Senescence Of Mesenchymal Stem Cells Research Articles

Modification of the Properties of Extracellular Matrix of Senescent Mesenchymal Stem Cells.

Cell senescence leads to changes in the secretory activity of mesenchymal stem cells (MSC), including proteins of extracellular matrix (ECM). Here we studied the regulatory properties of ECM of senescent MSC in a model with endothelial cells (EC). EC were seeded onto a decellularized extracellular matrix of senescent MSC. Changes in cell morphology and a decrease in cell growth were observed. In addition, increased production of inflammatory chemokines MCP-1 and GROα and reduced synthesis of proangiogenic growth factor FGF-2 were revealed. Analysis of ECM showed quantitative and qualitative changes, including fibronectin layer morphology, total protein content, and concentration of deposited growth factors such as VEGF. Thus, our work demonstrates that senescence of MSC can lead to modification of the effects of their ECM on EC activity.

Multi-omics analysis of human mesenchymal stem cells shows cell aging that alters immunomodulatory activity through the downregulation of PD-L1

Mesenchymal stem cells (MSCs) possess potent immunomodulatory activity and have been extensively investigated for their therapeutic potential in treating inflammatory disorders. However, the mechanisms underlying the immunosuppressive function of MSCs are not fully understood, hindering the development of standardized MSC-based therapies for clinical use. In this study, we profile the single-cell transcriptomes of MSCs isolated from adipose tissue (AD), bone marrow (BM), placental chorionic membrane (PM), and umbilical cord (UC). Our results demonstrate that MSCs undergo a progressive aging process and that the cellular senescence state influences their immunosuppressive activity by downregulating PD-L1 expression. Through integrated analysis of single-cell transcriptomic and proteomic data, we identify GATA2 as a regulator of MSC senescence and PD-L1 expression. Overall, our findings highlight the roles of cell aging and PD-L1 expression in modulating the immunosuppressive efficacy of MSCs and implicating perinatal MSC therapy for clinical applications in inflammatory disorders.

Multi-omic analysis of mandibuloacral dysplasia type A patient iPSC-derived MSC senescence reveals miR-311 as a novel biomarker for MSC senescence.

Mandibuloacral dysplasia type A (MADA) is a rare genetic progeroid syndrome associated with lamin A/C (LMNA) mutations. Pathogenic mutations of LMNA result in nuclear structural abnormalities, mesenchymal tissue damage and progeria phenotypes. However, it remains elusive how LMNA mutations cause mesenchymal-derived cell senescence and disease development. Here, we established an in vitro senescence model using induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) from MADA patients with homozygous LMNA p.R527C mutation. When expanded to passage 13 in vitro, R527C iMSCs exhibited marked senescence and attenuation of stemness potential, accompanied by immunophenotypic changes. Transcriptome and proteome analysis revealed that cell cycle, DNA replication, cell adhesion and inflammation might play important roles in senescence. In-depth evaluation of changes in extracellular vesicle (EV) derived iMSCs during senescence revealed that R527C iMSC-EVs could promote surrounding cell senescence by carrying pro-senescence microRNAs (miRNAs), including a novel miRNA called miR-311, which can serve as a new indicator for detecting chronic and acute mesenchymal stem cell (MSC) senescence and play a role in promoting senescence. Overall, this study advanced our understanding of the impact of LMNA mutations on MSC senescence and provided novel insights into MADA therapy as well as the link between chronic inflammation and aging development.

Repurpose dasatinib and quercetin: Targeting senescent cells ameliorates postmenopausal osteoporosis and rejuvenates bone regeneration.

Clinical therapies developed for estrogen-deficiency-driven postmenopausal osteoporosis (PMO) and related diseases, such as bone degeneration, show multiple adverse effects nowadays. Targeting senescent cells (SnCs) and the consequent senescence-associated secretory phenotype (SASP) with a combination of dasatinib and quercetin (DQ) is a recently developed novel therapy for multiple age-related diseases. Herein, we found that estrogen deficiency induced-bone loss was attributed to a pro-inflammatory microenvironment with SASP secretions and accelerated SnC accumulation, especially senescent mesenchymal stem cells (MSCs) characterized by exhaustion and dysfunction in middle aged rats. Systematically targeting SnCs with DQ strikingly ameliorated PMO and restored MSC function. Local administration of DQ and bone morphogenetic protein 2 (BMP2) in combination promoted osteogenic differentiation of MSCs and rejuvenated osteoporotic bone regeneration. Our results repurposed DQ as an attractive therapy for treating PMO and related diseases.

Decreased miR-17-92 Cluster Correlates with Senescent Features, Disrupted Oxidative Homeostasis, and Impaired Therapeutic Efficacy of Mesenchymal Stem Cells.

Aging and replicative cellular senescence are associated with the reduced therapeutic potential of mesenchymal stem cells (MSCs) on a variety of diseases. This study aimed to determine the mechanism in MSC senescence and further explore a modification strategy to reverse senescence-associated cell dysfunction in order to improve the therapeutic efficacy of MSCs on acute liver failure (ALF). We found that the adipose tissue-derived MSCs from old mice (oAMSCs) exhibited senescence phenotypes and showed reduced therapeutic efficacy in lipopolysaccharide and D-galactosamine-induced ALF, as shown by the increased hepatic necrosis, liver histology activity index scores, serum liver function indicator levels and inflammatory cytokine levels. The expression of miR-17-92 cluster members, especially miR-17 and miR-20a, was obviously decreased in oAMSCs and replicatively senescent AMSCs, and was consistent with the decreased oncogene c-Myc level during AMSC senescence and may mediate c-Myc stemness addiction. Further experiments revealed that c-Myc-regulated miR-17-92 expression contributed to increased p21 expression and redox system dysregulation during AMSC senescence. Furthermore, modification of AMSCs with the two key miRNAs in the miR-17-92 cluster mentioned above reversed the senescence features of oAMSCs and restored the therapeutic effect of senescent AMSCs on ALF. In conclusion, the cellular miR-17-92 cluster level is correlated with AMSC senescence and can be used both as an index for evaluating and as a modification target for improving the therapeutic potential of AMSCs.

GuiLu-ErXian Glue extract promotes mesenchymal stem cells (MSC)-Induced chondrogenesis via exosomes release and delays aging in the MSC senescence process

Ethnopharmacological relevanceThe treatment of osteoarthritis (OA) patients is a challenging problem. Mesenchymal stem cells (MSCs) are multipotent cells and play key roles in regenerative medicine for cartilage degeneration. GuiLu-ErXian Glue (GLEXG) is an herbal remedy widely used in traditional Chinese medicine to treat joint pain and disability in elderly OA patients. However, the mechanisms of how GLEXG affects MSCs-induced chondrogensis remains to be elucidated. Aim of the studyThe aim of this study was to investigate the effects of GLEXG on MSC-derived chondrogenesis, both in vitro and in vivo and its potential mechanisms. MethodsUsing human MSC (hMSCs) as in vitro model, the effects of HPLC-profiled GLEXG water extract on chondrogenic differentiation were investigated by 3D spheroid cultures under chondrogenesis-inducing medium (CIM) condition. The chondrogenesis process was evaluated by measuring the sphere sizes, chondrogenesis-related genes expression by reverse transcription real-time PCR that targeted type II/X collagens, SOX9, aggrecan, and protein expression by immunostaining. Anti-TGF-β1 neutralization antibody was used for mechanistic study. Mono-iodoacetate (MIA) induced OA joint was used to evaluate the effects of GLEXG on in vivo model. MSCs-derived exosomes were purified for proteomics study and senescence process was evaluated by cumulative population doublings and senescence-associated β-Galactosidase staining. ResultsThe results showed that GLEXG enhanced hMSCs chondrogenesis and upregulated RNA expression of type II/X collagen, SOX9 and aggrecan at 0.1 μg/mL, 0.3 μg/mL in vitro. In vivo, GLEXG at the dose of 0.3 μg intraarticular (i.a.) injection rescued the MIA-induced cartilage defect. Proteomics and ingenuity pathway analysis obtained from MSCs-released exosomes suggested that senescence pathway was less activated in GLEXG group than in vehicle group. Besides, GLEXG was able to increase cumulative population doubling and delayed hMSCs senescence process after four passages in cultures. Conclusionwe conclude that GLEXG promotes in vitro MSC-induced chondrogenesis possibly via exosomes release and delays aging in the MSC senescence process and that treatment with GLEXG (0.3 μg, i.a.) rescued cartilage defects in rat OA knee model.

Fisetin Attenuates Cellular Senescence Accumulation During Culture Expansion of Human Adipose-Derived Stem Cells.

Mesenchymal stem cells (MSCs) have long been viewed as a promising therapeutic for musculoskeletal repair. However, regulatory concerns including tumorgenicity, inconsistencies in preparation techniques, donor-to-donor variability, and the accumulation of senescence during culture expansion have hindered the clinical application of MSCs. Senescence is a driving mechanism for MSC dysfunction with advancing age. Often characterized by increased reactive oxygen species, senescence-associated heterochromatin foci, inflammatory cytokine secretion, and reduced proliferative capacity, senescence directly inhibits MSCs efficacy as a therapeutic for musculoskeletal regeneration. Furthermore, autologous delivery of senescent MSCs can further induce disease and aging progression through the secretion of the senescence-associated secretory phenotype (SASP) and mitigate the regenerative potential of MSCs. To alleviate these issues, the use of senolytic agents to selectively clear senescent cell populations has become popular. However, their benefits to attenuating senescence accumulation in human MSCs during the culture expansion process have not yet been elucidated. To address this, we analyzed markers of senescence during the expansion of human primary adipose-derived stem cells (ADSCs), a population of fat-resident MSCs commonly used in regenerative medicine applications. Next, we used the senolytic agent fisetin to determine if we can reduce these markers of senescence within our culture-expanded ADSC populations. Our results indicate that ADSCs acquire common markers of cellular senescence including increased reactive oxygen species, senescence-associated β-galactosidase, and senescence-associated heterochromatin foci. Furthermore, we found that the senolytic agent fisetin works in a dose-dependent manner and selectively attenuates these markers of senescence while maintaining the differentiation potential of the expanded ADSCs.

Stress-induced senescence in mesenchymal stem cells: Triggers, hallmarks, and current rejuvenation approaches

Mesenchymal stem cells (MSCs) have emerged as promising cell-based therapies in the treatment of degenerative and inflammatory conditions. However, despite accumulating evidence of the breadth of MSC functional potency, their broad clinical translation is hampered by inconsistencies in therapeutic efficacy, which is at least partly due to the phenotypic and functional heterogeneity of MSC populations as they progress towards senescence in vitro. MSC senescence, a natural response to aging and stress, gives rise to altered cellular responses and functional decline. This review describes the key regenerative properties of MSCs; summarises the main triggers, mechanisms, and consequences of MSC senescence; and discusses current cellular and extracellular strategies to delay the onset or progression of senescence, or to rejuvenate biological functions lost to senescence.

YAP Inhibition Alleviates Simulated Microgravity-Induced Mesenchymal Stem Cell Senescence via Targeting Mitochondrial Dysfunction.

Weightlessness in space leads to bone loss, muscle atrophy, and impaired immune defense in astronauts. Mesenchymal stem cells (MSCs) play crucial roles in maintaining the homeostasis and function of the tissue. However, how microgravity affects the characteristics MSCs and the related roles in the pathophysiological changes in astronauts remain barely known. Here we used a 2D-clinostat device to simulate microgravity. Senescence-associated-β-galactosidase (SA-β-gal) staining and the expression of senescent markers p16, p21, and p53 were used to evaluate the senescence of MSCs. Mitochondrial membrane potential (mΔΨm), reactive oxygen species (ROS) production, and ATP production were used to evaluate mitochondrial function. Western blot and immunofluorescence staining were used to investigate the expression and localization of Yes-associated protein (YAP). We found that simulated microgravity (SMG) induced MSC senescence and mitochondrial dysfunction. Mito-TEMPO (MT), a mitochondrial antioxidant, restored mitochondrial function and reversed MSC senescence induced by SMG, suggesting that mitochondrial dysfunction mediates SMG-induced MSC senescence. Further, it was found that SMG promoted YAP expression and its nuclear translocation in MSCs. Verteporfin (VP), an inhibitor of YAP, restored SMG-induced mitochondrial dysfunction and senescence in MSCs by inhibiting YAP expression and nuclear localization. These findings suggest that YAP inhibition alleviates SMG-induced MSC senescence via targeting mitochondrial dysfunction, and YAP may be a potential therapeutic target for the treatment of weightlessness-related cell senescence and aging.

Constitutively activated AMPKα1 protects against skeletal aging in mice by promoting bone-derived IGF-1 secretion.

Senile osteoporosis is characterized by age-related bone loss and bone microarchitecture deterioration. However, little is known to date about the mechanism that maintains bone homeostasis during aging. In this study, we identify adenosine monophosphate-activated protein kinase alpha 1 (AMPKα1) as a critical factor regulating the senescence and lineage commitment of mesenchymal stem cells (MSCs). A phospho-mutant mouse model shows that constitutive AMPKα1 activation prevents age-related bone loss and promoted MSC osteogenic commitment with increased bone-derived insulin-like growth factor 1 (IGF-1) secretion. Mechanistically, upregulation of IGF-1 signalling by AMPKα1 depends on cAMP-response element binding protein (CREB)-mediated transcriptional regulation. Furthermore, the essential role of the AMPKα1/IGF-1/CREB axis in promoting aged MSC osteogenic potential is confirmed using three-dimensional (3D) culture systems. Taken together, these results can provide mechanistic insight into the protective effect of AMPKα1 against skeletal aging by promoting bone-derived IGF-1 secretion.

Molecular Mobility of Polyrotaxane Surfaces Alleviates Oxidative Stress-Induced Senescence in Mesenchymal Stem Cells.

Polyrotaxane is a supramolecular assembly consisting of multiple cyclic molecules threaded by a linear polymer. One of the unique properties of polyrotaxane is molecular mobility, cyclic molecules moving along the linear polymer. Molecular mobility of polyrotaxane surfaces affects cell spreading, differentiation, and other cell-related aspects through changing subcellular localization of yes-associated proteins (YAPs). Subcellular YAP localization is also related to cell senescence derived from oxidative stress, which is known to cause cancer, diabetes, and heart disease. Herein, the effects of polyrotaxane surface molecular mobility on subcellular YAP localization and cell senescence following H2 O2 -induced oxidative stress are evaluated in human mesenchymal stem cells (HMSCs) cultured on polyrotaxane surfaces with different molecular mobilities. Oxidative stress promotes cytoplasmic YAP localization in HMSCs on high-mobility polyrotaxane surfaces; however, low-mobility polyrotaxane surfaces more effectively maintain nuclear YAP localization, exhibiting lower senescence-associated β-galactosidase activity and senescence-related gene expression and DNA damage than that seen with the high-mobility surfaces. These results suggest that the molecular mobility of polyrotaxane surfaces regulates subcellular YAP localization, thereby protecting HMSCs from oxidative stress-induced cell senescence. Applying the molecular mobility of polyrotaxane surfaces to implantable scaffolds can provide insights into the prevention and treatment of diseases caused by oxidative stress.

P53 negatively regulates the osteogenic differentiation in jaw bone marrow MSCs derived from diabetic osteoporosis

Patients with diabetic osteoporosis (DOP) often suffer from poor osseointegration of artificial implants, which is a challenge that affects implant outcomes. The osteogenic differentiation ability of human jaw bone marrow mesenchymal stem cells (JBMMSCs) is the key to implant osseointegration. Studies have shown that the microenvironment of hyperglycemia affects the osteogenic differentiation of mesenchymal stem cells (MSC), but the mechanism is still unclear. Therefore, the aim of this study was to isolate and culture JBMMSCs from surgically derived bone fragments from DOP patients and control patients to investigate the differences in their osteogenic differentiation ability and to elucidate its mechanisms. The results showed that the osteogenic ability of hJBMMSCs was significantly decreased in the DOP environment. Mechanism study showed that the expression of senescence marker gene P53 was significantly increased in DOP hJBMMSCs compared to control hJBMMSCs according to RNA-sequencing result. Further, DOP hJBMMSCs were found to display significant senescence using β-galactosidase staining, mitochondrial membrane potential and ROS assay, qRT-PCR and WB analysis. Overexpression of P53 in hJBMMSCs, knockdown of P53 in DOP hJBMMSCs, and knockdown followed by overexpression of P53 significantly affected the osteogenic differentiation ability of hJBMMSCs. These results suggest that MSC senescence is an important reason for decreasing osteogenic capacity in DOP patients. P53 is a key target in regulating hJBMMSCs aging, and knocking down P53 can effectively restore the osteogenic differentiation ability of DOP hJBMMSCs and promote osteosynthesis in DOP dental implants. It provided a new idea to elucidate the pathogenesis and treatment of diabetic bone metabolic diseases.

Senescence of mesenchymal stem cells: implications in extracellular vesicles, miRNAs and their functional and therapeutic potentials

Senescence is seen as the cellular counterpart of tissue and biological aging, with irreversible stagnation of cell growth, and changes in function and behavior. Mesenchymal stem cells (MSCs) are one of the key therapeutic tools in regenerative medicine, and their regenerative and therapeutic potential declines significantly with the increasing age of cell donors and prolonged continuous culture in vitro. MicroRNAs (miRNAs) are regarded as important players in regulating the expression and function of multiple genes and pathways. Emerging evidence suggests that extracellular vesicles (EVs) participate in a complex cell senescence network, at least partially by providing certain miRNAs. Therefore, MSC EVs and miRNAs are implicated in not only contributing to but also influenced by MSC senescence. Here, we will provide an overview of the recent results on roles and mechanisms of miRNAs, particularly EV-miRNAs, involved in MSC senescence, and discuss their implications in functional properties and therapeutic efficacy of MSCs and their EVs. Keywords: Extracellular vesicles, microRNAs, mesenchymal stem cells, senescence

Rapid and Live-Cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry.

Detection of cellular senescence is important quality analytics of cell therapy products, including mesenchymal stromal cells (MSCs). However, its detection is critically limited by the lack of specific markers and the destructive assays used to read out these markers. Here, we establish a rapid, live-cell assay for detecting senescent cells in heterogeneous mesenchymal stromal cell (MSC) cultures. We report that the T2 relaxation time measured by microscale Magnetic Resonance Relaxometry, which is related to intracellular iron accumulation, correlates strongly with senescence markers in MSC cultures under diverse conditions, including different passages and donors, size-sorted MSCs by inertial spiral microfluidic device, and drug-induced senescence. In addition, the live-cell and non-destructive method presented here has general applicability to other cells and tissues and can critically advance our understanding of cellular senescence.

An unbiased proteomics approach to identify the senescence-associated secretory phenotype of human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) derived from bone marrow can support skeletal tissue repair and regeneration owing to their self-renewing capacity, differentiation ability, and trophic functions. Bone marrow-derived MSCs undergo dramatic changes with aging, including the senescence-associated secretory phenotype (SASP) which may largely contribute to age-related changes in bone tissue leading to osteoporosis. A mass spectrometry-based proteomics approach was used to investigate the MSC SASP. Replicative senescence was achieved by exhaustive in vitro sub-cultivation and confirmed by standard proliferation criteria. Conditioned media from non-senescent and senescent MSCs underwent mass spectrometry. Proteomics and bioinformatics analyses enabled the identification of 95 proteins expressed uniquely in senescent MSCs. Protein ontology analysis revealed the enrichment of proteins linked to the extracellular matrix, exosomes, cell adhesion, and calcium ion binding. The proteomic analysis was independently validated by taking ten identified proteins with relevance to bone aging and confirming their increased abundance in conditioned media from replicatively senescent versus non-senescent MSCs (ACTα2, LTF, SOD1, IL-6, LTBP2, PXDN, SERPINE 1, COL1α1, THBS1, OPG). These target proteins were used to further investigate changes in the MSC SASP profile in response to other inducers of senescence, ionizing radiation (IR) and H2O2. Similar secreted protein expression profiles with replicatively senescent cells were seen with H2O2 treatment except for LTF and PXDN, which were increased by IR treatment. With both IR and H2O2 treatment there was a decrease in THBS1. In vivo investigation of these secreted proteins with aging was shown by significant changes in the abundance of OPG, COL1α1, IL-6, ACTα2, SERPINE 1, and THBS1 in the plasma of aged rats. This unbiased, comprehensive analysis of the changes in the MSC secretome with senescence defines the unique protein signature of the SASP in these cells and provides a better understanding of the aging bone microenvironment.

Eldecalcitol prevented OVX-induced osteoporosis through inhibiting BMSCs senescence by regulating the SIRT1-Nrf2 signal.

Background: Aging and oxidative stress are considered to be the proximal culprits of postmenopausal osteoporosis. Eldecalcitol (ED-71), a new active vitamin D derivative, has shown a good therapeutic effect on different types of osteoporosis, but the mechanism is unclear. This study focused on exploring whether ED-71 could prevent bone loss in postmenopausal osteoporosis by regulating the cell senescence of bone mesenchymal stem cells (BMSCs), and explaining its specific mechanism of action. Materials and methods: An ovariectomized (OVX) rat model was established and 30ng/kg ED-71 was administered orally once a day. The weight of rats was recorded regularly. Micro-computed tomography (CT) and histochemical staining were used to evaluate bone mass, histological parameters, and aging-related factors. Rat bone mesenchymal stem cells were extracted and cultivated in vitro. Aging cells were marked with senescence-associated β-gal (SA-β-gal) dyeing. The mRNA and protein levels of aging-related factors and SIRT1-Nrf2 signal were detected by RT-PCR, Western blot, and immunofluorescence staining. The reactive oxygen species (ROS) levels were detected by DCFH-DA staining. Results: Compared with the Sham group, the bone volume of the ovariectomized group rats decreased while their weight increased significantly. ED-71 prevented bone loss and inhibited weight gain in ovariectomized rats. More importantly, although the expression of aging-related factors in the bone tissue increased in the ovariectomized group, the addition of ED-71 reversed changes in these factors. After extracting and in vitro culturing bone mesenchymal stem cells, the proportion of aging bone mesenchymal stem cells was higher in the ovariectomized group than in the Sham group, accompanied by a significant decrease in the osteogenic capacity. ED-71 significantly improved the bone mesenchymal stem cells senescence caused by ovariectomized. In addition, ED-71 increased the expression of SIRT1 and Nrf2 in ovariectomized rat bone mesenchymal stem cells. Inhibition of SIRT1 or Nrf2 decreased the inhibitory effect of ED-71 on bone mesenchymal stem cells senescence. ED-71 also showed a suppression effect on the reactive oxygen species level in bone mesenchymal stem cells. Conclusion: Our results demonstrated that ED-71 could inhibit the cell senescence of bone mesenchymal stem cells in ovariectomized rats by regulating the SIRT1-Nrf2 signal, thereby preventing bone loss caused by osteoporosis.

Ginsenoside Rg2 Promotes the Proliferation and Stemness Maintenance of Porcine Mesenchymal Stem Cells through Autophagy Induction

Mesenchymal stem cells (MSCs) can be used as a cell source for cultivated meat production due to their adipose differentiation potential, but MSCs lose their stemness and undergo replicative senescence during expansion in vitro. Autophagy is an important mechanism for senescent cells to remove toxic substances. However, the role of autophagy in the replicative senescence of MSCs is controversial. Here, we evaluated the changes in autophagy in porcine MSCs (pMSCs) during long-term culture in vitro and identified a natural phytochemical, ginsenoside Rg2, that could stimulate pMSC proliferation. First, some typical senescence characteristics were observed in aged pMSCs, including decreased EdU-positive cells, increased senescence-associated beta-galactosidase activity, declined stemness-associated marker OCT4 expression, and enhanced P53 expression. Importantly, autophagic flux was impaired in aged pMSCs, suggesting deficient substrate clearance in aged pMSCs. Rg2 was found to promote the proliferation of pMSCs using MTT assay and EdU staining. In addition, Rg2 inhibited D-galactose-induced senescence and oxidative stress in pMSCs. Rg2 increased autophagic activity via the AMPK signaling pathway. Furthermore, long-term culture with Rg2 promoted the proliferation, inhibited the replicative senescence, and maintained the stemness of pMSCs. These results provide a potential strategy for porcine MSC expansion in vitro.

PPP3R1 Promotes MSCs Senescence by Inducing Plasma Membrane Depolarization and Increasing Ca2+ Influx.

Aging of mesenchymal stem cells(MSCs) has been widely reported to be strongly associated with aging-related diseases, including osteoporosis (OP). In particular, the beneficial functions of mesenchymal stem cells decline with age, limiting their therapeutic efficacy in age-related bone loss diseases. Therefore, how to improve mesenchymal stem cell aging to treat age-related bone loss is the current research focus. However, the underlying mechanism remains unclear. In this study, protein phosphatase 3, regulatory subunit B, alpha isoform, calcineurin B, type I (PPP3R1) was found to accelerate the senescence of mesenchymal stem cells, resulting in reduced osteogenic differentiation and enhanced adipogenic differentiation in vitro. Mechanistically, PPP3R1 induces changes in membrane potential to promote cellular senescence by polarizing to depolarizing, increasing Ca2+ influx and activating downstream NFAT/ATF3/p53 signaling. In conclusion, the results identify a novel pathway of mesenchymal stem cell aging that may lead to novel therapeutic approaches for age-related bone loss.

Empagliflozin-Pretreated Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Attenuated Heart Injury.

Small extracellular vesicles derived from mesenchymal stem cells (MSCs) play important roles in cardiac protection. Studies have shown that the cardiovascular protection of sodium-glucose cotransporter 2 inhibitor (SGLT2i) is independent of its hypoglycemic effect. This study is aimed at investigating whether small extracellular vesicles derived from MSCs pretreated with empagliflozin (EMPA) has a stronger cardioprotective function after myocardial infarction (MI) and to explore the underlying mechanisms. We evaluated the effects of EMPA on MSCs and the effects of EMPA-pretreated MSCs-derived small extracellular vesicles (EMPA-sEV) on myocardial apoptosis, angiogenesis, and cardiac function after MI in vitro and in vivo. The small extracellular vesicles of control MSCs (MSC-sEV) and EMPA-pretreated MSCs were extracted, respectively. Small extracellular vesicles were cocultured with apoptotic H9c2 cells induced by H2O2 or injected into the infarcted area of the Sprague-Dawley (SD) rat myocardial infarction model. EMPA increased the cell viability, migration ability, and inhibited apoptosis and senescence of MSCs. In vitro, EMPA-sEV inhibited apoptosis of H9c2 cells compared with the control group (MSC-sEV). In the SD rat model of MI, EMPA-sEV inhibited myocardial apoptosis and promoted angiogenesis in the infarct marginal areas compared with the MSC-sEV. Meanwhile, EMPA-sEV reduced infarct size and improved cardiac function. Through small extracellular vesicles (miRNA) sequencing, we found several differentially expressed miRNAs, among which miR-214-3p was significantly elevated in EMPA-sEV. Coculture of miR-214-3p high expression MSC-derived small extracellular vesicles with H9c2 cells produced similar protective effects. In addition, miR-214-3p was found to promote AKT phosphorylation in H9c2 cells. Our data suggest that EMPA-sEV significantly improve cardiac repair after MI by inhibiting myocardial apoptosis. miR-214-3p at least partially mediated the myocardial protection of EMPA-sEV through the AKT signaling pathway.

A passage-dependent network for estimating the in vitro senescence of mesenchymal stromal/stem cells using microarray, bulk and single cell RNA sequencing.

Long-term in vitro culture of human mesenchymal stem cells (MSCs) leads to cell lifespan shortening and growth stagnation due to cell senescence. Here, using sequencing data generated in the public domain, we have established a specific regulatory network of "transcription factor (TF)-microRNA (miRNA)-Target" to provide key molecules for evaluating the passage-dependent replicative senescence of mesenchymal stem cells for the quality control and status evaluation of mesenchymal stem cells prepared by different procedures. Short time-series expression miner (STEM) analysis was performed on the RNA-seq and miRNA-seq databases of mesenchymal stem cells from various passages to reveal the dynamic passage-related changes of miRNAs and mRNAs. Potential miRNA targets were predicted using seven miRNA target prediction databases, including TargetScan, miRTarBase, miRDB, miRWalk, RNA22, RNAinter, and TargetMiner. Then use the TransmiR v2.0 database to obtain experimental-supported transcription factor for regulating the selected miRNA. More than ten sequencing data related to mesenchymal stem cells or mesenchymal stem cells reprogramming were used to validate key miRNAs and mRNAs. And gene set variation analysis (GSVA) was performed to calculate the passage-dependent signature. The results showed that during the passage of mesenchymal stem cells, a total of 29 miRNAs were gradually downregulated and 210 mRNA were gradually upregulated. Enrichment analysis showed that the 29 miRNAs acted as multipotent regulatory factors of stem cells and participated in a variety of signaling pathways, including TGF-beta, HIPPO and oxygen related pathways. 210 mRNAs were involved in cell senescence. According to the target prediction results, the targets of these key miRNAs and mRNAs intersect to form a regulatory network of "TF-miRNA-Target" related to replicative senescence of cultured mesenchymal stem cells, across 35 transcription factor, 7 miRNAs (has-mir-454-3p, has-mir-196b-5p, has-mir-130b-5p, has-mir-1271-5p, has-let-7i-5p, has-let-7a-5p, and has-let-7b-5p) and 7 predicted targets (PRUNE2, DIO2, CPA4, PRKAA2, DMD, DDAH1, and GATA6). This network was further validated by analyzing datasets from a variety of mesenchymal stem cells subculture and lineage reprogramming studies, as well as qPCR analysis of early passages mesenchymal stem cells versus mesenchymal stem cells with senescence morphologies (SA-β-Gal+). The "TF-miRNA-Target" regulatory network constructed in this study reveals the functional mechanism of miRNAs in promoting the senescence of MSCs during in vitro expansion and provides indicators for monitoring the quality of functional mesenchymal stem cells during the preparation and clinical application.