A novel method for histological examination of hair follicles.

Intact and healthy hair follicles are important for hair growth after hair follicle transplantation. However, effective and practical evaluation methods for the quality of hair follicles are currently lacking. In the present study, we developed a novel fast staining method for histological examination of hair follicles. The whisker follicles from mice were used to explore the staining protocols, and the final protocol for the evaluation of human hair follicles was derived from animal experiments. After extraction, human hair follicles or mouse whisker follicles were permeabilized with 0.3% Triton X-100. Subsequently, hair follicles were processed by either hematoxylin or alkaline phosphatase staining. The integrity and growth state, including the status of hair follicle stem cells and blood vessels of the extracted hair follicles, were clearly identified under a light microscope. Unhealthy hair follicles from donors or hair follicles broken during extraction were easily revealed by this method. Importantly, it took less than half an hour to obtain images of an individual hair follicle. This method is simple and practical for evaluating the quality and status of hair follicles, providing a fast-screening procedure for hair follicle transplantation.

Copper promotes migration of adipose-derived stem cells by enhancing vimentin-Ser39 phosphorylation.

Mesenchymal stem cells (MSCs) are widely studied for their application in cell therapy. A noticed drawback of these cells in response to tissue injury is the low efficiency of homing. The present study was undertaken to explore a possible approach to promote the migration of MSCs. Primary cultures of rat adipose-derived stem cells (rADSCs) were cultured in standard L-DMEM media supplemented with or without copper (Cu) at its final concentration of 20 µM in cultures. The analyses of transwell and wound-healing assay revealed that Cu supplementation significantly promotes the migration of rADSCs in cultures. Further analysis found that Cu stimulated the phosphorylation of vimentin Ser39. Point mutation of vimentin Ser39 by substituting Ser with Ala prevented Cu-promoted migration of rADSCs. This study thus demonstrates that Cu promotes migration of rADSCs in cultures through at least in part Cu stimulation of vimentin Ser39 phosphorylation.

Copper levels affect targeting of hypoxia-inducible factor 1α to the promoters of hypoxia-regulated genes.

Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that regulates cellular responses to hypoxia. It controls the expression of both BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3) and insulin-like growth factor 2 (IGF2). Previous studies have demonstrated that in hypoxia, copper is required for the expression of BNIP3 but not for that of IGF2 Here, using ChIP assays, computational analyses, luciferase reporter assays, and real-time quantitative RT-PCR, we sought to better understand how copper regulates the differential target gene selectivity of HIF-1α. Human umbilical vein endothelial cells (HUVECs) were exposed to CoCl2 or hypoxia conditions to increase HIF-1α accumulation. The binding of HIF-1α to hypoxia-responsive element (HRE) sites in the BNIP3 or IGF2 gene promoter in high- or low-copper conditions was examined. Our analyses revealed three and two potential HRE sites in the BNIP3 and IGF2 promoters, respectively. We identified that HRE (-412/-404) in the BNIP3 promoter and HRE (-354/-347) in the IGF2 promoter are the critical binding sites of HIF-1α. Tetraethelenepentamine (TEPA)-mediated reduction in copper concentration did not affect hypoxia- or CoCl2-induced HIF-1α accumulation. However, the copper reduction did suppress the binding of HIF-1α to the HRE (-412/-404) in BNIP3 but not the binding of HIF-1α to the HRE (-354/-347) in IGF2 In summary, our findings uncovered the mechanistic basis for differential HIF-1α-mediated regulation of BNIP3 and IGF2, indicating that copper regulates target gene selectivity of HIF-1α at least in part by affecting HIF-1α binding to its cognate HRE in the promoters of these two genes.

Profiling of nuclear copper-binding proteins under hypoxic condition.

Under hypoxic condition, copper (Cu) accumulates in cell nuclei, and regulates the activity of hypoxia-inducible factor-1 (HIF-1) through Cu-binding proteins (CuBPs). To understand the CuBPs in the nucleus, proteomic approach was undertaken to explore the dynamic changes of the CuBPs in response to hypoxia. Human umbilical vein endothelial cells (HUVECs) were treated with dimethyloxalylglycine in a final concentration of 100 µM for 4 h to induce hypoxia, resulting in the accumulation of HIF-1α and Cu in the nucleus. Cu immobilized metal affinity chromatography was applied to extract the CuBPs, followed by identification using nanoliter-liquid chromatograpy combined with quadrupole time of flight tandem mass spectrometry (nanoLC-Q-TOF-MS/MS). There were 278 nuclear proteins that were found as CuBPs in the induced hypoxic group in contrast to 218 CuBPs in the control group. Functional annotation of these proteins in gene ontology category revealed that proteins participating in negative regulation of transcription from RNA polymerase II promoter were dramatically enriched by induced hypoixc treatment. Label-free quantitative proteomic approach identified quantitative changes of nuclear proteome; of 17 differentially expressed proteins, 8 were downregulated and 9 were upregulated in the induced hypoxic nuclei. Four of the 17 proteins were CuBPs, including ILF2 and TRA2B, both were downregulated, and LMNA and HSPB1, both were upregulated. We confirmed the protein change of ALB, LMNA and HSPB1 (HSP27) in real hypoxia, and suggested that the identified CuBPs could be the target for further study of Cu regulation of HIF-1 activity in the nucleus.

A novel knot method for individually measurable aortic constriction in rats.

A novel knot method in rats is reported that addresses several drawbacks in the current model of aortic constriction-induced heart hypertrophy. Using a rat model, we developed a two-step procedure that includes 1) measurement of individual aorta circumference using a surgical thread; and 2) constriction of the aorta using a thread with the desired length predefined by a knot at each end for a measurable reduction of the aortic circumference as referenced to the measurement in step 1. This knot approach produces a manageable gradient of aortic constriction in each rat, reaching a consistency among experimental animals that cannot be achieved by the traditional needle method. Notably, the animal model produced by our knot method showed cardiac hypertrophy and dysfunction with the severity proportional to the percentage reduction of the aorta circumference (50% vs. 60%). Additionally, our new procedure produced a lower mortality rate compared with the traditional needle method. Therefore, we recommend this knot method as an alternative procedure for aortic constriction with desired gradient in rats and larger-animal models.

Cytosolic H2O2 mediates hypertrophy, apoptosis, and decreased SERCA activity in mice with chronic hemodynamic overload.

Oxidative stress in the myocardium plays an important role in the pathophysiology of hemodynamic overload. The mechanism by which reactive oxygen species (ROS) in the cardiac myocyte mediate myocardial failure in hemodynamic overload is not known. Accordingly, our goals were to test whether myocyte-specific overexpression of peroxisomal catalase (pCAT) that localizes in the sarcoplasm protects mice from hemodynamic overload-induced failure and prevents oxidation and inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), an important sarcoplasmic protein. Chronic hemodynamic overload was caused by ascending aortic constriction (AAC) for 12 wk in mice with myocyte-specific transgenic expression of pCAT. AAC caused left ventricular hypertrophy and failure associated with a generalized increase in myocardial oxidative stress and specific oxidative modifications of SERCA at cysteine 674 and tyrosine 294/5. pCAT overexpression ameliorated myocardial hypertrophy and apoptosis, decreased pathological remodeling, and prevented the progression to heart failure. Likewise, pCAT prevented oxidative modifications of SERCA and increased SERCA activity without changing SERCA expression. Thus cardiac myocyte-restricted expression of pCAT effectively ameliorated the structural and functional consequences of chronic hemodynamic overload and increased SERCA activity via a post-translational mechanism, most likely by decreasing inhibitory oxidative modifications. In pressure overload-induced heart failure cardiac myocyte cytosolic ROS play a pivotal role in mediating key pathophysiologic events including hypertrophy, apoptosis, and decreased SERCA activity.

Fabrication of Vascular Grafts Using Poly(ε-Caprolactone) and Collagen-Encapsuled ADSCs for Interposition Implantation of Abdominal Aorta in Rhesus Monkeys.

The production of small-diameter artificial vascular grafts continues to encounter numerous challenges, with concerns regarding the degradation rate and endothelialization being particularly critical. In this study, porous PCL scaffolds were prepared, and PCL vascular grafts were fabricated by 3D bioprinting of collagen materials containing adipose-derived mesenchymal stem cells (ADSCs) on the internal wall of the porous PCL scaffold. The PCL vascular grafts were then implanted in the abdominal aorta of Rhesus monkeys for up to 640 days to analyze the degradation of the scaffolds and regeneration of the aorta. Changes in surface morphology, mechanical properties, crystallization property, and molecular weight of porous PCL revealed a similar degradation process of PCL in PBS at pH 7.4 containing Thermomyces lanuginosus lipase and in situ in the abdominal aorta of rhesus monkeys. The contrast of in vitro and in vivo degradation provided valuable reference data for predicting in vivo degradation based on in vitro enzymatic degradation of PCL for further optimization of PCL vascular graft fabrication. Histological analysis through hematoxylin and eosin (HE) staining and fluorescence immunostaining demonstrated that the PCL vascular grafts successfully induced vascular regeneration in the abdominal aorta over the 640-day period. These findings provided valuable insights into the regeneration processes of the implanted vascular grafts. Overall, this study highlights the significant potential of PCL vascular grafts for the regeneration of small-diameter blood vessels.

Implantation of Adipose-Derived Mesenchymal Stromal Cells (ADSCs)-Lining Prosthetic Graft Promotes Vascular Regeneration in Monkeys and Pigs.

BACKGROUND: Current replacement procedures for stenosis or occluded arteries using prosthetic grafts have serious limitations in clinical applications, particularly, endothelialization of the luminal surface is a long-standing unresolved problem. METHOD: We produced a cell-based hybrid vascular graft using a bioink engulfing adipose-derived mesenchymal stromal cells (ADSCs) and a 3D bioprinting process lining the ADSCs on the luminal surface of GORE-Tex grafts. The hybrid graft was implanted as an interposition conduit to replace a 3-cm-long segment of the infrarenal abdominal aorta in Rhesus monkeys. RESULTS: Complete endothelium layer and smooth muscle layer were fully developed within 21 days post-implantation, along with normalized collagen deposition and crosslinking in the regenerated vasculature in all monkeys. The regenerated blood vessels showed normal functionality for the longest observation of more than 1650 days. The same procedure was also conducted in miniature pigs for the interposition replacement of a 10-cm-long right iliac artery and showed the same long-term effective and safe outcome. CONCLUSION: This cell-based vascular graft is ready to undergo clinical trials for human patients.

Dietary copper supplementation reverses hypertrophic cardiomyopathy induced by chronic pressure overload in mice.

Sustained pressure overload causes cardiac hypertrophy and the transition to heart failure. We show here that dietary supplementation with physiologically relevant levels of copper (Cu) reverses preestablished hypertrophic cardiomyopathy caused by pressure overload induced by ascending aortic constriction in a mouse model. The reversal occurs in the continued presence of pressure overload. Sustained pressure overload leads to decreases in cardiac Cu and vascular endothelial growth factor (VEGF) levels along with suppression of myocardial angiogenesis. Cu supplementation replenishes cardiac Cu, increases VEGF, and promotes angiogenesis. Systemic administration of anti-VEGF antibody blunts Cu regression of hypertrophic cardiomyopathy. In cultured human cardiomyocytes, Cu chelation blocks insulin-like growth factor (IGF)-1- or Cu-stimulated VEGF expression, which is relieved by addition of excess Cu. Both IGF-1 and Cu activate hypoxia-inducible factor (HIF)-1alpha and HIF-1alpha gene silencing blocks IGF-1- or Cu-stimulated VEGF expression. HIF-1alpha coimmunoprecipitates with a Cu chaperone for superoxide dismutase-1 (CCS), and gene silencing of CCS, but not superoxide dismutase-1, prevents IGF-1- or Cu-induced HIF-1alpha activation and VEGF expression. Therefore, dietary Cu supplementation improves the condition of hypertrophic cardiomyopathy at least in part through CCS-mediated HIF-1alpha activation of VEGF expression and angiogenesis.

Metallothionein gene transfection reverses the phenotype of activated human hepatic stellate cells.

Metallothionein (MT) gene therapy leads to resolution of liver fibrosis in mouse model. The present study was undertaken to test the hypothesis that reversal of the phenotype of activated hepatic stellate cells (HSCs) contributes to the fibrinolysis effect of MT. Human HSC LX-2 cells were activated after they were cultured for 24 hours, as indicated by expression of α-smooth muscle actin (α-SMA) and collagen-I and depressed expression of collagenases. Transfection with a plasmid containing human MT-IIA gene in the activated HSCs effectively increased the protein level of MT. The expression of MT was accompanied by the reduction in protein levels of α-SMA and collagen-I and a decrease in their mRNA levels. Of importance, MT gene transfection resulted in upregulation of matrix metalloproteinases 1, 8, and 13, which are involved in the resolution of liver fibrosis. This study demonstrates that reversal of the phenotype of activated HSCs, particularly the upregulation of collagenases, is likely to be involved in the resolution of liver fibrosis observed in MT gene therapy.

Mitochondrial oxidative stress mediates angiotensin II-induced cardiac hypertrophy and Galphaq overexpression-induced heart failure.

RATIONALE: Mitochondrial dysfunction has been implicated in several cardiovascular diseases; however, the roles of mitochondrial oxidative stress and DNA damage in hypertensive cardiomyopathy are not well understood. OBJECTIVE: We evaluated the contribution of mitochondrial reactive oxygen species (ROS) to cardiac hypertrophy and failure by using genetic mouse models overexpressing catalase targeted to mitochondria and to peroxisomes. METHODS AND RESULTS: Angiotensin II increases mitochondrial ROS in cardiomyocytes, concomitant with increased mitochondrial protein carbonyls, mitochondrial DNA deletions, increased autophagy and signaling for mitochondrial biogenesis in hearts of angiotensin II-treated mice. The causal role of mitochondrial ROS in angiotensin II-induced cardiomyopathy is shown by the observation that mice that overexpress catalase targeted to mitochondria, but not mice that overexpress wild-type peroxisomal catalase, are resistant to cardiac hypertrophy, fibrosis and mitochondrial damage induced by angiotensin II, as well as heart failure induced by overexpression of Gαq. Furthermore, primary damage to mitochondrial DNA, induced by zidovudine administration or homozygous mutation of mitochondrial polymerase γ, is also shown to contribute directly to the development of cardiac hypertrophy, fibrosis and failure. CONCLUSIONS: These data indicate the critical role of mitochondrial ROS in cardiac hypertrophy and failure and support the potential use of mitochondrial-targeted antioxidants for prevention and treatment of hypertensive cardiomyopathy.

ABCE1 selectively promotes HIF-1α transactivation of angiogenic gene expression.

BACKGROUND: Copper (Cu), by inhibiting the factor inhibiting HIF-1 (FIH-1), promotes the transcriptional activity of hypoxia-inducible factor-1 (HIF-1). OBJECTIVE: The present study was undertaken to understand the molecular mechanism by which Cu inhibits FIH-1. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with dimethyloxalylglycine (DMOG) resulting in HIF-1α accumulation and the FIH-1 protein complexes were pulled down for candidate protein analysis. The metal binding sites were predicted by both MetalDetector V2.0 and Metal Ion-Binding Site Prediction Server, and then the actual ability to bind to Cu in vitro was tested by both Copper-Immobilized metal affinity chromatography (Cu-IMAC) and Isothermal Titration Calorimetry (ITC). Subsequently, subcellular localization was monitored by immunocytochemistry, GFP-fusion protein expression plasmid and Western blotting in the nuclear extract. The interaction of candidate protein with HIF-1α and FIH-1 was validated by Co-Immunoprecipitation (Co-IP). Finally, the effect of candidate protein on the FIH-1 structure and HIF-1α transcriptional activity was analyzed by the InterEvDock3 web server and real-time quantitative RT-PCR. RESULTS: ATP-binding cassette E1 (ABCE1) was present in the FIH-1 complexes and identified as a leading Cu-binding protein as indicated by a number of possible Cu binding sites. The ability of ABCE1 to bind Cu was demonstrated in vitro. ABCE1 entered the nucleus along with FIH-1 under hypoxic conditions. Protein interaction analysis revealed that ABCE1 prevented FIH-1 to bind iron ions, inhibiting FIH-1 enzymatic activity. ABCE1 silencing suppressed the expression of Cu-dependent HIF-1 target gene BNIP3, not that of Cu-independent IGF-2. CONCLUSION: The results demonstrate that ABCE1, as a Cu-binding protein, enters the nucleus under hypoxic conditions and inhibits FIH-1degradation of HIF-1α, thus promoting HIF-1 transactivation of angiogenic gene expression.

Alteration of endothelial permeability ensures cardiomyocyte survival from ischemic insult in the subendocardium of the heart.

Previous studies have shown that cardiomyocytes in the subendocardial region of myocardium survive from ischemic insult. This study was undertaken to explore possible mechanisms for the survival of these cardiomyocytes, focusing on changes in endothelial cells (ECs) and blood supply. C57/B6 mice were subjected to permanent ligation of left anterior descending (LAD) coronary artery to induce myocardial ischemia (MI). The hearts were harvested at 1, 4, and 7 days post MI and examined for histological changes. It was found that the survival of cardiomyocytes was associated with a preservation of ECs in the subendocardial region, as revealed by EC-specific tdTomato expression transgenic mice (Tie2tdTomato). However, the EC selective proteins, PECAM1 and VEGFR2, were significantly depressed in these ECs. Consequently, the ratio of PECAM1/tdTomato was significantly decreased, indicating a transformation from PECAM1+ ECs to PECAM1- ECs. Furthermore, EC junction protein, VE-cadherin, was not only depressed but also disassociated from PECAM1 in the same region. These changes led to an increase in EC permeability, as evidenced by increased blood infiltration in the subendocardial region. Thus, the increase in the permeability of ECs due to their transformation in the subendocardial region allows blood infiltration, creating a unique microenvironment and ensuring the survival of cardiomyocytes under ischemic conditions.

A clinical feasible stem cell encapsulation ensures an improved wound healing.

Cell encapsulation has proven to be promising in stem cell therapy. However, there are issues needed to be addressed, including unsatisfied yield, unmet clinically friendly formulation, and unacceptable viability of stem cells after cryopreservation and thawing. We developed a novel biosynsphere technology to encapsulate stem cells in clinically-ready biomaterials with controlled microsphere size. We demonstrated that biosynspheres ensure the bioviability and functionality of adipose-derived stromal cells (ADSCs) encapsulated, as delineated by a series of testing procedures. We further demonstrated that biosynspheres protect ADSCs from the hardness of clinically handling such as cryopreservation, thawing, high-speed centrifugation and syringe/nozzle injection. In a swine full skin defect model, we showed that biosynspheres were integrated to the destined tissues and promoted the repair of injured tissues with an accelerating healing process, less scar tissue formation and normalized deposition of collagen type I and type III, the ratio similar to that found in normal skin. These findings underscore the potential of biosynsphere as an improved biofabrication technology for tissue regeneration in clinical setting.

Gene expression patterns and related pathways in the hearts of rhesus monkeys subjected to prolonged myocardial ischemia.

After myocardial infarction (MI) occurs, progressive pathological cardiac remodeling results in heart dysfunction and even heart failure during the following months or years. The present study explored the molecular mechanisms underlying the late phase of MI at the global transcript level. A rhesus monkey model of myocardial ischemia induced by left anterior descending (LAD) artery ligation was established, and the heart tissue was collected eight weeks after ligation for transcriptome analysis by DNA microarray technology. Differentially expressed genes in the core infarcted area and remote infarcted area of the ischemic heart were detected with significance analysis of microarray (SAM), and related pathways were detected by Gene Ontology (GO)/pathway analysis. We found that compared to the sham condition, prolonged ischemia increased the levels of 941 transcripts, decreased the levels of 380 transcripts in the core infarcted area, and decreased the levels of 8 transcripts in the remote area in monkey heart tissue. Loss of coordination between the expression of genes, including natriuretic peptide A (NPPA), NPPB, and corin (Corin, serine peptidase), may aggravate cardiac remodeling. Furthermore, imbalance in the enriched significantly changed pathways, including fibrosis-related pathways, cardioprotective pathways, and the cardiac systolic pathway, likely also plays a key role in regulating the development of heart remodeling.

Zinc-glutathione in Chinese Baijiu prevents alcohol-associated liver injury.

Zinc depletion is associated with alcohol-associated liver injury. We tested the hypothesis that increasing zinc availability along with alcohol consumption prevents alcohol-associated liver injury. Zinc-glutathione (ZnGSH) was synthesized and directly added to Chinese Baijiu. Mice were administered a single gastric dose of 6 g/kg ethanol in Chinese Baijiu with or without ZnGSH. ZnGSH in Chinese Baijiu did not change the likeness of the drinkers but significantly reduced the recovery time from drunkenness along with elimination of high-dose mortality. ZnGSH in Chinese Baijiu decreased serum AST and ALT, suppressed steatosis and necrosis, and increased zinc and GSH concentrations in the liver. It also increased alcohol dehydrogenase and aldehyde dehydrogenase in the liver, stomach, and intestine and reduced acetaldehyde in the liver. Thus, ZnGSH in Chinese Baijiu prevents alcohol-associated liver injury by increasing alcohol metabolism timely with alcohol consumption, providing an alternative approach to the management of alcohol-associated drinking.

Alteration of Transcriptomic Profile and Antiseptic Efficacy of Adipose-Derived Mesenchymal Stromal/Stem Cells Under Different Culture Conditions.

Mesenchymal stromal/stem cells (MSCs) are a promising therapeutic agent for various diseases, including sepsis. However, translating MSC therapy to clinical applications remains challenging due to variations in the properties of MSCs under different preparation conditions. In this study, the gene expression profiles of human adipose-derived mesenchymal stromal/stem cells (ADSCs) under different culture conditions were compared in relation to their therapeutic efficacy for sepsis. Results showed that ADSCs cultured in media supplemented with human platelet lysates (hPL) (hPL-ADSCs) exhibited a smaller cell size and higher proliferative capacity, whereas ADSCs cultured in media supplemented with fetal bovine serum (FBS) (FBS-ADSCs) showed a broader and flatter shape. Both hPL-ADSCs and FBS-ADSCs exhibited a protective effect in a mouse model of sepsis; however, hPL-ADSCs displayed a better potency for immunosuppressive function, as evidenced by a better improvement of survival rate and further reduction of tissue injury and infectious biomarkers (alanine transaminase and procalcitonin). Furthermore, hPL-ADSCs caused a more anti-inflammatory transcriptomic shift, whereas FBS-ADSCs led to more depression of proinflammatory transcriptomic response. This study thus demonstrates that both hPL-ADSCs and FBS-ADSCs are effective for antiseptic therapy via different mechanisms of inflammatory manipulation, although hPL-ADSCs may imply a better preference.

Dose-specific efficacy of adipose-derived mesenchymal stem cells in septic mice.

BACKGROUND: Mesenchymal stem cells (MSCs) therapy for sepsis has been extensively studied in the past decade; however, the treatment regimen and mechanism of action of MSCs remain elusive. Here, we attempted to understand the efficacy and mechanism of action of MSCs on rescuing mice with sepsis. METHODS: A mouse model of sepsis was produced by cecal ligation and puncture (CLP). Allogeneic adipose-derived MSCs (ADSCs) were administered by intravenous infusion at 6 h after CLP, and dose-related effects of ADSCs on these mice were determined by survival rate, histopathological changes, biochemical and coagulation parameters, bacterial load, and plasma levels of endotoxin and inflammatory cytokines. The tissue distribution of intravenously infused ADSCs in septic mice was investigated by pre-labeling ADSCs with the lipophilic membrane dye PKH26. RNA sequencing analysis was performed to assess the transcriptional changes in peripheral blood mononuclear cells (PBMCs) and the liver. RESULTS: A significant therapeutic effect of ADSCs at a dose of 2 × 107 cells/kg in septic mice was evidenced by a remarkable reduction in mortality (35.89% vs. 8.89% survival rate), blood bacterial burden, systemic inflammation, and multiple organ damage. In contrast, ADSCs at a lower dose (1 × 107 cells/kg) failed to achieve any beneficial outcomes, while ADSCs at a higher dose (4 × 107 cells/kg) caused more early death within 24 h after CLP, retaining a steady survival rate of 21.42% thereafter. PKH26-labeled ADSCs were predominantly localized in the lungs of septic mice after intravenous infusion, with only a smaller proportion of PKH26-positive signals appearing in the liver and spleen. RNA sequencing analysis identified that insufficient phagocytic activity of PBMCs in addition to a hyperactivation of the hepatic immune response was responsible for the ineffectiveness of low-dose ADSCs therapy, and acute death caused by high-dose ADSCs infusion was associated with impaired coagulation signaling in PBMCs and exacerbated hepatic hypoxic injury. CONCLUSIONS: Our findings demonstrate a dose-specific effect of ADSCs on the treatment of sepsis due to dose-related interactions between exogenous stem cells and the host's microenvironment. Therefore, a precise dosing regimen is a prerequisite for ADSCs therapy for sepsis.

Protocol to assess fatal embolism risks from human stem cells.

Here, we present a protocol to identify the pro-embolic sub-population of human adipose-derived multipotent stromal cells (ADSCs) and predict fatal embolism risks from ADSC infusion. We describe steps for the collection, processing, and classification of ADSC single-cell RNA-seq data. We then detail the development of a mathematical model for predicting ADSC embolic risk. This protocol allows for the development of prediction models to enhance the assessment of cell quality and advance the clinical applications of stem cells. For complete details on the use and execution of this protocol, please refer to Yan et al. (2022).1.

Copper is required for cobalt-induced transcriptional activity of hypoxia-inducible factor-1.

Cobalt inhibits prolyl hydroxylases, leading to the accumulation of hypoxia-inducible factor-1α (HIF-1α) and a concomitant increase in the transcriptional activity of HIF-1. Therefore, cobalt has been under development as a drug for activating HIF-1 under some disease conditions. However, it has been shown that ischemic conditions resulted in the loss of copper, and the activation of HIF-1 would not occur unless copper was supplemented. The present study was undertaken to test the hypothesis that copper is also required for the cobalt activation of HIF-1 transcriptional activity. Human umbilical vein endothelial cells subjected to treatment with cobalt chloride (CoCl(2)) at concentrations above 25 µM for 2 h resulted in an accumulation of HIF-1α, which was determined by Western blot analysis, and an increase in the expression of vascular endothelial growth factor (VEGF), which was determined by real-time reverse transcription-polymerase chain reaction analysis for mRNA levels and enzyme-linked immunosorbent assay analysis for protein levels. The copper chelator tetraethylenepentamine at 25 µM did not significantly affect the accumulation of HIF-1α but blocked increases in VEGF mRNA and protein levels, an effect that could be reversed by the addition of 25 µM copper sulfate (CuSO(4)). In addition, gene silencing of the copper chaperone for Cu,Zn-superoxide dismutase blocked VEGF expression with little effect on cobalt-induced HIF-1α accumulation. The present study thus demonstrates that copper was required for cobalt-activated transcriptional activity of HIF-1, although copper did not affect cobalt-induced accumulation of HIF-1α in the cells.