Rongrong Wu

Leptin Signaling Is Required for Augmented Therapeutic Properties of Mesenchymal Stem Cells Conferred by Hypoxia Preconditioning

Hypoxia preconditioning enhances the therapeutic effect of mesenchymal stem cells (MSCs). However, the mechanism underlying hypoxia‐induced augmentation of the protective effect of MSCs on myocardial infarction (MI) is poorly understood. We show that hypoxia‐enhanced survival, mobility, and protection of cocultured cardiomyocytes were paralleled by increased expression of leptin and cell surface receptor CXCR4. The enhanced activities were abolished by either knockdown of leptin with a selective shRNA or by genetic deficiency of leptin or its receptor in MSCs derived, respectively, from ob/ob or db/db mice. To characterize the role of leptin in the regulation of MSC functions by hypoxia and its possible contribution to enhanced therapeutic efficacy, cell therapy using MSCs derived from wild‐type, ob/ob, or db/db mice was implemented in mouse models of acute MI. Augmented protection by hypoxia pretreatment was only seen with MSCs from wild‐type mice. Parameters that were differentially affected by hypoxia pretreatment included MSC engraftment, c‐Kit+ cell recruitment to the infarct, vascular density, infarct size, and long‐term contractile function. These data show that leptin signaling is an early and essential step for the enhanced survival, chemotaxis, and therapeutic properties of MSCs conferred by preculture under hypoxia. Leptin may play a physiological role in priming MSCs resident in the bone marrow endosteum for optimal response to systemic signaling molecules and subsequent tissue repair. Stem Cells 2014;32:2702–2713

SIRT1 ameliorates age-related senescence of mesenchymal stem cells via modulating telomere shelterin

Mesenchymal stem cells (MSCs) senescence is an age-related process that impairs the capacity for tissue repair and compromises the clinical use of autologous MSCs for tissue regeneration. Here, we describe the effects of SIRT1, a NAD+-dependent deacetylase, on age-related MSCs senescence. Knockdown of SIRT1 in young MSCs induced cellular senescence and inhibited cell proliferation whereas overexpression of SIRT1 in aged MSCs reversed the senescence phenotype and stimulated cell proliferation. These results suggest that SIRT1 plays a key role in modulating age-induced MSCs senescence. Aging-related proteins, P16 and P21 may be downstream effectors of the SIRT1-mediated anti-aging effects. SIRT1 protected MSCs from age-related DNA damage, induced telomerase reverse transcriptase (TERT) expression and enhanced telomerase activity but did not affect telomere length. SIRT1 positively regulated the expression of tripeptidyl peptidase 1 (TPP1), a component of the shelterin pathway that protects chromosome ends from DNA damage. Together, the results demonstrate that SIRT1 quenches age-related MSCs senescence by mechanisms that include enhanced TPP1 expression, increased telomerase activity and reduced DNA damage.

A Large-Scale Investigation of Hypoxia-Preconditioned Allogeneic Mesenchymal Stem Cells for Myocardial Repair in Nonhuman Primates: Paracrine Activity Without Remuscularization

Supplemental Digital Content is available in the text.

Enhanced Cardioprotection by Human Endometrium Mesenchymal Stem Cells Driven by Exosomal MicroRNA-21

Our group recently reported positive therapeutic benefit of human endometrium‐derived mesenchymal stem cells (EnMSCs) delivered to infarcted rat myocardium, an effect that correlated with enhanced secretion of protective cytokines and growth factors compared with parallel cultures of human bone marrow MSCs (BMMSCs). To define more precisely the molecular mechanisms of EnMSC therapy, in the present study, we assessed in parallel the paracrine and therapeutic properties of MSCs derived from endometrium, bone marrow, and adipose tissues in a rat model of myocardial infarction (MI). EnMSCs, BMMSCs, and adipose‐derived MSCs (AdMSCs) were characterized by fluorescence‐activated cell sorting (FACS). Paracrine and cytoprotective actions were assessed in vitro by coculture with neonatal cardiomyocytes and human umbilical vein endothelial cells. A rat MI model was used to compare cell therapy by intramyocardial injection of BMMSCs, AdMSCs, and EnMSCs. We found that EnMSCs conferred superior cardioprotection relative to BMMSCs or AdMSCs and supported enhanced microvessel density. Inhibitor studies indicated that the enhanced paracrine actions of EnMSCs were mediated by secreted exosomes. Analyses of exosomal microRNAs (miRs) by miR array and quantitative polymerase chain reaction revealed that miR‐21 expression was selectively enhanced in exosomes derived from EnMSCs. Selective antagonism of miR‐21 by anti‐miR treatment abolished the antiapoptotic and angiogenic effects of EnMSCs with parallel effects on phosphatase and tensin homolog (PTEN), a miR‐21 target and downstream Akt. The results of the present study confirm the superior cardioprotection by EnMSCs relative to BMMSCs or AdMSCs and implicates miR‐21 as a potential mediator of EnMSC therapy by enhancing cell survival through the PTEN/Akt pathway. The endometrium might be a preferential source of MSCs for cardiovascular cell therapy. Stem Cells Translational Medicine 2017;6:209–222

Human endometrial stem cells confer enhanced myocardial salvage and regeneration by paracrine mechanisms

Human endometrial stem cells (EnSCs) have the potential to be ‘off the shelf’ clinical reagents for the treatment of heart failure. Here, using an immunocompetent rat model of myocardial infarction (MI), we provide evidence that the functional benefits of EnSC transplantation are principally and possibly exclusively through a paracrine effect. Human EnSCs were delivered by intramyocardial injection into rats 30 min. after coronary ligation. EnSC therapy significantly preserved viable myocardium in the infarct zone and improved cardiac function at 28 days. Despite increased viable myocardium and vascular density, there was scant evidence of differentiation of EnSCs into any cardiovascular cell type. Cultured human EnSCs expressed a distinctive profile of cytokines that enhanced the survival, proliferation and function of endothelial cells in vitro. When injected into the peri‐infarct zone, human EnSCs activated AKT, ERK1/2 and STAT3 and inhibited the p38 signalling pathway. EnSC therapy decreased apoptosis and promoted cell proliferation and c‐kit+ cell recruitment in vivo. Myocardial protection and enhanced post‐infarction regeneration by EnSCs is mediated primarily by paracrine effects conferred by secreted cytokines that activate survival pathways and recruit endogenous progenitor stem cells. Menstrual blood provides a potentially limitless source of biologically competent ‘off the shelf’ EnSCs for allogeneic myocardial regenerative medicine.

Hypoxia Preconditioned Mesenchymal Stem Cells Prevent Cardiac Fibroblast Activation and Collagen Production via Leptin

Aims Activation of cardiac fibroblasts into myofibroblasts constitutes a key step in cardiac remodeling after myocardial infarction (MI), due to interstitial fibrosis. Mesenchymal stem cells (MSCs) have been shown to improve post-MI remodeling an effect that is enhanced by hypoxia preconditioning (HPC). Leptin has been shown to promote cardiac fibrosis. The expression of leptin is significantly increased in MSCs after HPC but it is unknown whether leptin contributes to MSC therapy or the fibrosis process. The objective of this study was to determine whether leptin secreted from MSCs modulates cardiac fibrosis. Methods Cardiac fibroblast (CF) activation was induced by hypoxia (0.5% O2). The effects of MSCs on fibroblast activation were analyzed by co-culturing MSCs with CFs, and detecting the expression of α-SMA, SM22α, and collagen IαI in CFs by western blot, immunofluorescence and Sirius red staining. In vivo MSCs antifibrotic effects on left ventricular remodeling were investigated using an acute MI model involving permanent ligation of the left anterior descending coronary artery. Results Co-cultured MSCs decreased fibroblast activation and HPC enhanced the effects. Leptin deficit MSCs from Ob/Ob mice did not decrease fibroblast activation. Consistent with this, H-MSCs significantly inhibited cardiac fibrosis after MI and mediated decreased expression of TGF-β/Smad2 and MRTF-A in CFs. These effects were again absent in leptin-deficient MSCs. Conclusion Our data demonstrate that activation of cardiac fibroblast was inhibited by MSCs in a manner that was leptin-dependent. The mechanism may involve blocking TGF-β/Smad2 and MRTF-A signal pathways.

Transplantation of SIRT1-engineered aged mesenchymal stem cells improves cardiac function in a rat myocardial infarction model

BACKGROUND Previous studies have demonstrated that biological aging has a negative influence on the therapeutic effects of mesenchymal stem cells (MSCs)-based therapy. Using a rat myocardial infarction (MI) model, we tested the hypothesis that silent mating type information regulation 2 homolog 1 (SIRT1) may ameliorate the phenotype and improve the function of aged MSCs and thus enhance the efficacy of aged MSCs-based therapy. METHODS Sixty female rats underwent left anterior descending coronary artery ligation and were randomly assigned to receiving: intramyocardial injection of cell culture medium (DMEM group); SIRT1 overexpression vector-treated aged MSCs (SIRT1-aged MSCs group) obtained from aged male SD rats or empty vector-treated aged MSCs (vector-aged MSCs group). Another 20 sham-operated rats that underwent open-chest surgery without coronary ligation or any other intervention served as controls. RESULTS SIRT1-aged MSC group exhibited enhanced blood vessel density in the border zone of MI hearts, which was associated with reduced cardiac remodeling, leading to improved cardiac performance. Consistent with the in vivo data, our in vitro experiments also demonstrated that SIRT1 overexpression ameliorated aged MSCs senescent phenotype and recapitulated the pro-angiogenesis property of MSCs and conferred the anti-stress response capabilities, as indicated by increases in pro-angiogenic factors, angiopoietin 1 (Ang1) and basic fibroblast growth factor (bFGF), expressions and a decrease in anti-angiogenic factor thrombospondin-1 (TBS1) at mRNA levels, and increases in Bcl-2/Bax ratio at protein level. CONCLUSIONS Up-regulating SIRT1 expression could enhance the efficacy of aged MSCs-based therapy for MI as it relates to the amelioration of senescent phenotype and hence improved biological function of aged MSCs.

Tongxinluo promotes mesenchymal stem cell tube formation in vitro

ObjectiveTo study whether Tongxinluo (TXL) can induce angiogenesis in bone marrow mesenchymal stem cells (MSCs), and to investigate the underlying mechanism.MethodsBone marrow MSCs were obtained from male Sprague-Dawley rats. We established an angiogenesis model in vitro via matrigel experiment. MSCs were seeded on matrigel coated 24-well plates, and treated by TXL 50 and 100 mg/L. After 24 h, we observed the tube formations of MSCs in the matrigel. Cell migration ability was examined by wound scratch test and transwell assay. Expressions of vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1), matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-2 (TIMP-2) were analyzed at the protein level by Western blot. Gelatin zymography assay was applied to investigating the MSC paracrine abilities of pro-MMP-2 and activated MMP-2.ResultsTXL promoted MSC tube formation in matrigel. The ratio of TXL 100 mg/L treated-MSC tubular length was increased 3.04-fold compared to the control group (P<0.05). Scratch test and transwell assay showed that TXL could improve the cell migration ability of MSCs. Western blot experiments showed that TXL promoted MSC synthesis of MMP-2, but it had no influence on the expressions of MMP-9 and TIMP-2. This effect was confirmed by gelatin zymography assay, which showed that TXL increased MSC secretion of pro-MMP-2 and activated MMP-2. VEGF expression of TXL treated-MSCs was increased compared to the control group. The expression of Flk-1 was not different among the groups.ConclusionsThis study demonstrates that TXL can promote the tube formation of MSCs, and the underlying mechanisms are associated with increased migration ability of MSCs and the up-regulation of MMP-2 and VEGF expressions.

MiR-211/STAT5A Signaling Modulates Migration of Mesenchymal Stem Cells to Improve its Therapeutic Efficacy.

Our previous study showed that the therapeutic effects of mesenchymal stem cells (MSCs) transplantation were improved by enhancing migration. MicroRNA‐211 (miR‐211) can modulate the migratory properties of some cell types by mechanisms that are not fully understood. This study was designed to investigate a possible role for miR‐211 in MSC migration, and whether genetic manipulation of miR‐211 in MSCs could be used to enhance its beneficial effects of cell transplantation. Transwell assays confirmed that MSCs migration of was significantly impaired by miR‐211 knockdown but enhanced by miR‐211 overexpression. MiR‐211 overexpressing MSCs also exhibited significantly increased cell engraftment in the peri‐infarct areas of female rat hearts 2 days after intravenous transplantation of male MSCs as shown by GFP tracking and SYR gene quantification. This conferred a significant decrease in infarct size and improved cardiac performance. By using a loss or gain of gene function approach, we demonstrated that miR‐211 targeted STAT5A to modulate MSCs migration, possibly by interacting with MAPK signaling. Furthermore, the beneficial effects of miR‐211 overexpression in MSCs were abolished by simultaneous overexpression of STAT5A whereas the negative effects of miR‐211 silencing on MSC migration were rescued by simultaneous downregulation of STAT5A. Finally, using ChIP‐PCR and luciferase assays, we provide novel evidence that STAT3 can directly bind to promoter elements that activate miR‐211 expression. STAT3/miR‐211/STAT5A signaling plays a key role in MSCs migration. Intravenous infusion of genetically modified miR‐211 overexpressing MSCs conveys enhanced protection from adverse post‐MI remodeling compared with unmodified MSCs. Stem Cells 2016;34:1846–1858

Concise Review: Optimized Strategies for Stem Cell‐Based Therapy in Myocardial Repair: Clinical Translatability and Potential Limitation

Ischemic heart diseases (IHDs) remain major public health problems with high rates of morbidity and mortality worldwide. Despite significant advances, current therapeutic approaches are unable to rescue the extensive and irreversible loss of cardiomyocytes caused by severe ischemia. Over the past 16 years, stem cell‐based therapy has been recognized as an innovative strategy for cardiac repair/regeneration and functional recovery after IHDs. Although substantial preclinical animal studies using a variety of stem/progenitor cells have shown promising results, there is a tremendous degree of skepticism in the clinical community as many stem cell trials do not confer any beneficial effects. How to accelerate stem cell‐based therapy toward successful clinical application attracts considerate attention. However, many important issues need to be fully addressed. In this Review, we have described and compared the effects of different types of stem cells with their dose, delivery routes, and timing that have been routinely tested in recent preclinical and clinical findings. We have also discussed the potential mechanisms of action of stem cells, and explored the role and underlying regulatory components of stem cell‐derived secretomes/exosomes in myocardial repair. Furthermore, we have critically reviewed the different strategies for optimizing both donor stem cells and the target cardiac microenvironments to enhance the engraftment and efficacy of stem cells, highlighting their clinical translatability and potential limitation. Stem Cells 2018;36:482–500