Translating mesenchymal stem cell and their exosome research into GMP compliant advanced therapy products: Promises, problems and prospects.

Mesenchymal stem cells (MSCs) are one of the few stem cell types used in clinical practice as therapeutic agents for immunomodulation and ischemic tissue repair, due to their unique paracrine capacity, multiple differentiation potential, active components in exosomes, and effective mitochondria donation. At present, MSCs derived from tissues such as bone marrow and umbilical cord are widely applied in preclinical and clinical studies. Nevertheless, there remain challenges to the maintenance of consistently good quality MSCs derived from different donors or tissues, directly impacting their application as advanced therapy products. In this review, we discuss the promises, problems, and prospects associated with translation of MSC research into a pharmaceutical product. We review the hurdles encountered in translation of MSCs and MSC-exosomes from the research bench to an advanced therapy product compliant with good manufacturing practice (GMP). These difficulties include how to set up GMP-compliant protocols, what factors affect raw material selection, cell expansion to product formulation, establishment of quality control (QC) parameters, and quality assurance to comply with GMP standards. To avoid human error and reduce the risk of contamination, an automatic, closed system that allows real-time monitoring of QC should be considered. We also highlight potential advantages of pluripotent stem cells as an alternative source for MSC and exosomes generation and manufacture.

Adverse clinical outcomes and immunosuppressive microenvironment of RHO-GTPase activation pattern in hepatocellular carcinoma.

BACKGROUND: Emerging evidence suggests that Rho GTPases play a crucial role in tumorigenesis and metastasis, but their involvement in the tumor microenvironment (TME) and prognosis of hepatocellular carcinoma (HCC) is not well understood. METHODS: We aim to develop a tumor prognosis prediction system called the Rho GTPases-related gene score (RGPRG score) using Rho GTPase signaling genes and further bioinformatic analyses. RESULTS: Our work found that HCC patients with a high RGPRG score had significantly worse survival and increased immunosuppressive cell fractions compared to those with a low RGPRG score. Single-cell cohort analysis revealed an immune-active TME in patients with a low RGPRG score, with strengthened communication from T/NK cells to other cells through MIF signaling networks. Targeting these alterations in TME, the patients with high RGPRG score have worse immunotherapeutic outcomes and decreased survival time in the immunotherapy cohort. Moreover, the RGPRG score was found to be correlated with survival in 27 other cancers. In vitro experiments confirmed that knockdown of the key Rho GTPase-signaling biomarker SFN significantly inhibited HCC cell proliferation, invasion, and migration. CONCLUSIONS: This study provides new insight into the TME features and clinical use of Rho GTPase gene pattern at the bulk-seq and single-cell level, which may contribute to guiding personalized treatment and improving clinical outcome in HCC.

The expression mechanism of programmed cell death 1 ligand 1 and its role in immunomodulatory ability of mesenchymal stem cells.

Programmed cell death 1 ligand 1 (PD-L1) is an important immunosuppressive molecule, which inhibits the function of T cells and other immune cells by binding to the receptor programmed cell death-1. The PD-L1 expression disorder plays an important role in the occurrence, development, and treatment of sepsis or other inflammatory diseases, and has become an important target for the treatment of these diseases. Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with multiple differentiation potential. In recent years, MSCs have been found to have a strong immunosuppressive ability and are used to treat various inflammatory insults caused by hyperimmune diseases. Moreover, PD-L1 is deeply involved in the immunosuppressive events of MSCs and plays an important role in the treatment of various diseases. In this review, we will summarize the main regulatory mechanism of PD-L1 expression, and discuss various biological functions of PD-L1 in the immune regulation of MSCs.

FGF21-Sirtuin 3 Axis Confers the Protective Effects of Exercise Against Diabetic Cardiomyopathy by Governing Mitochondrial Integrity.

BACKGROUND: Exercise is an effective nonpharmacological strategy to alleviate diabetic cardiomyopathy (DCM) through poorly defined mechanisms. FGF21 (fibroblast growth factor 21), a peptide hormone with pleiotropic benefits on cardiometabolic homeostasis, has been identified as an exercise responsive factor. This study aims to investigate whether FGF21 signaling mediates the benefits of exercise on DCM, and if so, to elucidate the underlying mechanisms. METHODS: The global or hepatocyte-specific FGF21 knockout mice, cardiomyocyte-selective ß-klotho (the obligatory co-receptor for FGF21) knockout mice, and their wild-type littermates were subjected to high-fat diet feeding and injection of streptozotocin to induce DCM, followed by a 6-week exercise intervention and assessment of cardiac functions. Cardiac mitochondrial structure and function were assessed by electron microscopy, enzymatic assays, and measurements of fatty acid oxidation and ATP production. Human induced pluripotent stem cell-derived cardiomyocytes were used to investigate the receptor and postreceptor signaling pathways conferring the protective effects of FGF21 against toxic lipids-induced mitochondrial dysfunction. RESULTS: Treadmill exercise markedly induced cardiac expression of ß-klotho and significantly attenuated diabetes-induced cardiac dysfunction in wild-type mice, accompanied by reduced mitochondrial damage and increased activities of mitochondrial enzymes in hearts. However, such cardioprotective benefits of exercise were largely abrogated in mice with global or hepatocyte-selective ablation of FGF21, or cardiomyocyte-specific deletion of ß-klotho. Mechanistically, exercise enhanced the cardiac actions of FGF21 to induce the expression of the mitochondrial deacetylase SIRT3 by AMPK-evoked phosphorylation of FOXO3, thereby reversing diabetes-induced hyperacetylation and functional impairments of a cluster of mitochondrial enzymes. FGF21 prevented toxic lipids-induced mitochondrial dysfunction and oxidative stress by induction of the AMPK/FOXO3/SIRT3 signaling axis in human induced pluripotent stem cell-derived cardiomyocytes. Adeno-associated virus-mediated restoration of cardiac SIRT3 expression was sufficient to restore the responsiveness of diabetic FGF21 knockout mice to exercise in amelioration of mitochondrial dysfunction and DCM. CONCLUSIONS: The FGF21-SIRT3 axis mediates the protective effects of exercise against DCM by preserving mitochondrial integrity and represents a potential therapeutic target for DCM. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03240978.

Investigating the Mechanisms of Bisdemethoxycurcumin in Ulcerative Colitis: Network Pharmacology and Experimental Verification.

Ulcerative colitis is a chronic inflammatory bowel disorder that is hard to cure once diagnosed. Bisdemethoxycurcumin has shown positive effects on inflammatory diseases. However, the underlying bioactive interaction between bisdemethoxycurcumin and ulcerative colitis is unclear. The objective of this study was to determine the core target and potential mechanism of action of bisdemethoxycurcumin as a therapy for ulcerative colitis. The public databases were used to identify potential targets for bisdemethoxycurcumin and ulcerative colitis. To investigate the potential mechanisms, the protein-protein interaction network, gene ontology analysis, and Kyoto encyclopedia of genes and genomes analysis have been carried out. Subsequently, experimental verification was conducted to confirm the findings. A total of 132 intersecting genes of bisdemethoxycurcumin, as well as ulcerative coli-tis-related targets, were obtained. SRC, EGFR, AKT1, and PIK3R1 were the targets of highest potential, and the PI3K/Akt and MAPK pathways may be essential for the treatment of ulcerative colitis by bisdemethoxycurcumin. Molecular docking demonstrated that bisdemethoxycurcumin combined well with SRC, EGFR, PIK3R1, and AKT1. Moreover, the in vitro experiments suggested that bisdemethoxycurcumin might reduce LPS-induced pro-inflammatory cytokines levels in RAW264.7 cells by suppressing PI3K/Akt and MAPK pathways. Our study provided a comprehensive overview of the potential targets and molecular mechanism of bisdemethoxycurcumin against ulcerative colitis. Furthermore, it also provided a theoretical basis for the clinical treatment of ulcerative colitis, as well as compelling evidence for further study on the mechanism of bisdemethoxycurcumin in the treatment of ulcerative colitis.

Distinct Disease Severity Between Children and Older Adults With Coronavirus Disease 2019 (COVID-19): Impacts of ACE2 Expression, Distribution, and Lung Progenitor Cells.

BACKGROUND: Children and older adults with coronavirus disease 2019 (COVID-19) display a distinct spectrum of disease severity yet the risk factors aren't well understood. We sought to examine the expression pattern of angiotensin-converting enzyme 2 (ACE2), the cell-entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the role of lung progenitor cells in children and older patients. METHODS: We retrospectively analyzed clinical features in a cohort of 299 patients with COVID-19. The expression and distribution of ACE2 and lung progenitor cells were systematically examined using a combination of public single-cell RNA-seq data sets, lung biopsies, and ex vivo infection of lung tissues with SARS-CoV-2 pseudovirus in children and older adults. We also followed up patients who had recovered from COVID-19. RESULTS: Compared with children, older patients (>50 years.) were more likely to develop into serious pneumonia with reduced lymphocytes and aberrant inflammatory response (P = .001). The expression level of ACE2 and lung progenitor cell markers were generally decreased in older patients. Notably, ACE2 positive cells were mainly distributed in the alveolar region, including SFTPC positive cells, but rarely in airway regions in the older adults (P < .01). The follow-up of discharged patients revealed a prolonged recovery from pneumonia in the older (P < .025). CONCLUSIONS: Compared to children, ACE2 positive cells are generally decreased in older adults and mainly presented in the lower pulmonary tract. The lung progenitor cells are also decreased. These risk factors may impact disease severity and recovery from pneumonia caused by SARS-Cov-2 infection in older patients.

Convalescent plasma is of limited clinical benefit in critically ill patients with coronavirus disease-2019: a cohort study.

BACKGROUND: Recently, convalescent plasma (CP) transfusion was employed for severe or critically ill patients with coronavirus disease-2019. However, the benefits of CP for patients with different conditions are still in debate. To contribute clinical evidence of CP on critically ill patients, we analyze the characteristics and outcomes of patients with or without CP transfusion. METHODS: In this cohort study, 14 patients received CP transfusion based on the standard treatments, whereas the other 10 patients received standard treatments as control. Clinical characteristics and outcomes were analyzed. The cumulative survival rate was calculated by Kaplan-Meier survival analysis. RESULTS: Data analysis was performed on 24 patients (male/female: 15/9) with a median age of 64.0 (44.5-74.5) years. Transient fever was reported in one patient. The cumulative mortality was 21% (3/14) in patients receiving CP transfusion during a 28-day observation, whereas one dead case (1/10) was reported in the control group. No significant difference was detected between groups in 28-day mortality (P = 0.615) and radiological alleviation of lung lesions (P = 0.085). CONCLUSION: In our current study, CP transfusion was clinically safe based on the safety profile; however, the clinical benefit was not significant in critically ill patients with more comorbidities at the late stage of disease during a 28-day observation.

PSCs Reveal PUFA-Provoked Mitochondrial Stress as a Central Node Potentiating RPE Degeneration in Bietti's Crystalline Dystrophy.

Bietti's crystalline dystrophy (BCD) is an incurable retinal disorder caused by the polypeptide 2 of cytochrome P450 family 4 subfamily V (CYP4V2) mutations. Patients with BCD present degeneration of retinal pigmented epithelial (RPE) cells and consequent blindness. The lack of appropriate disease models and patients' RPE cells limits our understanding of the pathological mechanism of RPE degeneration. In this study, using CYP4V2 mutant pluripotent stem cells as disease models, we demonstrated that RPE cells with CYP4V2 mutations presented a disrupted fatty acid homeostasis, which were characterized with excessive accumulation of poly-unsaturated fatty acid (PUFA), including arachidonic acid (AA) and eicosapentaenoic acid (EPA). The PUFA overload increased mitochondrial reactive oxygen species, impaired mitochondrial respiratory functions, and triggered mitochondrial stress-activated p53-independent apoptosis in CYP4V2 mutant RPE cells. Restoration of the mutant CYP4V2 using adeno-associated virus 2 (AAV2) can effectively reduce PUFA deposition, alleviate mitochondria oxidative stresses, and rescue RPE cell death in BCD RPE cells. Taken together, our results highlight a role of PUFA-induced mitochondrial damage as a central node to potentiate RPE degeneration in BCD patients. AAV2-mediated gene therapy may represent a feasible strategy for the treatment of BCD.

Overexpression of ERBB4 rejuvenates aged mesenchymal stem cells and enhances angiogenesis via PI3K/AKT and MAPK/ERK pathways.

The age-related functional exhaustion limits potential efficacy of mesenchymal stem cells (MSC) in treating cardiovascular disease. Therefore, rejuvenation of aged MSC in the elderly population is of great interest. We have previously reported that Erb-B2 receptor tyrosine kinase 4 ( ERBB4) plays a critical role in regulating MSC survival under hypoxia. The aim of this study was to investigate whether ERBB4 rejuvenates aged MSC and how ERBB4 enhances therapeutic efficacy of aged MSC in treating myocardial infarction (MI). Compared with vector aged MSC (aged-MSC), ERBB4-engineered aged MSC (ER4-aged-MSC) conferred resistance to oxidative stress-induced cell death and ameliorated the senescent phenotype in vitro. Four weeks after MI, the ER4-aged-MSC group exhibited enhanced blood vessel density, reduced cardiac remodeling and apoptosis with improved heart function compared with the aged-MSC group. Overexpression of ERBB4 caused an increase in phosphorylated v-akt murine thymoma viral oncogene homolog 1 (AKT), and phosphorylated ERK expression under hypoxia. ER4-aged-MSC secreted higher levels of angiopoietin, epithelial neutrophil activating peptide 78, VEGF, and fibroblast growth factor 2, and enhanced tube formation in HUVEC. The impact of ERBB4 on protein expression, proangiogenesis, cell behavior, and cytokine secretion was abolished by inhibiting PI3K/AKT and MAPK/ERK signaling pathway.-Liang, X., Ding, Y., Lin, F., Zhang, Y., Zhou, X., Meng, Q., Lu, X., Jiang, G., Zhu, H., Chen, Y., Lian, Q., Fan, H., Liu, Z. Overexpression of ERBB4 rejuvenates aged mesenchymal stem cells and enhances angiogenesis via PI3K/AKT and MAPK/ERK pathways.

Vascular progenitor cell senescence in patients with Marfan syndrome.

Vascular progenitor cells (VPCs) present in the adventitia of the vessel wall play a critical role in the regulation of vascular repair following injury. This study aimed to assess the function of VPCs isolated from patients with Marfan syndrome (MFS). VPCs were isolated from control and MFS donors and characterized. Compared with control-VPCs, MFS-VPCs exhibited cellular senescence as demonstrated by increased cell size, higher SA-ß-gal activity and elevated levels of p53 and p21. RNA sequencing showed that several cellular process-related pathways including cell cycle and cellular senescence were significantly enriched in MFP-VPCs. Notably, the expression level of TGF-ß1 was much higher in MFS-VPCs than control-VPCs. Treatment of control-VPCs with TGF-ß1 significantly enhanced mitochondrial reactive oxidative species (ROS) and induced cellular senescence whereas inhibition of ROS reversed these effects. MFS-VPCs displayed increased mitochondrial fusion and decreased mitochondrial fission. Treatment of control-VPCs with TGF-ß1 increased mitochondrial fusion and reduced mitochondrial fission. Nonetheless, treatment of mitofusin2 (Mfn2)-siRNA inhibited TGF-ß1-induced mitochondrial fusion and cellular senescence. Furthermore, TGF-ß1-induced mitochondrial fusion was mediated by the AMPK signalling pathway. Our study shows that TGF-ß1 induces VPC senescence in patients with MFS by mediating mitochondrial dynamics via the AMPK signalling pathway.

Mesenchymal stem cells alleviate oxidative stress-induced mitochondrial dysfunction in the airways.

BACKGROUND: Oxidative stress-induced mitochondrial dysfunction can contribute to inflammation and remodeling in patients with chronic obstructive pulmonary disease (COPD). Mesenchymal stem cells protect against lung damage in animal models of COPD. It is unknown whether these effects occur through attenuating mitochondrial dysfunction in airway cells. OBJECTIVE: We sought to examine the effect of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) on oxidative stress-induce mitochondrial dysfunction in human airway smooth muscle cells (ASMCs) in vitro and in mouse lungs in vivo. METHODS: ASMCs were cocultured with iPSC-MSCs in the presence of cigarette smoke medium (CSM), and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis were measured. Conditioned medium from iPSC-MSCs and transwell cocultures were used to detect any paracrine effects. The effect of systemic injection of iPSC-MSCs on airway inflammation and hyperresponsiveness in ozone-exposed mice was also investigated. RESULTS: Coculture of iPSC-MSCs with ASMCs attenuated CSM-induced mitochondrial ROS, apoptosis, and ΔΨm loss in ASMCs. iPSC-MSC-conditioned medium or transwell cocultures with iPSC-MSCs reduced CSM-induced mitochondrial ROS but not ΔΨm or apoptosis in ASMCs. Mitochondrial transfer from iPSC-MSCs to ASMCs was observed after direct coculture and was enhanced by CSM. iPSC-MSCs attenuated ozone-induced mitochondrial dysfunction, airway hyperresponsiveness, and inflammation in mouse lungs. CONCLUSION: iPSC-MSCs offered protection against oxidative stress-induced mitochondrial dysfunction in human ASMCs and in mouse lungs while reducing airway inflammation and hyperresponsiveness. These effects are, at least in part, dependent on cell-cell contact, which allows for mitochondrial transfer, and paracrine regulation. Therefore iPSC-MSCs show promise as a therapy for oxidative stress-dependent lung diseases, such as COPD.

Deferiprone inhibits iron overload-induced tissue factor bearing endothelial microparticle generation by inhibition oxidative stress induced mitochondrial injury, and apoptosis.

Iron overload-induced cardiovascular toxicity is one of the most common causes of morbidity and mortality in beta-thalassemia major patients. We have previously shown that iron overload-induced systemic arterial changes characterized by endothelial dysfunction are associated with increased endothelial microparticle (EMP) release. In this study, we further demonstrate how EMP release is associated with iron-induced mitochondrial injury and apoptosis of endothelial cells. Iron increased the production of reactive oxygen species (ROS) and calcium influx into mitochondria [Ca2+]m. Iron also disturbed mitochondrial respiration function and eventually led to loss of mitochondrial membrane potential (ΔΨm). A significant increase in apoptotic cells and EMPs were found under iron treatment. EMPs contained tissue factor (TF), which has potential clinical impact on thromboembolic phenomenon. Then, we investigated the salvaging effect of deferiprone (L1) on endothelial cell damage and EMP release. We found that L1 could inhibit iron-induced ROS generation, and decrease mitochondrial damage with the resultant effect of less endothelial cell apoptosis and EMP release. L1 could protect endothelial cells from iron-induced toxic effects and minimize EMP release, which could be potentially helpful in a subgroup of thalassemia patients who have increased thromboembolic complications.

Human iPSC-MSC-Derived Xenografts Modulate Immune Responses by Inhibiting the Cleavage of Caspases.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-ß1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732.

NLRP3 inflammasome induced liver graft injury through activation of telomere-independent RAP1/KC axis.

Acute-phase inflammation plays a critical role in liver graft injury. Inflammasomes, multi-molecular complexes in the cytoplasm, are responsible for initiating inflammation. Here, we aimed to explore the role of inflammasomes in liver graft injury and further to investigate the regulatory mechanism. In a clinical liver transplant cohort, we found that intragraft expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes was significantly up-regulated post-transplantation. Importantly, overexpression of NLRP3 was strongly associated with poor liver function characterized by high levels of ALT, AST, and urea, as well as neutrophil infiltration after transplantation. The significant correlation between NLRP3 and IL-1ß mRNA levels led us to focus on one of the associated upstream regulators, telomere-independent repressor activator protein 1 (RAP1), which was further proved to be co-localized with NLRP3 in neutrophils. In the liver of a mouse model (hepatic ischaemia/reperfusion and hepatectomy model) and isolated neutrophils from RAP1-/- mice, the expression levels of NLRP3 and keratinocyte chemoattractant (KC) were significantly down-regulated in contrast to those in wild types. The levels of ALT and AST, as well as the neutrophil infiltration, were also decreased by RAP1 deficiency. In our clinical validation, intragraft KC expression was associated with NLRP3 and co-localized with RAP1 in neutrophils. Furthermore, NLRP3 inflammasomes were up-regulated by recombinant KC in the isolated neutrophils and liver of the mouse model. Our data demonstrated that NLRP3 inflammasomes, activated by the RAP1/KC axis, played a critical role in initiating inflammation during the early stage of liver graft injury. Targeting RAP1/KC/NLRP3 inflammasomes may offer a new therapeutic strategy against liver graft injury. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Absence of NUCKS augments paracrine effects of mesenchymal stem cells-mediated cardiac protection.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) contribute to myocardial repair after myocardial infarction (MI) by secreting a panel of growth factors and cytokines. This study was to investigate the potential mechanisms of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) in regulation of the profiles of BM-MSCs secretion and compare the therapeutic efficacy of NUCKS-/-- and wide type-BM-MSCs (WT-BM-MSCs) on MI. The secretion profiles between NUCKS-/-- and WT-BM-MSCs under hypoxia (1%O2) were analyzed. Gene function analysis showed that compared with WT-BM-MSCs-conditioned medium (CdM), some genes over-presented in NUCKS-/--BM-MSCs-CdM were closely associated with inflammatory response, regulation of cell proliferation, death, migration and secretion. Notably, VEGFa in NUCKS-/--BM-MSCs-CdM was higher than that of WT-BM-MSCs-CdM. WT-BM-MSCs and NUCKS-/--BM-MSCs were transplanted into the peri-infarct region in mice of MI. At 4 weeks after cell transplantation, NUCKS-/-- or WT-BM-MSCs group significantly improved heart function and vessels density and reduced infarction size and apoptosis of cardiomyocytes. Furthermore, NUCKS-/--BM-MSCs provided better cardioprotective effects than WT-BM-MSCs against MI. Our study demonstrates that depletion of NUCKS enhances the therapeutic efficacy of BM-MSCs for MI via regulating the secretion.

iPSC-derived mesenchymal stem cells exert SCF-dependent recovery of cigarette smoke-induced apoptosis/proliferation imbalance in airway cells.

Mesenchymal stem cells (MSCs) have emerged as a potential cell-based therapy for pulmonary emphysema in animal models. Our previous study demonstrated that human induced pluripotent stem cell-derived MSCs (iPSC-MSCs) were superior over bone marrow-derived MSCs (BM-MSCs) in attenuating cigarette smoke (CS)-induced airspace enlargement possibly through mitochondrial transfer. This study further investigated the effects of iPSC-MSCs on inflammation, apoptosis, and proliferation in a CS-exposed rat model and examined the effects of the secreted paracrine factor from MSCs as another possible mechanism in an in vitro model of bronchial epithelial cells. Rats were exposed to 4% CS for 1 hr daily for 56 days. At days 29 and 43, human iPSC-MSCs or BM-MSCs were administered intravenously. We observed significant attenuation of CS-induced elevation of circulating 8-isoprostane and cytokine-induced neutrophil chemoattractant-1 after iPSC-MSC treatment. In line, a superior capacity of iPSC-MSCs was also observed in ameliorating CS-induced infiltration of macrophages and neutrophils and apoptosis/proliferation imbalance in lung sections over BM-MSCs. In support, the conditioned medium (CdM) from iPSC-MSCs ameliorated CS medium-induced apoptosis/proliferation imbalance of bronchial epithelial cells in vitro. Conditioned medium from iPSC-MSCs contained higher level of stem cell factor (SCF) than that from BM-MSCs. Deprivation of SCF from iPSC-MSC-derived CdM led to a reduction in anti-apoptotic and pro-proliferative capacity. Taken together, our data suggest that iPSC-MSCs may possess anti-apoptotic/pro-proliferative capacity in the in vivo and in vitro models of CS-induced airway cell injury partly through paracrine secretion of SCF.

Fibroblast growth factor 21 prevents atherosclerosis by suppression of hepatic sterol regulatory element-binding protein-2 and induction of adiponectin in mice.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. It acts as a key downstream target of both peroxisome proliferator-activated receptor α and γ, the agonists of which have been used for lipid lowering and insulin sensitization, respectively. However, the role of FGF21 in the cardiovascular system remains elusive. METHODS AND RESULTS: The roles of FGF21 in atherosclerosis were investigated by evaluating the impact of FGF21 deficiency and replenishment with recombinant FGF21 in apolipoprotein E(-/-) mice. FGF21 deficiency causes a marked exacerbation of atherosclerotic plaque formation and premature death in apolipoprotein E(-/-) mice, which is accompanied by hypoadiponectinemia and severe hypercholesterolemia. Replenishment of FGF21 protects against atherosclerosis in apolipoprotein E(-/-)mice via 2 independent mechanisms, inducing the adipocyte production of adiponectin, which in turn acts on the blood vessels to inhibit neointima formation and macrophage inflammation, and suppressing the hepatic expression of the transcription factor sterol regulatory element-binding protein-2, thereby leading to reduced cholesterol synthesis and attenuation of hypercholesterolemia. Chronic treatment with adiponectin partially reverses atherosclerosis without obvious effects on hypercholesterolemia in FGF21-deficient apolipoprotein E(-/-) mice. By contrast, the cholesterol-lowering effects of FGF21 are abrogated by hepatic expression of sterol regulatory element-binding protein-2. CONCLUSIONS: FGF21 protects against atherosclerosis via fine tuning the multiorgan crosstalk among liver, adipose tissue, and blood vessels.

Loss of ATOH8 Increases Stem Cell Features of Hepatocellular Carcinoma Cells.

BACKGROUND & AIMS: Levels of atonal homolog 8 (ATOH8) are reduced in 48% of hepatitis B virus-associated hepatocellular carcinoma cells (HCCs). ATOH8 downregulation is associated with loss of tumor differentiation, indicating an effect mediated by cancer stem cells. We investigated the effects of loss of ATOH8 in human hepatocellular carcinoma (HCC) cells and cell lines. METHODS: HCC and adjacent nontumor tissues were collected, from 2001 through 2012, from 242 patients undergoing hepatectomy at Sun Yat-Sen University Cancer Center in China; 83% of HCCs were associated with hepatitis B virus (HBV) infection. CD133(+) cells were isolated from tumor tissues by flow cytometry. Experiments were performed in HBV-positive and HBV-negative HCC cell lines, the immortalized liver cell line LO2, and 8 other HCC cell lines. ATOH8 was expressed from lentiviral vectors in PLC8024 and Huh7 cells; levels were knocked down with small interfering RNAs in QSG7701 cells. Cells carrying empty vectors were used as controls. Gene regulation by ATOH8 was assessed in mobility shift and luciferase reporter assays. Cells were analyzed in proliferation, foci formation, and colony formation assays. The tumorigenic and chemo-resistant potential of cells were investigated by assessing growth of xenograft tumors in immunocompromised mice. Metastatic features of cells were assessed in Matrigel invasion assays and wound healing analyses. RESULTS: Levels of ATOH8 mRNA were reduced by more than 4-fold, compared to nontumor tissues, in 118 of 242 HCC samples (48.8%). Patients with tumor reductions in ATOH8 had significantly shorter times of disease-free survival (mean, 41.4 months) than patients with normal tissue levels (mean, 52.6 months). ATOH8 expression was reduced in HepG2, Huh7, PLC8024 and CRL8064 HCC cells, as well as CD133(+) cells isolated from human HCC samples. Transgenic expression of ATOH8 in HCC cell lines significantly reduced proliferation and foci colony formation, as well as their invasive and migratory abilities. Transgenic expression of ATOH8 reduced the ability of HBV-positive PLC8024 cells to form tumors in mice, compared to control cells. Cells with ATOH8 knockdown formed xenograft tumors more rapidly, in more mice, than control cells. ATOH8 repressed transcription of stem-cell associated genes including OCT4, NANOG, and CD133. Knockdown of ATOH8 in CD133-negative QSG7701 cells caused them to express CD133; acquire self-renewal, differentiation, chemo-resistance properties; form more xenograft tumors in mice; and generate induced pluripotent stem cells (based on staining for alkaline phosphatase and their ability to form embryoid bodies and teratomas). Alternatively, expression of ATOH8 in PLC8024 and Huh7 cells significantly reduced the numbers of cells expressing CD133, and increased the chemo-sensitivity of Huh7 cells to 5-fluorouracil (5-FU) and cisplatin, in vitro and in mice. CONCLUSIONS: ATOH8 appears to be a tumor suppressor that induces stem-cell features and chemoresistance in HCC cells. Strategies to restore its levels and activities might be developed to treat patients with liver cancer.

Insensitivity of Human iPS Cells-Derived Mesenchymal Stem Cells to Interferon-γ-induced HLA Expression Potentiates Repair Efficiency of Hind Limb Ischemia in Immune Humanized NOD Scid Gamma Mice.

Adult mesenchymal stem cells (MSCs) are immunoprivileged cells due to the low expression of major histocompatibility complex (MHC) II molecules. However, the expression of MHC molecules in human-induced pluripotent stem cells (iPSCs)-derived MSCs has not been investigated. Here, we examined the expression of human leukocyte antigen (HLA) in human MSCs derived from iPSCs, fetuses, and adult bone marrow (BM) after stimulation with interferon-γ (IFN-γ), compared their repair efficacy, cell retention, inflammation, and HLA II expression in immune humanized NOD Scid gamma (NSG) mice of hind limb ischemia. In the absence of IFN-γ stimulation, HLA-II was expressed only in BM-MSCs after 7 days. Two and seven days after stimulation, high levels of HLA-II were observed in BM-MSCs, intermediate levels were found in fetal-MSCs, and very low levels in iPSC-MSCs. The levels of p-STAT1, interferon regulatory factor 1, and class II transactivator exhibited similar phenomena. Moreover, p-STAT1 antagonist significantly reversed the high expression of HLA-II in BM-MSCs. Compared to adult BM-MSCs, transplanting iPSC-MSCs into hu-PBMNC NSG mice revealed markedly more survival iPSC-MSCs, less inflammatory cell accumulations, and better recovery of hind limb ischemia. The expression of HLA-II in MSCs in the ischemia limbs was detected in BM-MSCs group but not in iPSC-MSCs group at 7 and 21 days after transplantation. Our results demonstrate that, compared to adult MSCs, human iPSC-MSCs are insensitive to proinflammatory IFN-γ-induced HLA-II expression and iPSC-MSCs have a stronger immune privilege after transplantation. It may attribute to a better therapeutic efficacy in allogeneic transplantation.

Thrombopoietin Protects Cardiomyocytes from Iron-Overload Induced Oxidative Stress and Mitochondrial Injury.

BACKGROUND/AIMS: Thalassaemia accompanied with iron-overload is common in Hong Kong. Iron-overload induced cardiomyopathy is the commonest cause of morbidity and mortality in patients with ß-thalassaemia. Chronic iron-overload due to blood transfusion can cause cardiac failure. Decreased antioxidant defence and increased ROS production may lead to oxidative stress and cell injury. Iron-overload may lead to heart tissue damage through lipid peroxidation in response to oxidative stress, and a great diversity of toxic aldehydes are formed when lipid hydroperoxides break down in heart and plasma. METHODS: Iron entry into embryonic heart H9C2 cells was determined by calcein assay using a fluorometer. Reactive oxygen species (ROS) production in cells treated with FeCl3 or thrombopoietin (TPO) was monitored by using the fluorescent probe H2DCFDA. Changes in mitochondrial membrane potential of H9C2 cells were quantified by using flow cytometry. RESULTS: We demonstrated that iron induced oxidative stress and apoptosis in cardiomyocytes, and that iron increased ROS production and reduced cell viability in a dose-dependent manner. Iron treatment increased the proportion of cells with JC-1 monomers, indicating a trend of drop in the mitochondrial membrane potential. TPO exerted a cardio-protective effect on iron-induced apoptosis. CONCLUSIONS: These findings suggest that iron-overload leads to the generation of ROS and further induces apoptosis in cardiomyocytes via mitochondrial pathways. TPO might exert a protective effect on iron-overload induced apoptosis via inhibiting oxidative stress and suppressing the mitochondrial pathways in cardiomyocytes.