risk of sepsis through bacterial transmission is one of the most serious problems in platelet transfusion. In processing platelet concentrates (PCs), several methods have been put into practice to minimize the risk of bacterial transmission, such as stringent monitoring by cultivation assays and inactivation treatment by photoirradiation with or without chemical agents. As another potential option, we applied a light-emitting diode (LED) with a peak emission wavelength of 265 nm, which has been shown to be effective for water, to disinfect PCs. In a bench-scale UV-LED exposure setup, a 10-min irradiation, corresponding to an average fluence of 9.2 mJ/cm2, resulted in >2.0 log, 1.0 log, and 0.6 log inactivation (mean, n = 6) of Escherichia coli, Staphylococcus aureus, and Bacillus cereus, respectively, in non-diluted plasma PCs. After a 30-min exposure, platelet counts decreased slightly (18 ± 7%: mean ± SD, n = 7); however, platelet surface expressions of CD42b, CD61, CD62P, and PAC-1 binding did not change significantly (P>0.005), and agonist-induced aggregation and adhesion/aggregation under flow conditions were well maintained. Our findings indicated that the 265 nm UV-LED has high potential as a novel disinfection method to ensure the microbial safety of platelet transfusion.

Minor impact of patient alloantibodies against human platelet antigen (HPA)-15 in the effectiveness of platelet transfusion: A pilot study.

BACKGROUND: Alloantibodies against human platelet antigen (HPA)-15 are sometimes detected in patients with platelet transfusion refractoriness (PTR); however, little is known about their impact on PTR. STUDY DESIGN AND METHODS: Two patients who possessed HPA-15 alloantibodies (Patient 1, anti-HPA-15b; Patient 2, anti-HPA-15a) and human leukocyte antigen (HLA) antibodies were enrolled. The efficacy of HPA-15-compatible vs -incompatible platelet transfusion was compared by focusing on ABO- and HLA-matched transfusions on the basis of the 24-hour corrected count increment (CCI-24 hours) for platelets. The titers of HPA-15 antibodies in the patients' sera were also monitored. RESULTS: The patients received 71 and 12 ABO-compatible, HLA-matched platelet transfusions, respectively, during the monitoring periods. Among these transfusions, CCI-24 hours could be calculated in 27 and 10 transfusions, respectively, and the HPA-15 genotype of the donors was determined. There were no significant differences in the CCI-24 hours between the HPA-15 compatible and incompatible transfusions in both patients (P = .30 and .56, respectively, Mann-Whitney U test). There was no significant change in the HPA-15b antibody titer in Patient 1 during the monitoring period, while the HPA-15a antibody level in Patient 2 was undetectable at the end of the monitoring period, although the titer was low at the beginning. CONCLUSION: The efficacy of HPA-15-incompatible platelet transfusions was not necessarily inferior to that of HPA-15 compatible ones. Although the case number was limited, our results suggest that HPA-15 antibodies do not have a significant impact on the effects of platelet transfusion.

The Kg-antigen, RhAG with a Lys164Gln mutation, gives rise to haemolytic disease of the newborn.

The Kg-antigen was first discovered in an investigation of a mother whose infant had haemolytic disease of the newborn (HDN). The antibody against the Kg-antigen is believed to be responsible for HDN. The Kg-antigen is provisionally registered under the number 700045, according to the Red Cell Immunogenetics and Blood Group Terminology. However, the molecular nature of the Kg-antigen has remained a mystery for over 30 years. In this study, a monoclonal antibody against the Kg-antigen and the recombinant protein were developed that allowed for the immunoprecipitation analysis. Immunoprecipitants from the propositus' red blood cell ghosts were subjected to mass spectrometry analysis, and DNA sequence analysis of the genes was also performed. A candidate for the Kg-antigen was molecularly isolated and confirmed to be a determinant of the Kg-antigen by cell transfection and flow cytometry analyses. The Kg-antigen and the genetic mutation were then screened for in a Japanese population. The molecular nature of the Kg-antigen was shown to be RhAG with a Lys164Gln mutation. Kg phenotyping further clarified that 0.22% of the Japanese population studied was positive for the Kg-antigen. These findings provide important information on the Kg-antigen, which has been clinically presumed to give rise to HDN.

Frequency of allotype "b" in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high-resolution melt analysis.

BACKGROUND: Antibodies against human platelet antigens (HPAs) cause thrombocytopenias. It is thus important to know the frequency of "b" allotypes in each HPA system for the diagnosis and treatment of anti-HPA antibody-mediated thrombocytopenia. STUDY DESIGN AND METHODS: Genomic DNA was extracted from peripheral blood cells obtained from 2170 blood donors in Japan and was subjected to high-resolution melt (HRM) analysis using polymerase chain reaction for each of the HPA genes, using 23 primer pairs. For genotyping, the resulting amplicons were classified based on their HRM curves. In some cases, direct sequence analysis was performed after HRM analysis to determine nucleotide substitutions. In cases where amino acid substitutions were predicted, protein expression levels were examined in a cell line using 293T cells. RESULTS: The frequencies of each of the HPA-b genotypes were as follows: HPA-1b, 0.4%; HPA-2b, 11.8%; HPA-3b, 41.3%; HPA-4b, 0.8%; HPA-5b, 4.3%; HPA-6b, 1.9%; HPA-15b, 48.8%; HPA-21b, 0.6%; and "b" allotype in the other HPA systems, 0.0%. Twenty-eight variants were found; nine of them were predicted to cause amino acid substitution. However, expression analysis revealed that they did not affect protein expression levels on the cell surface. CONCLUSION: Nine HPA systems are of primary importance in Japan in potentially triggering thrombocytopenia via the HPA antibodies. Similar studies in other countries or races, together with ours, could provide basic information for clinicians in multiethnic societies.

A more efficient preparation system for HLA-eliminated platelets.

BACKGROUND AND OBJECTIVES: Although HLA-eliminated platelets can facilitate transfusions to patients possessing HLA antibodies, no such products are currently available commercially perhaps because the platelet collection rate is not yet economically viable. We have improved this process' efficiency by employing a hollow-fibre system at the last step of the production process after an acid and a reaction buffer have been washed out conventionally by centrifugation. MATERIALS AND METHODS: HLA-eliminated platelets were prepared via four distinct steps: chilled on ice, treated with an acid solution, diluted and finally washed using the hollow-fibre system. The efficiency of this platelet recovery process was determined. The resulting products' platelet characteristics, including a capacity for HLA expression, were evaluated in vitro and compared in detail to their corresponding originals. RESULTS: The average efficiency of platelet recovery was 91%. Although the expression levels of CD62P, a molecular marker for platelet activation, were approximately threefold higher on new platelets than on the original platelets, their HLA expression levels were lower. The phagocytosis assay, with monoclonal antibodies and cognate HLA antibody-containing sera, suggested that HLA-ABC molecules on the cell surface were sufficiently removed. The platelet functions, including the agonist-induced aggregability and adherence/aggregability of the collagen-coated plates under certain conditions, were conserved and not significantly different from the original ones. CONCLUSION: We propose a novel preparation system for producing HLA-eliminated platelets without centrifugation, which ensures a highly efficient, and therefore, much more economical method of platelet recovery that also retains their key functionality.

Sensitivity and specificity of passive immune-basophil activation test to detect allergic transfusion reactions.

BACKGROUND: The basophil activation test (BAT), performed with patient blood samples and supernatants from transfused blood, was developed to elucidate the mechanistic relationship between transfusion and the resultant allergic transfusion reactions (ATRs). This test cannot be performed on myelosuppressed patients and neonates because of the absence of basophils. Therefore, we devised the passive immune basophil activation test (pi-BAT) using patients' plasma and residual transfused blood as sources of immunoglobulin E and allergen, respectively, and the basophils of healthy volunteers served as a source of the responder cells. The sensitivity and specificity of the pi-BAT, however, remained largely unknown. STUDY DESIGN AND METHODS: In this study, the pi-BAT was performed on 31 patients with nonhemolytic transfusion reactions including nine non-ATR and 22 ATR (12 mild and 10 moderate-to-severe) cases to examine its sensitivity and specificity. RESULTS: Nine of the 10 cases with moderate-to-severe ATR tested positive, whereas all the non-ATR cases negative, strongly indicating immunoglobulin E and allergens are involved in the pathogenesis underlying the blood transfusion-triggered adverse effects. CONCLUSION: Thus, we propose that pi-BAT can be used to detect moderate-to-severe ATRs and their underlying mechanisms.

A revised road map for the commitment of human cord blood CD34-negative hematopoietic stem cells.

We previously identified CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells as primitive hematopoietic stem cells (HSCs) in human cord blood. In this study, we develop a prospective ultra-high-resolution purification method by applying two positive markers, CD133 and GPI-80. Using this method, we succeed in purifying single long-term repopulating CD34- HSCs with self-renewing capability residing at the apex of the human HSC hierarchy from cord blood, as evidenced by a single-cell-initiated serial transplantation analysis. The gene expression profiles of individual CD34+ and CD34- HSCs and a global gene expression analysis demonstrate the unique molecular signature of CD34- HSCs. We find that the purified CD34- HSCs show a potent megakaryocyte/erythrocyte differentiation potential in vitro and in vivo. Megakaryocyte/erythrocyte progenitors may thus be generated directly via a bypass route from the CD34- HSCs. Based on these data, we propose a revised road map for the commitment of human CD34- HSCs in cord blood.