Adipose-Derived Mesenchymal Stromal/Stem Cell Line Prevents Hepatic Ischemia/Reperfusion Injury in Rats by Inhibiting Inflammasome Activation.

Mesenchymal stromal/stem cells (MSCs) have shown potential in the treatment of degenerative diseases, including ischemia/reperfusion injury (IRI), which occurs during organ transplantation and represents the main cause of post-transplant graft dysfunction. However, MSCs have heterogeneous characteristics, and studies of MSCs therapy have shown a variety of outcomes. To establish a new effective MSCs therapy, we developed an adipose-derived mesenchymal stromal/stem cell line (ASCL) and compared its therapeutic effects on primary adipose-derived MSCs (ASCs) using a hepatocyte co-culture model of hypoxia/reoxygenation in vitro and a rat model of hepatic IRI in vivo. The results showed that both ASCL and ASCs protect against hypoxia by improving hepatocyte viability, inhibiting reactive oxygen species release, and upregulating transforming growth factor-ß in vitro. In vivo, ASCL or ASCs were infused into the spleen 24 h before the induction of rat hepatic IRI. The results showed that ASCL significantly improved the survival outcomes compared with the control (normal saline infusion) with the significantly decreased serum levels of liver enzymes and less damage to liver tissues compared with ASCs. Both ASCL and ASCs suppressed NOD-like receptor family pyrin domain-containing 3 inflammasome activation and subsequently reduced the release of activated IL-1ß and IL-18, which is considered an important mechanism underlying ASCL and ASCs infusion in hepatic IRI. In addition, ASCL can promote the release of interleukin-1 receptor antagonist, which was previously reported as a key factor in hampering the inflammatory cascade during hepatic IRI. Our results suggest ASCL as a new candidate for hepatic IRI treatment due to its relatively homogeneous characteristics.

Platelet production using adipose-derived mesenchymal stem cells: Mechanistic studies and clinical application.

Megakaryocytes (MKs) are platelet progenitor stem cells found in the bone marrow. Platelets obtained from blood draws can be used for therapeutic applications, especially platelet transfusion. The needs for platelet transfusions for clinical situation is increasing, due in part to the growing number of patients undergoing chemotherapy. Platelets obtained from donors, however, have the disadvantages of a limited storage lifespan and the risk of donor-related infection. Extensive effort has therefore been directed at manufacturing platelets ex vivo. Here, we review ex vivo technologies for MK development, focusing on human adipose tissue-derived mesenchymal stem/stromal cell line (ASCL)-based strategies and their potential clinical application. Bone marrow and adipose tissues contain mesenchymal stem/stromal cells that have an ability to differentiate into MKs, which release platelets. Taking advantage of this mechanism, we developed a donor-independent system for manufacturing platelets for clinical application using ASCL established from adipose-derived mesenchymal stem/stromal cells (ASCs). Culture of ASCs with endogenous thrombopoietin and its receptor c-MPL, and endogenous genes such as p45NF-E2 leads to MK differentiation and subsequent platelet production. ASCs compose heterogeneous cells, however, and are not suitable for clinical application. Thus, we established ASCLs, which expand into a more homogeneous population, and fulfill the criteria for mesenchymal stem cells set by the International Society for Cellular Therapy. Using our ASCL culture system with MK lineage induction medium without recombinant thrombopoietin led to peak production of platelets within 12 days, which may be sufficient for clinical application.

Megakaryocytes and platelets from a novel human adipose tissue-derived mesenchymal stem cell line.

The clinical need for platelet transfusions is increasing; however, donor-dependent platelet transfusions are associated with practical problems, such as the limited supply and the risk of infection. Thus, we developed a manufacturing system for platelets from a donor-independent cell source: a human adipose-derived mesenchymal stromal/stem cell line (ASCL). The ASCL was obtained using an upside-down culture flask method and satisfied the minimal criteria for defining mesenchymal stem cells (MSCs) by The International Society for Cellular Therapy. The ASCL showed its proliferation capacity for ≥2 months without any abnormal karyotypes. The ASCL was cultured in megakaryocyte induction media. ASCL-derived megakaryocytes were obtained, with a peak at day 8 of culture, and ASCL-derived platelets (ASCL-PLTs) were obtained, with a peak at day 12 of culture. We observed that CD42b+ cells expressed an MSC marker (CD90) which is related to cell adhesion. Compared with peripheral platelets, ASCL-PLTs exhibit higher levels of PAC1 binding, P-selectin surface exposure, ristocetin-induced platelet aggregation, and ADP-induced platelet aggregation, as well as similar levels of fibrinogen binding and collagen-induced platelet aggregation. ASCL-PLTs have lower epinephrine-induced platelet aggregation. The pattern of in vivo kinetics after infusion into irradiated immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice was similar to that of platelet concentrates. ASCL-PLTs have similar characteristics to those of peripheral platelets and might have an additional function as MSCs. The establishment of the ASCL and its differentiation into ASCL-PLTs do not require gene transfer, and endogenous thrombopoietin is used for differentiation. The present protocol is a simple method that does not require feeder cells, further enhancing the clinical application of our approach.

Studies of a microchip flow-chamber system to characterize whole blood thrombogenicity in healthy individuals.

INTRODUCTION: A whole blood flow-chamber system, the Total Thrombus-formation Analysis System (T-TAS), was developed for quantitative analysis of platelet thrombus formation (PTF) using microchips with thrombogenic surfaces (collagen, PL chip; collagen plus tissue thromboplastin, AR chip) under shear stress conditions. We evaluated the usefulness of the T-TAS for assessing individual thrombogenicity compared with other platelet function tests. MATERIALS AND METHODS: Blood samples from 31 healthy volunteers were applied to the T-TAS to measure PTF starting time (T10: time to reach 10 kPa), occlusion time (T60 for PL chip; T80 for AR chip), and area under the curve (AUC10, area under curve until 10 min for PL chip; AUC30, 30 min for AR chip) under various shear rates (1000, 1500, 2000s(-1) for PL chip; 300 s(-1) for AR chip). Platelet functions were also tested using platelet aggregometry, the PFA-100 (collagen and epinephrine [C/EPI], collagen and adenosine diphosphate [C/ADP]), and the VerifyNow P2Y12 assay. RESULTS: Individual pressure waveforms, including PTF starting and ending points, varied among healthy subjects. In the PL chip, T10 and AUC10 showed a shear-dependent correlation with C/EPI or C/ADP. VerifyNow P2Y12 values were not significantly associated with the parameters of the T-TAS. Platelet counts were correlated with all AR measurements, and mostly with PL measurements. CONCLUSION: The results of the T-TAS were associated with those of the PFA-100 in many respects, indicating that its characteristics are related to shear-induced PTF. The T-TAS showed few correlations with platelet aggregometry and the VerifyNow P2Y12 assay. The T-TAS may allow for the measurement of comprehensive parameters of individual thrombogenicity under whole blood flow conditions.

OP9 bone marrow stroma cells differentiate into megakaryocytes and platelets.

Platelets are essential for hemostatic plug formation and thrombosis. The mechanisms of megakaryocyte (MK) differentiation and subsequent platelet production from stem cells remain only partially understood. The manufacture of megakaryocytes (MKs) and platelets from cell sources including hematopoietic stem cells and pluripotent stem cells have been highlighted for studying the platelet production mechanisms as well as for the development of new strategies for platelet transfusion. The mouse bone marrow stroma cell line OP9 has been widely used as feeder cells for the differentiation of stem cells into MK lineages. OP9 cells are reported to be pre-adipocytes. We previously reported that 3T3-L1 pre-adipocytes differentiated into MKs and platelets. In the present study, we examined whether OP9 cells differentiate into MKs and platelets using MK lineage induction (MKLI) medium previously established to generate MKs and platelets from hematopoietic stem cells, embryonic stem cells, and pre-adipocytes. OP9 cells cultured in MKLI medium had megakaryocytic features, i.e., positivity for surface markers CD41 and CD42b, polyploidy, and distinct morphology. The OP9-derived platelets had functional characteristics, providing the first evidence for the differentiation of OP9 cells into MKs and platelets. We then analyzed gene expressions of critical factors that regulate megakaryopoiesis and thrombopoiesis. The gene expressions of p45NF-E2, FOG, Fli1, GATA2, RUNX1, thrombopoietin, and c-mpl were observed during the MK differentiation. Among the observed transcription factors of MK lineages, p45NF-E2 expression was increased during differentiation. We further studied MK and platelet generation using p45NF-E2-overexpressing OP9 cells. OP9 cells transfected with p45NF-E2 had enhanced production of MKs and platelets. Our findings revealed that OP9 cells differentiated into MKs and platelets in vitro. OP9 cells have critical factors for megakaryopoiesis and thrombopoiesis, which might be involved in a mechanism of this differentiation. p45NF-E2 might also play important roles in the differentiation of OP9 cells into MK lineages cells.

Effects of VerifyNow P2Y12 test and CYP2C19*2 testing on clinical outcomes of patients with cardiovascular disease: a systematic review and meta-analysis.

The VerifyNow P2Y12 test is a whole-blood, light transmission-based optical detection assay that measures adenosine diphosphate-induced platelet aggregation in a cartridge containing fibrinogen-coated beads. The usefulness of this device to assess the effect of clopidogrel on platelet function was recently highlighted for predicting cardiovascular (CV) events. We therefore performed a meta-analysis to analyze whether adverse clinical outcomes of patients with CV disease increase in association with high on-treatment platelet reactivity (HPR) using the VerifyNow P2Y12 test. We also investigated the effect of the CYP2C19*2 polymorphism on the occurrence of CV events among clopidogrel-treated patients. Relevant studies were searched for in the PubMed database and Cochrane Central Register of Controlled Trials database, and selected if they included data about platelet function assessed using the VerifyNow P2Y12 test or CYP2C19*2 polymorphism in clopidogrel-treated patients with CV disease, and the occurrence of CV events within 6-12 months. In our meta-analysis, 4817 subjects from eight studies and 5307 subjects from seven studies were included for review of platelet function and CYP2C19*2 polymorphism, respectively. Overall, 2237 (46.4%) patients had HPR and these patients had significantly higher odds of CV events compared to patients without HPR (odds ratio(OR) = 3.05, 95% confidence interval: 2.33-3.98, p < 0.001). Heterogeneity among studies was not significant (Cochran's Q-test, p = 0.80 and I(2) = 0%). In the secondary investigation, 1926 (36.3%) patients had at least one CYP2C19*2 allele. Heterogeneity in the OR of the CV events between the CYP2C19*2 allele carriers and non-carriers was observed among the seven studies (Cochran's Q-test, p = 0.01 and I(2) = 56.5%), and the range of the ORs among the seven studies was 0.58-16.6. In conclusion, HPR assessed by VerifyNow P2Y12 test was associated with increased adverse clinical outcomes of clopidogrel-treated patients with CV disease.

Induction of functional platelets from mouse and human fibroblasts by p45NF-E2/Maf.

Determinant factors leading from stem cells to megakaryocytes (MKs) and subsequently platelets have yet to be identified. We now report that a combination of nuclear factor erythroid-derived 2 p45 unit (p45NF-E2), Maf G, and Maf K can convert mouse fibroblast 3T3 cells and adult human dermal fibroblasts into MKs. To screen MK-inducing factors, gene expressions were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells known to differentiate into MKs. 3T3 cells transfected with candidate factors were cultured in a defined MK lineage induction medium. Among the tested factors, transfection with p45NF-E2/MafG/MafK lead to the highest frequency of CD41-positive cells. Adult human dermal fibroblasts transfected with these genes were cultured in MK lineage induction medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 90% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of CD41-positive, platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip, and human CD41-positive platelets were incorporated into thrombi on the chip, demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.

Effect of chronic kidney disease on platelet reactivity to dual-antiplatelet therapy in patients treated with drug-eluting stents.

We investigated the effect of chronic kidney disease (CKD) on platelet function in patients receiving dual-antiplatelet therapy after drug-eluting stent (DES) implantation. We examined 19 patients with CKD and 18 patients without CKD who underwent percutaneous coronary intervention (PCI) with DES. All of the patients had been on chronic aspirin treatment. Blood samples were obtained 20-24 h after a loading dose (300 mg) of clopidogrel. Platelet function was evaluated by measuring the closure time (CT) of a collagen/epinephrine (CEPI) or collagen/adenosine diphosphate (CADP) cartridge in the PFA-100 system. Maximum aggregation of agonist (epinephrine, ADP, collagen, or ristocetin)-induced platelet aggregation was also examined by light transmittance aggregometry. The frequency of the poor responders among CKD (n = 9, 47.4%) patients was significantly higher than that among non-CKD patients (n = 2, 11.1%, P = 0.016) as assessed using a CEPI-CT cartridge. Multiple logistic regression analysis revealed that CKD (odds ratio 7.39, 95% confidence interval 1.38-62.09, P = 0.0183] was the only independent determinant of the poor responders. There were no significant differences between the two groups in the value of CADP-CT or in maximum aggregation of epinephrine-, ADP-, collagen-, and ristocetin-induced platelet aggregation. Compared with non-CKD patients, CKD patients had lower response to aspirin therapy as assessed by the PFA-100 system with a CEPI cartridge. On the other hand, the platelet response to dual-antiplatelet therapy was not different between CKD and non-CKD patients.

Culture of megakaryocytes and platelets from subcutaneous adipose tissue and a preadipocyte cell line.

The molecular mechanisms whereby stem cells develop into platelet-producing megakaryocytes (MKs) are not yet fully understood. Within this chapter we describe a two-step in vitro culture system in which MKs and platelets are generated from primary subcutaneous adipose tissues and the preadipocyte cell line 3T3L1. The cells are first cultured in an adipocyte induction medium for 10-12 days, followed by 8-14 days culture in a MK differentiation medium. Adipose tissue-derived MKs and platelets display a number of morphological and functional characteristics (e.g., secretory granules, open canalicular membranes) comparable with the native cell type. The use of subcutaneous adipose tissue to produce a large number of platelets is advantageous because this tissue is easily obtained and available in large quantities. Thus, this in vitro culture system may prove useful in both regenerative medicine, but it may also be used in understanding fundamental research questions within MK and platelet research, including further elucidation of the pathways that cause cells to differentiate along the MK lineage ultimately leading to platelet production.

GSK-3ß negatively regulates megakaryocyte differentiation and platelet production from primary human bone marrow cells in vitro.

Glycogen synthase kinase (GSK)-3, a constitutively active serine-threonine kinase, acts as a key regulator of major signaling pathways, including the Wnt, Hedgehog, and Notch pathways. Although a number of studies have demonstrated that GSK-3 plays a critical role in several cellular processes, such as differentiation, growth, and apoptosis, the effects of GSK-3 on platelet production have not been explored. There are two GSK-3 isoforms, GSK-3α and GSK-3ß. GSK-3ß is more highly expressed in platelets. In the present study, primary human bone marrow cells were cultured for 12 days in megakaryocyte lineage induction (MKLI) media to induce their differentiation into megakaryocyte (MK) lineage cells, in the presence or absence (+/-) of TWS119, a GSK-3ß inhibitor, during MK differentiation from stem cells and subsequent platelet production. MK maturation, MK production, and subsequent platelet production were markedly enhanced in cells cultured in TWS119 (+) compared with cells cultured in TWS119 (-). These effects on MK lineage cells were thrombopoietin (TPO)-dependent. We next performed the experiment focusing on the inhibitory effect of GSK-3ß on platelet production. Bone marrow cell-derived CD41 (+)/CD42b (+)/propidium iodide (-) cells in the large (MK)-sized cell population (day 8), as living mature MKs, were further cultured in the MKLI media in TWS119 (+/-) for 6 days. Platelet production from mature MKs in TWS119 (+) was approximately two-fold higher than that in TWS119 (-). The mature MKs were cultured in MKLI media in TWS119, in TPO (+/-), and platelet production was markedly decreased in TPO (-). This indicated that the GSK-3ß inhibition affects thrombopoiesis under these conditions with TPO. To identify the target(s) of GSK-3ß inhibition during differentiation into MK lineage cells, we performed a differential gene expression study and subsequent pathway analysis of the large (MK)-sized CD41 (+)/propidium iodide (-) cells cultured in TWS119 (+/-) for 3 days. The results of the analysis indicated that GSK-3ß inhibition during differentiation into MK lineage cells affected factors related to transcription, apoptosis, cell division, cell cycle, blood coagulation, lipid transport, keratin filament, metabolic processes, and the Wnt signaling and transforming growth factor-ß signaling pathways. These observations suggest that GSK-3ß inhibition and TPO treatment affect both megakaryopoiesis and thrombopoiesis in an in vitro differentiation system for primary human bone marrow cells.

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro.

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.

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.

Alpha 2A adrenergic receptor polymorphism is associated with plasma von Willebrand factor levels in a general population.

High levels of plasma von Willebrand factor are a proposed risk factor for atherothrombotic disorders. Previously, ABO blood type and the von Willebrand factor -1793C/G polymorphism were shown to affect interindividual variations in plasma von Willebrand factor levels. We previously reported that polymorphisms of the alpha 2A adrenergic receptor, an epinephrine receptor, were associated with platelet function as assessed by platelet function analyzer-100. The measurement value of platelet function analyzer-100 has been reportedly associated with plasma von Willebrand factor levels. Also, it was demonstrated that epinephrine administration increases plasma von Willebrand factor levels in vivo. Thus, the present study investigated the association between plasma von Willebrand factor levels and genetic polymorphisms as follows: ABO blood type, von Willebrand factor -1051G/A (linked with -1793C/G), alpha 2A adrenergic receptor 1780A/G, and alpha 2A adrenergic receptor 2372A/G. Study subjects were genetically unrelated Japanese men (n = 277) recruited at their regular medical examinations. Genotyping of the polymorphisms was performed using the single nucleotide primer extension-based method. Plasma von Willebrand factor levels were measured as ristocetin-cofactor activities. The O blood type and alpha 2A adrenergic receptor 2372AA genotype were significantly associated with lower von Willebrand factor levels, though von Willebrand factor -1051G/A polymorphism did not affect them. In stratification analysis of the group according to blood type O and non-O, the significant association between the 2372AA genotype and lower plasma von Willebrand factor levels was observed in non-O subjects, but not O subjects. In conclusion, the alpha 2A adrenergic receptor 2372A/G polymorphism is associated with plasma von Willebrand factor levels in a general population.

Coronary artery disease and a functional polymorphism of hTERT.

Genetic variation, a -1327T/C polymorphism, of human telomerase reverse transcriptase (hTERT) is associated with leukocyte telomere length in healthy subjects, but clinical significances of this functional polymorphism are not clear. Recently, the relationship between the telomere system and coronary artery disease (CAD) was reported. We investigated the association between the -1327T/C polymorphism and (a) susceptibility to CAD and (b) telomere length in CAD patients. In a case-control study, 104 patients confirmed by coronary angiography and 115 age- and sex-matched controls were enrolled. There was a higher frequency of the -1327C/C genotype in CAD patients (51.9%) compared with controls (36.5%, p = 0.0218). Among the 104 CAD patients, leukocyte telomere length in the -1327C/C genotype (7.62+/-2.19 kb, mean+/-SD) was shorter than that in the -1327T/C and -1327T/T genotypes (8.74+/-2.92, p = 0.0287). These findings suggest that the -1327C/C genotype is a genetic risk factor for CAD and relates to shorter telomere length among CAD patients.

Identification of ADRA2A polymorphisms related to shear-mediated platelet function.

alpha2A adrenergic receptor (ADRA2A) on platelets interacts with epinephrine, which has a key role in regulating platelet functions. There is familial clustering of inter-individual variations in the epinephrine-induced platelet aggregation, the molecular basis of which, however, has not been fully understood. In this study, we screened the sequence variations in the transcriptional region of ADRA2A gene and analyzed the relationship between the two common polymorphisms and platelet function using epinephrine/collagen cartridge in the platelet function analyzer-100 system, in a healthy Japanese male population (n=211). Among the identified 16 sequence variations including five novel variations, 1780GG genotype was associated with longer closure time which represents low platelet function under high shear-stress conditions (p=0.0478). We also observed enhanced effect of the combination of 1780GG and 2372AA genotypes on longer closure time (p=0.0319). These findings suggest that 1780A/G and 2372A/G polymorphisms are associated with platelet function in interactions with collagen/epinephrine.