miR‐155‐5p inhibition rejuvenates aged mesenchymal stem cells and enhances cardioprotection following infarction

Aging impairs the functions of human mesenchymal stem cells (MSCs), thereby severely reducing their beneficial effects on myocardial infarction (MI). MicroRNAs (miRNAs) play crucial roles in regulating the senescence of MSCs; however, the underlying mechanisms remain unclear. Here, we investigated the significance of miR‐155‐5p in regulating MSC senescence and whether inhibition of miR‐155‐5p could rejuvenate aged MSCs (AMSCs) to enhance their therapeutic efficacy for MI. Young MSCs (YMSCs) and AMSCs were isolated from young and aged donors, respectively. The cellular senescence of MSCs was evaluated by senescence‐associated β‐galactosidase (SA‐β‐gal) staining. Compared with YMSCs, AMSCs exhibited increased cellular senescence as evidenced by increased SA‐β‐gal activity and decreased proliferative capacity and paracrine effects. The expression of miR‐155‐5p was much higher in both serum and MSCs from aged donors than young donors. Upregulation of miR‐155‐5p in YMSCs led to increased cellular senescence, whereas downregulation of miR‐155‐5p decreased AMSC senescence. Mechanistically, miR‐155‐5p inhibited mitochondrial fission and increased mitochondrial fusion in MSCs via the AMPK signaling pathway, thereby resulting in cellular senescence by repressing the expression of Cab39. These effects were partially reversed by treatment with AMPK activator or mitofusin2‐specific siRNA (Mfn2‐siRNA). By enhancing angiogenesis and promoting cell survival, transplantation of anti‐miR‐155‐5p‐AMSCs led to improved cardiac function in an aged mouse model of MI compared with transplantation of AMSCs. In summary, our study shows that miR‐155‐5p mediates MSC senescence by regulating the Cab39/AMPK signaling pathway and miR‐155‐5p is a novel target to rejuvenate AMSCs and enhance their cardioprotective effects.     

Myc‐regulated microRNAs attenuate embryonic stem cell differentiation

Myc proteins are known to have an important function in stem cell maintenance. As Myc has been shown earlier to regulate microRNAs (miRNAs) involved in proliferation, we sought to determine whether c‐Myc also affects embryonic stem (ES) cell maintenance and differentiation through miRNAs. Using a quantitative primer‐extension PCR assay we identified miRNAs, including, miR‐141, miR‐200, and miR‐429 whose expression is regulated by c‐Myc in ES cells, but not in the differentiated and tumourigenic derivatives of ES cells. Chromatin immunoprecipitation analyses indicate that in ES cells c‐Myc binds proximal to genomic regions encoding the induced miRNAs. We used expression profiling and seed homology to identify genes specifically downregulated both by these miRNAs and by c‐Myc. We further show that the introduction of c‐Myc‐induced miRNAs into murine ES cells significantly attenuates the downregulation of pluripotency markers on induction of differentiation after withdrawal of the ES cell maintenance factor LIF. In contrast, knockdown of the endogenous miRNAs accelerate differentiation. Our data show that in ES cells c‐Myc acts, in part, through a subset of miRNAs to attenuate differentiation.     

Induction of hepatocyte‐like cells from human umbilical cord‐derived mesenchymal stem cells by defined microRNAs

Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl₄‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.     

Differential expression of basal microRNAs’ patterns in human dental pulp stem cells

MicroRNAs (miRNAs) are small non‐coding RNAs that regulate translation of mRNA into protein and play a crucial role for almost all biological activities. However, the identification of miRNAs from mesenchymal stem cells (MSCs), especially from dental pulp, is poorly understood. In this study, dental pulp stem cells (DPSCs) were characterized in terms of their proliferation and differentiation capacity. Furthermore, 104 known mature miRNAs were profiled by using real‐time PCR. Notably, we observed 19 up‐regulated miRNAs and 29 significantly down‐regulated miRNAs in DPSCs in comparison with bone marrow MSCs (BM‐MSCs). The 19 up‐regulated miRNAs were subjected to ingenuity analysis, which were composed into 25 functional networks. We have chosen top 2 functional networks, which comprised 10 miRNA (hsa‐miR‐516a‐3p, hsa‐miR‐125b‐1‐3p, hsa‐miR‐221‐5p, hsa‐miR‐7, hsa‐miR‐584‐5p, hsa‐miR‐190a, hsa‐miR‐106a‐5p, hsa‐mir‐376a‐5p, hsa‐mir‐377‐5p and hsa‐let‐7f‐2‐3p). Prediction of target mRNAs and associated biological pathways regulated by each of this miRNA was carried out. We paid special attention to hsa‐miR‐516a‐3p and hsa‐miR‐7‐5p as these miRNAs were highly expressed upon validation with qRT‐PCR analysis. We further proceeded with loss‐of‐function analysis with these miRNAs and we observed that hsa‐miR‐516a‐3p knockdown induced a significant increase in the expression of WNT5A. Likewise, the knockdown of hsa‐miR‐7‐5p increased the expression of EGFR. Nevertheless, further validation revealed the role of WNT5A as an indirect target of hsa‐miR‐516a‐3p. These results provide new insights into the dynamic role of miRNA expression in DPSCs. In conclusion, using miRNA signatures in human as a prediction tool will enable us to elucidate the biological processes occurring in DPSCs.     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

Trichostatin a modulates intracellular reactive oxygen species through SOD2 and FOXO1 in human bone marrow‐mesenchymal stem cells

Engraft cells are often exposed to oxidative stress and inflammation; therefore, any factor that can provide the stem cells resistance to these stresses may yield better efficacy in stem cell therapy. Studies indicate that histone deacetylase (HDACs) inhibitors alleviate damage induced by oxidative stress. In this study, we investigated whether regulation of reactive oxygen species (ROS) occurs through the HDAC inhibitor trichostatin A (TSA) in human bone marrow‐mesenchymal stem cells (hBM‐MSCs). Intracellular ROS levels increased following exposure to hydrogen peroxide (H₂O₂), and were suppressed by TSA treatment. Levels of the antioxidant enzyme superoxide dismutase 2 (SOD2) increased following treatment with 200 nM TSA and to a lesser level at 1–5 μM TSA. Cell protective effects against oxidative stress were significantly increased in TSA‐MSCs after treatment with low doses of TSA (50–500 nM) and decreased with high doses of TSA (5–10 μM). Consistent results were obtained with immunoblot analysis for caspase3. Investigation of Forkhead box O1 (FOXO1), superoxide dismutase 2 (SOD2), and p53 levels to determine intracellular signaling by TSA in oxidative stress‐induced MSCs demonstrated that expression of phosphorylated‐FOXO1 and phosphorylated‐SOD2 decreased in H₂O₂‐treated MSCs while levels of p53 increased. These effects were reversed by the treatment of 200 nM TSA. These results suggest that the main function of ROS modulation by TSA is activated through SOD2 and FOXO1. Thus, optimal treatment with TSA may protect hBM‐MSCs against oxidative stress. Copyright © 2014 John Wiley & Sons, Ltd.     

miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin

Inflammation and apoptosis play important roles in the initiation and progression of acute lung injury (ALI). Our previous study has shown that progranulin (PGRN) exerts lung protective effects during LPS‐induced ALI. Here, we have investigated the potential roles of PGRN‐targeting microRNAs (miRNAs) in regulating inflammation and apoptosis in ALI and have highlighted the important role of PGRN. LPS‐induced lung injury and the protective roles of PGRN in ALI were first confirmed. The function of miR‐34b‐5p in ALI was determined by transfection of a miR‐34b‐5p mimic or inhibitor in intro and in vivo. The PGRN level gradually increased and subsequently significantly decreased, reaching its lowest value by 24 hr; PGRN was still elevated compared to the control. The change was accompanied by a release of inflammatory mediators and accumulation of inflammatory cells in the lungs. Using bioinformatics analysis and RT‐PCR, we demonstrated that, among 12 putative miRNAs, the kinetics of the miR‐34b‐5p levels were closely associated with PGRN expression in the lung homogenates. The gain‐ and loss‐of‐function analysis, dual‐luciferase reporter assays, and rescue experiments confirmed that PGRN was the functional target of miR‐34b‐5p. Intravenous injection of miR‐34b‐5p antagomir in vivo significantly inhibited miR‐34b‐5p up‐regulation, reduced inflammatory cytokine release, decreased alveolar epithelial cell apoptosis, attenuated lung inflammation, and improved survival by targeting PGRN during ALI. miR‐34b‐5p knockdown attenuates lung inflammation and apoptosis in an LPS‐induced ALI mouse model by targeting PGRN. This study shows that miR‐34b‐5p and PGRN may be potential targets for ALI treatments.     

Mesenchymal stem cells deliver exogenous miR‐21 via exosomes to inhibit nucleus pulposus cell apoptosis and reduce intervertebral disc degeneration

Although mesenchymal stem cells (MSCs) transplantation into the IVD (intervertebral disc) may be beneficial in inhibiting apoptosis of nucleus pulposus cells (NPCs) and alleviating IVD degeneration, the underlying mechanism of this therapeutic process has not been fully explained. The purpose of this study was to explore the protective effect of MSC‐derived exosomes (MSC‐exosomes) on NPC apoptosis and IVD degeneration and investigate the regulatory effect of miRNAs in MSC‐exosomes and associated mechanisms for NPC apoptosis. MSC‐exosomes were isolated from MSC medium, and its anti‐apoptotic effect was assessed in a cell and rat model. The down‐regulated miRNAs in apoptotic NPCs were identified, and their contents in MSC‐exosomes were detected. The target genes of eligible miRNAs and possible downstream pathway were investigated. Purified MSC‐exosomes were taken up by NPCs and suppressed NPC apoptosis. The levels of miR‐21 were down‐regulated in apoptotic NPCs while MSC‐exosomes were enriched in miR‐21. The exosomal miR‐21 could be transferred into NPCs and alleviated TNF‐α induced NPC apoptosis by targeting phosphatase and tensin homolog (PTEN) through phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Intradiscal injection of MSC‐exosomes alleviated the NPC apoptosis and IVD degeneration in the rat model. In conclusion, MSC‐derived exosomes prevent NPCs from apoptotic process and alleviate IVD degeneration, at least partly, via miR‐21 contained in exosomes. Exosomal miR‐21 restrains PTEN and thus activates PI3K/Akt pathway in apoptotic NPCs. Our work confers a promising therapeutic strategy for IVD degeneration.     

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.     

miR‐10a restores human mesenchymal stem cell differentiation by repressing KLF4

miRNAs have recently been shown to play a significant role in human aging. However, data demonstrating the effects of aging‐related miRNAs in human mesenchymal stem cells (hMSCs) are limited. We observed that hMSC differentiation decreased with aging. We also identified that miR‐10a expression was significantly decreased with age by comparing the miRNA expression of hMSCs derived from young and aged individuals. Therefore, we hypothesized that the downregulation of miR‐10a may be associated with the decreased differentiation capability of hMSCs from aged individuals. Lentiviral constructs were used to up‐ or downregulate miR‐10a in young and old hMSCs. Upregulation of miR‐10a resulted in increased differentiation to adipogenic, osteogenic, and chondrogenic lineages and in reduced cell senescence. Conversely, downregulation of miR‐10a resulted in decreased cell differentiation and increased cell senescence. A chimeric luciferase reporter system was generated, tagged with the full‐length 3′‐UTR region of KLF4 harboring the seed‐matched sequence with or without four nucleotide mutations. These constructs were cotransfected with the miR‐10a mimic into cells. The luciferase activity was significantly repressed by the miR‐10a mimic, proving the direct binding of miR‐10a to the 3′‐UTR of KLF4. Direct suppression of KLF4 in aged hMSCs increased cell differentiation and decreased cell senescence. In conclusion, miR‐10a restores the differentiation capability of aged hMSCs through repression of KLF4. Aging‐related miRNAs may have broad applications in the restoration of cell dysfunction caused by aging. J. Cell. Physiol. 228: 2324–2336, 2013. © The Authors. Published by Wiley Periodicals, Inc.     

A Novel Regulatory Mechanism for Differentiation of Mesenchymal Stem Cell: Redox State of DJ‐1 Matters

Reactive oxygen species (ROS) are multifunctional gas transmitters with diverse biological actions (adverse vs beneficial) dependent on their level. The differentiation of vascular stem cells into smooth muscle cells (SMCs) might be involved in the pathogenesis of cardiovascular disorders including hypertension and atherosclerosis. Therefore, controlling the differentiation of vascular stem cells is a potential strategy for the treatment of vascular diseases. Nonetheless, it remains to be revealed whether ROS could mediate the differentiation of mesenchymal stem cells (MSCs) into SMCs. In addition, there are no redox (reduction–oxidation)‐sensitive molecules identified, which are responsible for the ROS‐induced differentiation of MSCs. In article number 1700208, Baek et al. [Proteomics 2017, 17, Issue 21] found that ROS mediate the differentiation of MSCs into SMCs through the modification of redox states of a multifunctional ROS‐responsive protein, DJ‐1, revealing a novel regulatory mechanism for differentiation of MSCs into SMCs and shedding light into the future development of stem‐cell‐targeted pharmacotherapy.     

Secreted microvesicular miR‐31 inhibits osteogenic differentiation of mesenchymal stem cells

Damage to cells and tissues is one of the driving forces of aging and age‐related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self‐renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor‐age‐dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR‐31 as a crucial component. We demonstrated that miR‐31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR‐31 is secreted within senescent cell‐derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled‐3. Therefore, we suggest that microvesicular miR‐31 in the plasma of elderly might play a role in the pathogenesis of age‐related impaired bone formation and that miR‐31 might be a valuable plasma‐based biomarker for aging and for a systemic environment that does not favor cell‐based therapies whenever osteogenesis is a limiting factor.     

Human umbilical cord mesenchymal stem cells facilitate the up‐regulation of miR‐153‐3p,whereby attenuating MGO‐induced peritoneal fibrosis in rats

MiRNAs contribute greatly to epithelial to mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs), which is a crucial step in peritoneal fibrosis (PF). In this study, we tried to profile whether miRNA expression differences exist after human umbilical cord mesenchymal stem cells (hUCMSCs) treatment in PF rats and investigate the possible role of miR‐153‐3p involved in anti‐EMT process. We randomly assigned 34 rats into three groups: control group (Group Control), MGO‐induced PF rats (Group MGO) and hUCMSCs‐treated rats (Group MGO + hUCMSCs). MiRNA microarrays and real‐time PCR analyses were conducted in three groups. α‐SMA, Snail1 and E‐cadherin expression were detected by Western blot. Luciferase reporter assays were used to detect the effects of miR‐153‐3p overexpression on Snai1 in rat peritoneal mesothelial cells (RPMCs). We identified differentially expressed miRNAs related to EMT, in which miR‐153‐3p demonstrated the greatest increase in Group MGO + hUCMSCs. Transient cotransfection of miR‐153‐3p mimics with luciferase expression plasmids resulted in a significant repression of Snai1 3′‐untranslated region luciferase activity in RPMCs. These studies suggest that miR‐153‐3p is a critical molecule in anti‐EMT effects of hUCMSCs in MGO‐induced PF rats. MiR‐153‐3p might exert its beneficial effect through directly targeting Snai1.     

Induced miR‐99a expression represses Mtor cooperatively with miR‐150 to promote regulatory T‐cell differentiation

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4⁺ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T‐cell‐expressed miRNAs in naive mouse CD4⁺ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR‐100, miR‐99a and miR‐10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR‐99a cooperated with miR‐150 to repress the expression of the Th17‐promoting factor mTOR. The comparably low expression of miR‐99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR‐150 could only repress Mtor in the presence of miR‐99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs.