Gap Junction-Mediated Transport of Metabolites Between Stem Cells and Vascular Endothelial Cells.

We have previously demonstrated that small molecular transfer, such as glucose, between hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs) and vascular endothelial cells via gap junctions constitutes an important mechanism of stem cell therapy. Cell metabolites are high-potential small-molecule candidates that can be transferred to small molecules between stem cells and vascular endothelial cells. Here, we investigated the differences in metabolite levels between stem cells (HSCs and MSCs), vascular endothelial cells, and the levels of circulating non-hematopoietic white blood cells (WBCs). The results showed remarkable differences in metabolite concentrations between cells. Significantly higher concentrations of adenosine triphosphate (ATP), guanosine triphosphate (GTP), total adenylate or guanylate levels, glycolytic intermediates, and amino acids were found in HSCs compared with vascular endothelial cells. In contrast, there was no significant difference in the metabolism of MSCs and vascular endothelial cells. From the results of this study, it became clear that HSCs and MSCs differ in their metabolites. That is, metabolites that transfer between stem cells and vascular endothelial cells differ between HSCs and MSCs. HSCs may donate various metabolites, several glycolytic and tricarboxylic acid cycle metabolites, and amino acids to damaged vascular endothelial cells as energy sources and activate the energy metabolism of vascular endothelial cells. In contrast, MSCs and vascular endothelial cells regulate each other under normal conditions. As the existing MSCs cannot ameliorate the dysregulation during insult, exogenous MSCs administered by cell therapy may help restore normal metabolic function in the vascular endothelial cells by taking up excess energy sources from the lumens of blood vessels. Results of this study suggested that the appropriate timing of cell therapy is different between HSCs and MSCs.

Pre-Clinical Proof of Concept: Intra-Carotid Injection of Autologous CD34-Positive Cells for Chronic Ischemic Stroke.

This study was conducted to evaluate the safety and efficacy of human peripheral blood CD34 positive (CD34+) cells transplanted into a murine chronic stroke model to obtain pre-clinical proof of concept, prior to clinical testing. Granulocyte colony stimulating factor (G-CSF) mobilized human CD34+ cells [1 × 104 cells in 50 µl phosphate-buffered saline (PBS)] were intravenously (iv) or intra-carotid arterially (ia) transplanted 4 weeks after the induction of stroke (chronic stage), and neurological function was evaluated. In this study, severe combined immune deficiency (SCID) mice were used to prevent excessive immune response after cell therapy. Two weeks post cell therapy, the ia CD34+ cells group demonstrated a significant improvement in neurological functions compared to the PBS control. The therapeutic effect was maintained 8 weeks after the treatment. Even after a single administration, ia transplantation of CD34+ cells had a significant therapeutic effect on chronic stroke. Based on the result of this pre-clinical proof of concept study, a future clinical trial of autologous peripheral blood CD34+ cells administration in the intra-carotid artery for chronic stroke patients is planned.

Increased RNA Transcription of Energy Source Transporters in Circulating White Blood Cells of Aged Mice.

Circulating white blood cells (WBC) contribute toward maintenance of cerebral metabolism and brain function. Recently, we showed that during aging, transcription of metabolism related genes, including energy source transports, in the brain significantly decreased at the hippocampus resulting in impaired neurological functions. In this article, we investigated the changes in RNA transcription of metabolism related genes (glucose transporter 1 [Glut1], Glut3, monocarboxylate transporter 4 [MCT4], hypoxia inducible factor 1-α [Hif1-α], prolyl hydroxylase 3 [PHD3] and pyruvate dehydrogenase kinase 1 [PDK1]) in circulating WBC and correlated these with brain function in mice. Contrary to our expectations, most of these metabolism related genes in circulating WBC significantly increased in aged mice, and correlation between their increased RNA transcription and impaired neurological functions was observed. Bone marrow mononuclear transplantation into aged mice decreased metabolism related genes in WBC with accelerated neurogenesis in the hippocampus. In vitro analysis revealed that cell-cell interaction between WBC and endothelial cells via gap junction is impaired with aging, and blockade of the interaction increased their transcription in WBC. Our findings indicate that gross analysis of RNA transcription of metabolism related genes in circulating WBC has the potential to provide significant information relating to impaired cell-cell interaction between WBC and endothelial cells of aged mice. Additionally, this can serve as a tool to evaluate the change of the cell-cell interaction caused by various treatments or diseases.

Bone Marrow Mononuclear Cells Transplantation and Training Increased Transplantation of Energy Source Transporters in Chronic Stroke.

OBJECTIVES: Bone marrow mononuclear cells (BM-MNC) show a significant therapeutic effect in combination with training even in the chronic phase of stroke. However, the mechanism of this combination therapy has not been investigated. Here, we examined its effects on brain metabolism in chronic stroke mice. MATERIALS AND METHODS: BM-MNC (1x105 cells in 100 µL of phosphate-buffered saline) were intravenously transplanted at 4 weeks (chronic stage) after the middle cerebral artery occlusion. At 3 h and 10 weeks after the administration of BM-MNC, we evaluated transcription changes of the metabolism-related genes, hypoxia inducible factor 1-α (Hif-1α), prolyl hydroxylase 3 (Phd3), pyruvate dehydrogenase kinase 1 (Pdk1), Na+/K+-ATPase (Atp1α1‒3), connexins, glucose transporters, and monocarboxylate transporters, in the brain during chronic phase of stroke using quantitative polymerase chain reaction. RESULTS: The results showed transcriptional activation of the metabolism-related genes in the contralateral cortex at 3 h after BM-MNC transplantation. Behavioral tests were performed after cell therapy, and the brain metabolism of mice with improved motor function was examined at 10 weeks after cell therapy. The therapeutic efficacy of the combination therapy with BM-MNC transplantation and training was evident in the form of transcriptional activation of ipsilateral anterior cerebral artery (ACA) cortex. CONCLUSIONS: BM-MNC transplantation combined with training for chronic stroke activated gene expression in both the ipsilateral and the contralateral side.

Gap junction-mediated cell-cell interaction between transplanted mesenchymal stem cells and vascular endothelium in stroke.

We have shown previously that transplanted bone marrow mononuclear cells (BM-MNC), which are a cell fraction rich in hematopoietic stem cells, can activate cerebral endothelial cells via gap junction-mediated cell-cell interaction. In the present study, we investigated such cell-cell interaction between mesenchymal stem cells (MSC) and cerebral endothelial cells. In contrast to BM-MNC, for MSC we observed suppression of vascular endothelial growth factor uptake into endothelial cells and transfer of glucose from endothelial cells to MSC in vitro. The transfer of such a small molecule from MSC to vascular endothelium was subsequently confirmed in vivo and was followed by suppressed activation of macrophage/microglia in stroke mice. The suppressive effect was absent by blockade of gap junction at MSC. Furthermore, gap junction-mediated cell-cell interaction was observed between circulating white blood cells and MSC. Our findings indicate that gap junction-mediated cell-cell interaction is one of the major pathways for MSC-mediated suppression of inflammation in the brain following stroke and provides a novel strategy to maintain the blood-brain barrier in injured brain. Furthermore, our current results have the potential to provide a novel insight for other ongoing clinical trials that make use of MSC transplantation aiming to suppress excess inflammation, as well as other diseases such as COVID-19 (coronavirus disease 2019).

Intravenous Bone Marrow Mononuclear Cells Transplantation Improves the Effect of Training in Chronic Stroke Mice.

There is no effective treatment for chronic stroke if the acute or subacute phase is missed. Rehabilitation alone cannot easily achieve a dramatic recovery in function. In contrast to significant therapeutic effects of bone marrow mononuclear cells (BM-MNC) transplantation for acute stroke, mild and non-significant effects have been shown for chronic stroke. In this study, we have evaluated the effect of a combination of BM-MNC transplantation and neurological function training in chronic stroke. The effect of BM-MNC on neurological functional was tested four weeks after permanent middle cerebral artery occlusion (MCAO) insult in mice. BM-MNC (1 × 105cells in 100 µl PBS) were injected into the vein of MCAO model mice, followed by behavioral tests as functional evaluations. Interestingly, there was a significant therapeutic effect of BM-MNC only when repeated training was performed. This suggested that cell therapy alone was not sufficient for chronic stroke treatment; however, training with cell therapy was effective. The combination of these differently targeted therapies provided a significant benefit in the chronic stroke mouse model. Therefore, targeted cell therapy via BM-MNC transplantation with appropriate training presents a promising novel therapeutic option for patients in the chronic stroke period.

Intravenous Bone Marrow Mononuclear Cells Transplantation in Aged Mice Increases Transcription of Glucose Transporter 1 and Na+/K+-ATPase at Hippocampus Followed by Restored Neurological Functions.

We recently reported that intravenous bone marrow mononuclear cell (BM-MNC) transplantation in stroke improves neurological function through improvement of cerebral metabolism. Cerebral metabolism is known to diminish with aging, and the reduction of metabolism is one of the presumed causes of neurological decline in the elderly. We report herein that transcription of glucose transporters, monocarboxylate transporters, and Na+/K+-ATPase is downregulated in the hippocampus of aged mice with impaired neurological functions. Intravenous BM-MNC transplantation in aged mice stimulated the transcription of glucose transporter 1 and Na+/K+-ATPase α1 followed by restoration of neurological function. As glucose transporters and Na+/K+-ATPases are closely related to cerebral metabolism and neurological function, our data indicate that BM-MNC transplantation in aged mice has the potential to restore neurological function by activating transcription of glucose transporter and Na+/K+-ATPase. Furthermore, our data indicate that changes in transcription of glucose transporter and Na+/K+-ATPase could be surrogate biomarkers for age-related neurological impairment as well as quantifying the efficacy of therapies.

Bone Marrow Mononuclear Cells Activate Angiogenesis via Gap Junction-Mediated Cell-Cell Interaction.

Background and Purpose- Bone marrow mononuclear cells (BM-MNCs) are a rich source of hematopoietic stem cells and have been widely used in experimental therapies for patients with ischemic diseases. Activation of angiogenesis is believed to be one of major BM-MNC mode of actions, but the essential mechanism by which BM-MNCs activate angiogenesis have hitherto been elusive. The objective of this study is to reveal the mechanism how BM-MNCs activate angiogenesis. Methods- We have evaluated the effect of direct cell-cell interaction between BM-MNC and endothelial cell on uptake of VEGF (vascular endothelial growth factor) into endothelial cells in vitro. Cerebral ischemia model was used to evaluate the effects of direct cell-cell interaction with transplanted BM-MNC on endothelial cell at ischemic tissue. Results- The uptake of VEGF into endothelial cells was increased by BM-MNC, while being inhibited by blockading the gap junction. Low-molecular-weight substance was transferred from BM-MNC into endothelial cells via gap junctions in vivo, followed by increased expression of hypoxia-inducible factor-1α and suppression of autophagy in endothelial cells. The concentration of glucose in BM-MNC cytoplasm was significantly higher than in endothelial cells, and transfer of glucose homologue from BM-MNC to endothelial cells was observed. Conclusions- Our findings demonstrated cell-cell interaction via gap junction is the prominent pathway for activation of angiogenesis at endothelial cells after ischemia and provided novel paradigm that energy source supply by stem cell to injured cell is one of the therapeutic mechanisms of cell-based therapy. Visual Overview- An online visual overview is available for this article.

Up-regulation of CD64 Expression on Monocytes in Patients With Active Adult-Onset Still Disease: A Possible Biomarker of Disease Activity.

OBJECTIVES: In this study, we investigated whether monocyte CD64 (mCD64) expression is correlated with disease activity in patients with adult-onset Still disease (AOSD) and whether it could be used to distinguish between active and inactive disease states. METHODS: We reviewed a series of 10 patients with a definite diagnosis of AOSD, recruited from January 2013 to December 2016. We used flow cytometry to quantitatively measure mCD64 expression levels in patients presenting with active and inactive disease states and statistically analyzed the corresponding changes. RESULTS: The mean ± SD values of mCD64 expression levels in patients with active and inactive disease states were 77,148.3 ± 39,066.3 and 19,225.8 ± 7006.2 molecules/cell, respectively, indicating significantly higher mCD64 expression in the active state than in the inactive state (p = 0.005). Receiver operating characteristic analysis with a cutoff value of 31,796.0 molecules/cell was applied to distinguish active from inactive disease states; the sensitivity and specificity were both 100%. In these patients, only the mCD64 expression levels changed in parallel with disease activity under tocilizumab treatment; other conventional biomarkers measured showed no changes. CONCLUSIONS: Monocyte CD64 expression could be used to clearly distinguish between active and inactive AOSD. Thus, mCD64 could be a promising biomarker for evaluating the disease activity of AOSD, even in patients receiving tocilizumab treatment.

Clot-Derived Contaminants in Transplanted Bone Marrow Mononuclear Cells Impair the Therapeutic Effect in Stroke.

Background and Purpose- The beneficial effects of bone marrow mononuclear cell (BM-MNC) transplantation in preclinical experimental stroke have been reliably demonstrated. However, only overall modest effects in clinical trials were observed. We have investigated and reported a cause of the discrepancy between the preclinical and clinical studies. Methods- To investigate the possible cause of low efficacy of BM-MNC transplantation in experimental stroke, we have focused on blood clot formation, which is not uncommon in human bone marrow aspirates. To evaluate the effects of clot-derived contaminants in transplanted BM-MNC on stroke outcome, a murine stroke model was used. Results- We show that BM-MNC separated by an automatic cell isolator (Sepax2), which does not have the ability to remove clots, did not attenuate brain atrophy after stroke. In contrast, manually isolated, clot-free BM-MNC exerted therapeutic effects. Clot-derived contaminants were also transplanted intravenously to poststroke mice. We found that the transplanted contaminants were trapped at the peristroke area, which were associated with microglial/macrophage activation. Conclusions- Clot-derived contaminants in transplanted BM-MNC nullify therapeutic effects in experimental stroke. This may explain neutral results in clinical trials, especially in those using automated stem cell separators that lack the ability to remove clot-derived contaminants. Visual Overview- An online visual overview is available for this article.

Diffusion-weighted Whole-body Imaging with Background Body Signal Suppression (DWIBS) as a Novel Imaging Modality for Disease Activity Assessment in Takayasu's Arteritis.

A 26-year-old woman with Takayasu's arteritis (TAK) experienced back and neck pain during tocilizumab (TCZ) treatment. The levels of C-reactive protein were normal, and ultrasonography revealed no significant changes. Diffusion-weighted whole-body imaging with background body signal suppression (DWIBS) showed signal enhancement in the walls of several arteries. Contrast computed tomography showed arterial inflammation in the same lesion. After increasing the dose of prednisolone and TCZ, all signal enhancements decreased and continued to decrease, as observed on days 76 and 132. Thus, DWIBS may be a novel imaging modality for assessing the disease activity of TAK, particularly during follow-up.

Decreased levels of circulating CD34(+) cells are associated with coronary heart disease in Japanese patients with type 2 diabetes.

AIMS/INTRODUCTION: Circulating progenitor cells, including CD34 positive (CD34(+)) cells, play a key role in neovascularisation and the maintenance of vascular endothelial function. Several lines of evidence show an association between decreased levels of circulating CD34(+) cells and cardiovascular disease. However, the contribution of circulating CD34(+) cells to the occurrence of cardiovascular events in diabetic patients remains unclear. MATERIALS AND METHODS: In the present study with a median follow up of 4.6 years, we analyzed the level of circulating CD34(+) cells in 192 patients with type 2 diabetes. The outcome variables were coronary heart disease (CHD) events (cardiovascular death, unstable angina, myocardial infarction, percutaneous coronary intervention or coronary artery bypass grafting) and cerebrovascular disease events (cerebral infarction, cerebral hemorrhage or transient ischemic attack). RESULTS: Decreased levels of circulating CD34(+) cells were associated with a significantly higher incidence of CHD based on Kaplan-Meier analysis (P = 0.0052). After adjusting for age, sex, dyslipidemia, hypertension, glycated hemoglobin, history of cardiovascular disease, body mass index, and statin and renin angiotensin system inhibitors use, decreased levels of CD34(+) cells were significantly associated with the incidence of CHD events (hazard ratio of low tertile 2.61, 95% confidence interval 1.22-5.96; P = 0.013, reference; high tertile). CONCLUSIONS: Decreased levels of circulating CD34(+) cells might predict CHD events in patients with diabetes, and this could be useful for identifying patients with diabetes at high risk of cardiovascular events.

Microstructural abnormality in white matter, regulatory T lymphocytes, and depressive symptoms after stroke.

BACKGROUND: The purpose of the present study was to investigate the existence of microstructure abnormalities in the white matter circuit in stroke patients and its relationship to depressive episodes. To target the prevention of depression, we also investigated the relationship between lymphocyte subsets and cerebral abnormalities in patients. METHODS: Participants included 18 patients with acute ischemic stroke and 22 healthy control subjects. Diffusion tensor imaging was performed. Whole-brain voxel-based analysis was used to compare fractional anisotropy (FA) between groups. Blood samples were obtained, and the lymphocyte subsets were evaluated using flow cytometry. Follow-up examinations were conducted on 12 patients at 6 months. RESULTS: FA was decreased in the bilateral anterior limb of the internal capsule in stroke patients. At the 6-month follow-up examination, there was a significant increase in FA, which was associated with a lower depression scale score. Patients showed a decreased percentage of circulated regulatory T lymphocytes, and the degree of reduction was related to the decrease in the FA value in the internal capsule. CONCLUSIONS: FA reductions in the anterior limb of the internal capsule cause abnormality in the frontal-subcortical circuits and confer a biological vulnerability, which in combination with environmental stressors results in the onset of depression. Our findings also demonstrated the possibility of preventing post-stroke depression by targeting the role of regulatory T lymphocytes in brain tissue repair and regeneration after stroke.

Human umbilical cord provides a significant source of unexpanded mesenchymal stromal cells.

BACKGROUND AIMS: Human mesenchymal stromal cells (MSC) have considerable potential for cell-based therapies, including applications for regenerative medicine and immune suppression in graft-versus-host disease (GvHD). However, harvesting cells from the human body can cause iatrogenic disorders and in vitro expansion of MSC carries a risk of tumorigenesis and/or expansion of unexpected cell populations. METHODS: Given these problems, we have focused on umbilical cord, a tissue obtained with few ethical problems that contains significant numbers of MSC. We have developed a modified method to isolate MSC from umbilical cord, and investigated their properties using flow cytometry, mRNA analysis and an in vivo GvHD model. RESULTS: Our study demonstrates that, using umbilical cord, large numbers of MSC can be safely obtained using a simple procedure without in vitro expansion, and these non-expanded MSC have the potential to suppress GvHD. CONCLUSIONS: Our results suggest that the combined banking of umbilical cord-derived MSC and identical cord blood-derived hematopoietic stem cell banking, where strict inspection of the infectious disease status of donors is performed, as well as further benefits of HLA-matched mesenchymal cells, could become one of the main sources of cells for cell-based therapy against various disorders.

Mouse ES cells maintained in different pluripotency-promoting conditions differ in their neural differentiation propensity.

Prior to differentiation, embryonic stem (ES) cells in culture are maintained in a so-called "undifferentiated" state, allowing derivation of multiple downstream cell lineages when induced in a directed manner, which in turn grants these cells their "pluripotent" state. The current work is based on a simple observation that the initial culture condition for maintaining mouse ES cells in an "undifferentiated" state does impact on the differentiation propensity of these cells, in this case to a neuronal fate. We point out the importance in judging the "pluripotency" of a given stem cell culture, as this clearly demonstrated that the "undifferentiated" state of these cells is not necessarily a "pluripotent" state, even for a widely used mouse ES cell line. We partly attribute this difference in the initial value of ES cells to the naïve-to-primed status of pluripotency, which in turn may affect early events of the differentiation in vitro.

Random BAC FISH of monocot plants reveals differential distribution of repetitive DNA elements in small and large chromosome species.

BAC FISH (fluorescence in situ hybridization using bacterial artificial chromosome probes) is a useful cytogenetic technique for physical mapping, chromosome marker screening, and comparative genomics. As a large genomic fragment with repetitive sequences is inserted in each BAC clone, random BAC FISH without adding competitive DNA can unveil complex chromosome organization of the repetitive elements in plants. Here we performed the comparative analysis of the random BAC FISH in monocot plants including species having small chromosomes (rice and asparagus) and those having large chromosomes (hexaploid wheat, onion, and spider lily) in order to understand a whole view of the repetitive element organization in Poales and Asparagales monocots. More unique and less dense dispersed signals of BAC FISH were observed in species with smaller chromosomes in both the Poales and Asparagales species. In the case of large-chromosome species, 75-85% of the BAC clones were detected as dispersed repetitive FISH signals along entire chromosomes. The BAC FISH of Lycoris did not even show localized repetitive patterns (e.g., centromeric localization) of signals.

Reduced ischemic brain injury by partial rejuvenation of bone marrow cells in aged rats.

Circulating bone marrow-derived immature cells, including endothelial progenitor cells, have been implicated in homeostasis of the microvasculature. Decreased levels of circulating endothelial progenitor cells, associated with aging and/or cardiovascular risk factors, correlate with poor clinical outcomes in a range of cardiovascular diseases. Herein, we transplanted bone marrow cells from young stroke-prone spontaneously hypertensive rats (SHR-SP) into aged SHR-SP, the latter not exposed to radiation or chemotherapy. Analysis of recipient peripheral blood 28 days after transplantation revealed that 5% of circulating blood cells were of donor origin. Cerebral infarction was induced on day 30 posttransplantation. Animals transplanted with bone marrow from young SHR-SP displayed an increase in density of the microvasculature in the periinfarction zone, reduced ischemic brain damage and improved neurologic function. In vitro analysis revealed enhanced activation of endothelial nitric oxide synthase and reduced activation p38 microtubule-associated protein (MAP) kinase, the latter associated with endothelial apoptosis, in cultures exposed to bone marrow-derived mononuclear cells from young animals versus cells from aged counterparts. Our findings indicate that partial rejuvenation of bone marrow from aged rats with cells from young animals enhances the response to ischemic injury, potentially at the level of endothelial/vascular activation, providing insight into a novel approach ameliorate chronic vascular diseases.

Injury-induced neural stem/progenitor cells in post-stroke human cerebral cortex.

Increasing evidence points to accelerated neurogenesis after stroke, and support of such endogenous neurogenesis has been shown to improve stroke outcome in experimental animal models. The present study analyses post-stroke cerebral cortex after cardiogenic embolism in autoptic human brain. Induction of nestin- and musashi-1-positive cells, potential neural stem/progenitor cells, was observed at the site of ischemic lesions from day 1 after stroke. These two cell populations were present at distinct locations and displayed different temporal profiles of marker expression. However, no surviving differentiated mature neural cells were observed by 90 days after stroke in the previously ischemic region. Consistent with recent reports of neurogenesis in the cerebral cortex after induction of stroke in rodent models, the present current data indicate the presence of a regional regenerative response in human cerebral cortex. Furthermore, observations underline the potential importance of supporting survival and differentiation of endogenous neural stem/progenitor cells in post-stroke human brain.

Circulating CD34-positive cells have prognostic value for neurologic function in patients with past cerebral infarction.

Increasing evidence points to a role for circulating endothelial progenitors, including populations of CD34-positive (CD34(+)) cells present in peripheral blood, in vascular homeostasis and neovascularization. In this report, circulating CD34(+) cells in individuals with a history of cerebral infarction were correlated with changes in neurologic function over a period of 1 year. Patients with decreased levels of CD34(+) cells displayed significant worsening in neurologic function, evaluated by the Barthel Index and Clinical Dementia Rating. These results support the hypothesis that levels of circulating CD34(+) cells have prognostic value for neural function, consistent with their potential role in maintaining cerebral circulation.