Senescence Of Mesenchymal Stem Cells Research Articles

RNA sequencing profiles reveal dynamic signaling and glucose metabolic features during bone marrow mesenchymal stem cell senescence

BackgroundStem cell senescence is considered as a significant driver of organismal aging. As individuals age, the number of stem cells is declined, and the ability to proliferate and survive is also weakened. It has been reported that metabolism plays an important role in stem cell self-renewal, multilineage differentiation, senescence and fate determination, which has aroused widespread concerns. However, whether metabolism-related genes or signalling pathways are involved in physiological aging remain largely undetermined.ResultsIn the current study, we showed 868 up-regulated and 2006 down-regulated differentially expressed genes (DEGs) in bone marrow mesenchymal stem cells (MSCs) from old rats in comparison with that from young rats by performing RNA sequence. And DEGs functions and pathways were further selected by function enrichment analysis. The results indicated that the high expression of DEGs might participate in cell differentiation, growth factor binding and etc., while the down-regulated DEGs were majorly enriched in metabolism process, such as the cellular metabolic process and mitochondria. Then, we screened and verified DEGs related to glucose metabolism and investigated the glycolysis levels. We identified that glucose uptake, lactate secretion, ATP production and relative extracellular acidification rates (ECAR) were all diminished in MSCs from old rats. More importantly, we conducted microRNA prediction on the key DEGs of glycolysis to elucidate the potential molecular mechanisms of glucose metabolism affecting MSC senescence.ConclusionsOur study unravelled the profiles of DEGs in age-associated MSC senescence and their functions and pathways. We also clarified DEGs related to glucose metabolism and down-regulated glycolysis level in age-associated MSC senescence. This study will uncover the metabolic effects on regulating stem cell senescence, and provide novel therapeutic targets for ameliorating age-associated phenotypes.

Zuogui Wan slowed senescence of bone marrow mesenchymal stem cells by suppressing Wnt/β-catenin signaling

Ethnopharmacological relevanceIn traditional Chinese medicine (TCM), Zuogui Wan (ZGW) is a classical prescription for senile disorders and delay aging. Modern studies show that ZGW promotes central nerve cell regeneration, prevents and cures osteoporosis, enhances the body's antioxidant capacity, regulates the body's immune function, and promotes mesenchymal stem cells (MSCs) proliferation. Aim of the study: It has been shown that MSCs aging is closely associated with organism's aging and age-related disorders. The study aimed to define the effects of ZGW on the aging bone marrow mesenchymal stem cells (BMSCs) and to identify the mechanisms of ZGW delaying BMSCs senescence. Materials and methods: Network pharmacology analysis combined with GEO data mining, molecular docking and experimental validation were used to evaluate the mechanisms by which ZGW delays MSCs senescence (MSCS). LC-MS was used for quality control analysis of ZGW. Results: PPI network analysis revealed that EGF, TNF, JUN, MMPs, IL-6, MAPK8, and MYC are components of the core PPI network. GO and KEGG analyses revealed that oxidative stress, regulation of response to DNA damage stimuli, and Wnt signaling were significantly enriched. GEO database validation also indicated that Wnt signaling closely correlated with MSCs aging. Molecular docking analysis of the top-13 active components in the “ZGW-Targets-MSCS” network indicated that most components have strong affinity for key proteins in Wnt signaling, suggesting that modulation of Wnt signaling is an important mechanism of ZGW activity against MSCS. Further experimental validation found that ZGW indeed regulates Wnt signaling and suppresses the expression of age-related factors to enhance cell proliferation, ameliorate DNA damage, and reduce senescence-related secretory phenotype (SASP) secretion, thereby maintaining multidirectional differentiation of rat BMSCs. Similar results were obtained using the Wnt inhibitor, XAV-939. Conclusions: Together, our data show that ZGW slows BMSCs aging by suppressing Wnt signaling.

Role of Lysosomal Acidification Dysfunction in Mesenchymal Stem Cell Senescence.

Mesenchymal stem cell (MSC) transplantation has been widely used as a potential treatment for a variety of diseases. However, the contradiction between the low survival rate of transplanted cells and the beneficial therapeutic effects has affected its clinical use. Lysosomes as organelles at the center of cellular recycling and metabolic signaling, play essential roles in MSC homeostasis. In the first part of this review, we summarize the role of lysosomal acidification dysfunction in MSC senescence. In the second part, we summarize some of the potential strategies targeting lysosomal proteins to enhance the therapeutic effect of MSCs.

CD26 Inhibition Potentiates the Therapeutic Effects of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells by Delaying Cellular Senescence.

Mesenchymal stem cells (MSCs) are recognized as potential treatments for multiple degenerative and inflammatory disorders as a number of animal and human studies have indicated their therapeutic effects. There are also several clinically approved medicinal products that are manufactured using these cells. For such large-scale manufacturing requirements, the in vitro expansion of harvested MSCs is essential. Multiple subculturing of MSCs, however, provokes cellular senescence processes which is known to deteriorate the therapeutic efficacy of the cells. Strategies to rejuvenate or selectively remove senescent MSCs are therefore highly desirable for fostering future clinical applications of these cells. In this present study, we investigated gene expression changes related to cellular senescence of MSCs derived from umbilical cord blood and found that CD26, also known as DPP4, is significantly upregulated upon cellular aging. We further observed that the inhibition of CD26 by genetic or pharmacologic means delayed the cellular aging of MSCs with their multiple passaging in culture. Moreover, the sorting and exclusion of CD26-positive MSCs from heterogenous cell population enhanced in vitro cell attachment and reduced senescence-associated cytokine secretion. CD26-negative MSCs also showed superior therapeutic efficacy in mouse lung emphysema model. Our present results collectively suggest CD26 is a potential novel target for the rejuvenation of senescent MSCs for their use in manufacturing MSC-based applications.

Exploring Microenvironment Strategies to Delay Mesenchymal Stem Cell Senescence.

Mesenchymal stem cells (MSCs) have recently emerged as an important candidate for cell therapy and tissue regeneration. However, some limitations in translational research and therapies still exist, such as insufficient cell supply, inadequate differentiation potential, and decreased immune capacity, all of which result from replicative senescence during long-term in vitro culture. In vitro, stem cells lack a protective microenvironment owing to the absence of physical and biochemical cues compared with the in vivo niche, which provides dynamic physicochemical and biological cues. This difference results in accelerated aging after long-term in vitro culture. Therefore, it remains a great challenge to delay replicative senescence in culture. Constructing a microenvironment to delay replicative senescence of MSCs by maintaining their phenotypes, properties, and functions is a feasible strategy to solve this problem, and has made measurable progress both in preclinical studies and in clinical trials. Here, we review the current knowledge on the characteristics of senescent MSCs, explore the molecular mechanisms of MSCs senescence, describe the niche of MSCs, and discuss some current microenvironment strategies to delay MSCs replicative senescence that can broaden their range of therapeutic applications.

TLR4 downregulation by the RNA-binding protein PUM1 alleviates cellular aging and osteoarthritis.

Dysfunction of mRNA or RNA-binding proteins (RBPs) causes cellular aging and age-related degenerative diseases; however, information regarding the mechanism through which RBP-mediated posttranscriptional regulation affects cellular aging and related disease processes is limited. In this study, PUM1 was found to be associated with the self-renewal capacity and aging process of human mesenchymal stem cells (MSC). PUM1 interacted with the 3'-untranslated region of Toll-like receptor 4 (TLR4) to suppress TLR4 mRNA translation and regulate the activity of nuclear factor-κB (NF-κB), a master regulator of the aging process in MSCs. PUM1 overexpression protected MSCs against H2O2-induced cellular senescence by suppressing TLR4-mediated NF-κB activity. TLR4-mediated NF-κB activation is a key regulator in osteoarthritis (OA) pathogenesis. PUM1 overexpression enhanced the chondrogenic potential of MSCs even under the influence of inflammation-inducing factors, such as lipopolysaccharide (LPS) or interleukin-1β (IL-1β), whereas the chondrogenic potential was reduced following the PUM1 knockdown-mediated TLR4 activation. PUM1 levels decreased under inflammatory conditions in vitro and during OA progression in human and mouse disease models. PUM1 knockdown in human chondrocytes promoted chondrogenic phenotype loss, whereas PUM1 overexpression protected the cells from inflammation-mediated disruption of the chondrogenic phenotype. Gene therapy using a lentiviral vector encoding mouse PUM1 showed promise in preserving articular cartilage integrity in OA mouse models. In conclusion, PUM1 is a novel suppressor of MSC aging, and the PUM1-TLR4 regulatory axis represents a potential therapeutic target for OA.

NAP1L2 drives mesenchymal stem cell senescence and suppresses osteogenic differentiation.

Senescence of bone marrow mesenchymal stem cells (BMSCs) impairs stemness and osteogenic differentiation, but the key regulators for senescence and the related osteogenesis are not well defined. Herein, we screened the gene expression profiles of human BMSCs from young and old donors and identified that elevation of the nucleosome assembly protein 1‐like 2 (NAP1L2) expression was correlated with BMSC senescence and impaired osteogenesis. Elevated NAP1L2 expression was observed in replicative cell senescence and induced cell senescence in vitro, and in age‐related senescent human and mouse BMSCs in vivo, concomitant with significantly augmented chromatin accessibility detected by ATAC‐seq. Loss‐ and gain‐of‐functions of NAP1L2 affected activation of NF‐κB pathway, status of histone 3 lysine 14 acetylation (H3K14ac), and chromatin accessibility on osteogenic genes in BMSCs. Mechanistic studies revealed that NAP1L2, a histone chaperone, recruited SIRT1 to deacetylate H3K14ac on promoters of osteogenic genes such as Runx2, Sp7, and Bglap and suppressed the osteogenic differentiation of BMSCs. Importantly, molecular docking analysis showed a possible bond between NAP1L2 and an anti‐aging reagent, the nicotinamide mononucleotide (NMN), and indeed, administration of NMN alleviated senescent phenotypes of BMSCs. In vivo and clinical evidence from aging mice and patients with senile osteoporosis also confirmed that elevation of NAP1L2 expression was associated with suppressed osteoblastogenesis. Taken together, our findings suggest that NAP1L2 is a regulator of both BMSC cell senescence and osteogenic differentiation, and provide a new theoretical basis for aging‐related disease.

Pulmonary Mesenchymal Stem Cells in Mild Cases of COVID-19 Are Dedicated to Proliferation; In Severe Cases, They Control Inflammation, Make Cell Dispersion, and Tissue Regeneration

Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in virtually all tissues; they have potent self-renewal capacity and differentiate into multiple cell types. For many reasons, these cells are a promising therapeutic alternative to treat patients with severe COVID-19 and pulmonary post-COVID sequelae. These cells are not only essential for tissue regeneration; they can also alter the pulmonary environment through the paracrine secretion of several mediators. They can control or promote inflammation, induce other stem cells differentiation, restrain the virus load, and much more. In this work, we performed single-cell RNA-seq data analysis of MSCs in bronchoalveolar lavage samples from control individuals and COVID-19 patients with mild and severe clinical conditions. When we compared samples from mild cases with control individuals, most genes transcriptionally upregulated in COVID-19 were involved in cell proliferation. However, a new set of genes with distinct biological functions was upregulated when we compared severely affected with mild COVID-19 patients. In this analysis, the cells upregulated genes related to cell dispersion/migration and induced the γ-activated sequence (GAS) genes, probably triggered by IFNGR1 and IFNGR2. Then, IRF-1 was upregulated, one of the GAS target genes, leading to the interferon-stimulated response (ISR) and the overexpression of many signature target genes. The MSCs also upregulated genes involved in the mesenchymal-epithelial transition, virus control, cell chemotaxis, and used the cytoplasmic RNA danger sensors RIG-1, MDA5, and PKR. In a non-comparative analysis, we observed that MSCs from severe cases do not express many NF-κB upstream receptors, such as Toll-like (TLRs) TLR-3, -7, and -8; tumor necrosis factor (TNFR1 or TNFR2), RANK, CD40, and IL-1R1. Indeed, many NF-κB inhibitors were upregulated, including PPP2CB, OPTN, NFKBIA, and FHL2, suggesting that MSCs do not play a role in the “cytokine storm” observed. Therefore, lung MSCs in COVID-19 sense immune danger and act protectively in concert with the pulmonary environment, confirming their therapeutic potential in cell-based therapy for COVID-19. The transcription of MSCs senescence markers is discussed.

Uridine relieves MSCs and chondrocyte senescence in vitvo and exhibits the potential to treat osteoarthritis in vivo

ABSTRACT Osteoarthritis (OA) is a degenerative disease of extremely high incidence in the elderly. Therefore, anti-aging may be an important prerequisite for treating OA. The senescence of chondrocytes and mesenchymal stem cells (MSCs) is one of the important factors that causes OA. Here, the effect of uridine (which is a functional food derived from plants or animals) on senescence of chondrocytes and MSCs was evaluated in in vivo and in vitro experiments. For this, we established the senescence model of chondrocyte and MSCs in vitro, and established the OA model in vivo, and a series of experiments (such as CLSM, ELISA, Western blot, etc.) were conducted to evaluate the effect of uridine on chondrocyte and MSCs senescence. The results showed that uridine could alleviate chondrocyte and MSCs senescence in vitro by evaluating a series of aging markers. Furthermore, uridine could also relieve OA in vivo. In summary, in the present work, we found that uridine can alleviate chondrocyte and MSCs senescence in in vitro and in vivo experiments. Uridine has shown great potential in the treatment of OA in vivo, suggesting that uridine could be used to treat and prevent OA induced by aging, and has potential clinical applications in future.

Zuogui Wan Slowed Senescence of Mesenchymal Stem Cells by Suppressing Wnt/β-Catenin Signaling

Zuogui Wan Slowed Senescence of Mesenchymal Stem Cells by Suppressing Wnt/β-Catenin Signaling

Inhibition of IKKβ/NF-κB signaling facilitates tendinopathy healing by rejuvenating inflamm-aging induced tendon-derived stem/progenitor cell senescence

Degenerative rotator cuff tendinopathy (RCT) is a chronic tendon disease caused by degeneration and inflammation, which often affects the elderly population. Mesenchymal stem cell senescence is generally recognized as an important pathophysiological mechanism in many age-related skeletal diseases. Herein, we collected human tendon-derived stem/progenitor cells (TSPCs) from degenerative supraspinatus tendons and found that TSPC senescence is closely related to RCT. We further identified that nuclear factor κB (NF-κB) pathway activation is involved in age-related inflammation (inflamm-aging) of degenerative RCT. Moreover, whole genome RNA sequencing revealed that in vitro inhibition of the I kappa B kinase β (IKKβ)/NF-κB signaling pathway could reverse the aged TSPC phenotype with decreased TSPC senescence and increased tenogenic potential. To achieve effective in vivo inhibition of IKKβ/NF-κB signaling, we fabricated IKKβ small interfering RNA (siRNA)-loaded gold nanoclusters (AuNC-siRNA) for efficient and convenient intra-articular delivery of IKKβ siRNA. We found that AuNC-siRNA prevented inflamm-aging-induced TSPC senescence and dysfunction in a degenerative RCT aged rat model. Together, these data show that inflamm-aging causes degenerative RCT through inducing TSPC senescence, which can be reversed by blocking the IKKβ/NF-κB pathway in vivo. Thus, our study provides a promising therapeutic strategy for degenerative RCT via intra-articular delivery of IKKβ siRNA using AuNCs.

TWIST1 controls cellular senescence and energy metabolism in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are promising cells for regenerative medicine therapies because they can differentiate towards multiple cell lineages. However, the occurrence of cellular senescence and the acquiring of the senescence-associated secretory phenotype (SASP) limit their clinical use. Since the transcription factor TWIST1 influences expansion of MSCs, its role in regulating cellular senescence was investigated. The present study demonstrated that silencing of TWIST1 in MSCs increased the occurrence of senescence, characterised by a SASP profile different from irradiation-induced senescent MSCs. Knowing that senescence alters cellular metabolism, cellular bioenergetics was monitored by using the Seahorse XF apparatus. Both TWIST1-silencing-induced and irradiation-induced senescent MSCs had a higher oxygen consumption rate compared to control MSCs, while TWIST1-silencing-induced senescent MSCs had a low extracellular acidification rate compared to irradiation-induced senescent MSCs. Overall, data indicated how TWIST1 regulation influenced senescence in MSCs and that TWIST1 silencing-induced senescence was characterised by a specific SASP profile and metabolic state.

Effects of intentionally treated water on the growth of mesenchymal stem cells: An exploratory study

ObjectiveThis study explored if human primary mesenchymal stem cells (MSCs), derived from two donors and cultivated in a medium made with intentionally treated water, would exhibit more growth and pluripotency than MSCs from the same source but grown in untreated (control) water. DesignTo create the treated water, three Buddhist monks directed their attention toward commercially bottled water while holding the intention that the water would enhance the growth of MSCs. Under double-blind conditions, cell culture growth mediums were prepared with the treated and untreated water, which was in turn used to grow the primary MSCs. Primary cells obtained from two donors were designated as Cells #1 and Cells #2. The prediction was that treated water would result in increased cell proliferation, that more cells would enter the cell cycle growth phase, and that there would be increased expression of genes (NANOG, OCT4 and SOX2) associated with improved cell growth and decreased expression of genes (p16, p21, and p53) associated with a decline in cell growth. The improved growth hypothesis was directional, thus one-tailed p-values were used to evaluate the results. ResultsProliferation averaged across Cells #1 and #2 showed overall increased growth in treated as compared to control water (p = 0.0008). Cells #1 and #2 considered separately had differences in the same direction but only Cells #2 showed a significant difference on day 6 (p = 0.01). For cell cycle, there was a significantly greater percentage of Cells #2 in the S interphase with treated vs. control water (p = 0.04). For the gene expression analysis, when considering the average across the two donor cells, only the NANOG gene expression was in the predicted direction (p = 0.01); by contrast, the p16 gene expression was significantly opposite to the predicted direction (p = 0.005, one-tailed, post-hoc). For Cells #1 considered separately, no differences were significant except for p16, which resulted in an effect opposite to the predicted outcome (p = 0.05). For Cells #2, three genes were significantly in the predicted directions: NANOG (p = 0.0008), OCT4 (p = 0.005), and P53 (p = 0.05); p16 was significantly opposite to the prediction (p = 0.001). ConclusionIntentionally treated water appeared to have some biological effects on the growth, pluripotency and senescence of human MSCs. This was especially the case in one of the two donor cells tested, but the effects were not consistently in the predicted direction. As an exploratory study, caution is warranted in interpreting these outcomes, and adjustment for multiple testing would likely reduce some of the weaker effects to nonsignificant. But given the double-blind protocol, as well as several more significant outcomes in the predicted directions, further research is warranted.

Age-related changes in the energy of human mesenchymal stem cells.

Aging is a physiological process that leads to a higher risk for the most devastating diseases. There are a number of theories of human aging proposed, and many of them are directly or indirectly linked to mitochondria. Here, we used mesenchymal stem cells (MSCs) from young and older donors to study age-related changes in mitochondrial metabolism. We have found that aging in MSCs is associated with a decrease in mitochondrial membrane potential and lower NADH levels in mitochondria. Mitochondrial DNA content is higher in aged MSCs, but the overall mitochondrial mass is decreased due to increased rates of mitophagy. Despite the higher level of ATP in aged cells, a higher rate of ATP consumption renders them more vulnerable to energy deprivation compared to younger cells. Changes in mitochondrial metabolism in aged MSCs activate the overproduction of reactive oxygen species in mitochondria which is compensated by a higher level of the endogenous antioxidant glutathione. Thus, energy metabolism and redox state are the drivers for the aging of MSCs/mesenchymal stromal cells.

PBX1 Attenuates Hair Follicle-Derived Mesenchymal Stem Cell Senescence and Apoptosis by Alleviating Reactive Oxygen Species-Mediated DNA Damage Instead of Enhancing DNA Damage Repair.

Tissues and organs undergo structural deterioration and functional decline during aging. DNA damage is considered a major cause of stem cell senescence. Although stem cells develop sophisticated DNA repair systems, when the intrinsic and extrinsic insults exceed the DNA repair capacity, cellular senescence, and age-related diseases inevitably occur. Therefore, the prevention and alleviation of DNA damage is an alternative to DNA repair in attenuating stem cell senescence and preventing age-related diseases. Pre-B-cell leukaemia homeobox 1 (PBX1) participates in maintaining the pluripotency of human embryonic and haematopoietic stem cells. Our recent studies showed that PBX1 promotes hair follicle-derived mesenchymal stem cell (HF-MSC) proliferation, decreases cellular senescence and apoptosis, and enhances induced pluripotent stem cell generation. Whether PBX1 attenuates HF-MSC senescence and apoptosis by alleviating DNA damage or by enhancing DNA repair remains unknown. In this study, we aimed to determine the effects of PBX1 on the intrinsic ROS or extrinsic H2O2-induced cellular senescence of HF-MSCs. To this end, we generated HF-MSCs overexpressing either PBX1, or poly (ADP-ribose) polymerase 1, or both. Our results showed that PBX1 overexpression attenuates HF-MSC senescence and apoptosis by alleviating reactive oxygen species (ROS)-mediated DNA damage instead of enhancing DNA repair. This is the first study to report that PBX1 attenuates stem cell senescence and apoptosis by alleviating DNA damage. It provides new insight into the mechanism of stem cell senescence and lays the foundation for the development of strategies for age-related disease prevention and treatment, and in particular, hair follicle repair and regeneration.

BMP-2 Induced Signaling Pathways and Phenotypes: Comparisons Between Senescent and Non-senescent Bone Marrow Mesenchymal Stem Cells.

The use of BMP-2 in orthopedic surgery is limited by uncertainty surrounding its effects on the differentiation of mesenchymal stem cells (MSCs) and how this is affected by cellular aging. This study compared the effects of recombinant human BMP-2 (rhBMP-2) on osteogenic and adipogenic differentiation between senescent and non-senescent MSCs. Senescent and non-senescent MSCs were cultured in osteogenic and adipogenic differentiation medium containing various concentrations of rhBMP-2. The phenotypes of these cells were compared by performing a calcium assay, adipogenesis assay, staining, real-time PCR, western blotting, and microarray analysis. rhBMP-2 induced osteogenic differentiation to a lesser extent (P < 0.001 and P = 0.005 for alkaline phosphatase activity and Ca2+ release) in senescent MSCs regardless of dose-dependent increase in both cells. However, the induction of adipogenic differentiation by rhBMP-2 was comparable between them. There was no difference between these two groups of cells in the adipogenesis assay (P = 0.279) and their expression levels of PPARγ were similar. Several genes such as CHRDL1, NOG, SMAD1, SMAD7, and FST encoding transcription factors were proposed to underlie the different responses of senescent and non-senescent MSCs to rhBMP-2 in microarray analyses. Furthermore, inflammatory, adipogenic, or cell death-related signaling pathways such as NF-kB or p38-MAPK pathways were upregulated by BMP-2 in senescent MSCs, whereas bone forming signaling pathways involving BMP, SMAD, and TGF- ß were upregulated in non-senescent MSCs as expected. This phenomenon explains bone forming dominance by non-senescent MSCs and possible frequent complications such as seroma, osteolysis, or neuritis in senescent MSCs during BMP-2 use in orthopedic surgery.

Curcumin Alleviates the Senescence of Canine Bone Marrow Mesenchymal Stem Cells during In Vitro Expansion by Activating the Autophagy Pathway.

Senescence in mesenchymal stem cells (MSCs) not only hinders the application of MSCs in regenerative medicine but is also closely correlated with biological aging and the development of degenerative diseases. In this study, we investigated the anti-aging effects of curcumin (Cur) on canine bone marrow-derived MSCs (cBMSCs), and further elucidated the potential mechanism of action based on the modulation of autophagy. cBMSCs were expanded in vitro with standard procedures to construct a cell model of premature senescence. Our evidence indicates that compared with the third passage of cBMSCs, many typical senescence-associated phenotypes were observed in the sixth passage of cBMSCs. Cur treatment can improve cBMSC survival and retard cBMSC senescence according to observations that Cur (1 μM) treatment can improve the colony-forming unit-fibroblasts (CFU-Fs) efficiency and upregulated the mRNA expression of pluripotent transcription factors (SOX-2 and Nanog), as well as inhibiting the senescence-associated beta-galactosidase (SA-β-gal) activities and mRNA expression of the senescence-related markers (p16 and p21) and pro-inflammatory molecules (tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)). Furthermore, Cur (0.1 μM~10 μM) was observed to increase autophagic activity, as identified by upregulation of microtubule-associated protein 1 light chain 3 (LC3), unc51-like autophagy-activating kinase-1 (ULK1), autophagy-related gene (Atg) 7 and Atg12, and the generation of type II of light chain 3 (LC3-II), thereby increasing autophagic vacuoles and acidic vesicular organelles, as well as causing a significant decrease in the p62 protein level. Moreover, the autophagy activator rapamycin (RAP) and Cur were found to partially ameliorate the senescent features of cBMSCs, while the autophagy inhibitor 3-methyladenine (3-MA) was shown to aggravate cBMSCs senescence and Cur treatment was able to restore the suppressed autophagy and counteract 3-MA-induced cBMSC senescence. Hence, our study highlights the important role of Cur-induced autophagy and its effects for ameliorating cBMSC senescence and provides new insight for delaying senescence and improving the therapeutic potential of MSCs.

Increasing cytosolic Ca2+ levels restore cell proliferation and stem cell potency in aged MSCs

Aging is an inescapable complex physiological but extendable process, and all cells, including stem cells, are altered over time. Diverse mechanism(s) could modulate stem cell number, their proliferation rate, and promote tissue repair during aging that leads to longevity. However, the factors that could restore aging stem cell potency and would lead to healthy aging are not fully identified. Here we show that maintaining cytosolic Ca2+ levels was essential for modulating stem cells function in aged mesenchymal stem cells (MSCs). Increasing external Ca2+ induced spindle shape stem cell morphology and maintained stem cell surface marker expression in aged bone marrow-derived MSCs. Similarly, stem cell survival and proliferation of aged MSCs was dependent on cytosolic Ca2+ levels. Importantly, Ca2+ entry potentiated cell cycle progression, and stem cell potential was increased in cells incubated with higher external Ca2+. Moreover, blocking Ca2+ entry using SKF 96365, decreased stem cell survival and its proliferation but, treatment with 2-APB did not significantly affected cell proliferation, rather only modulated cell viability. Evaluation of Ca2+ entry channels, showed that TRPC1/Orai1/Orai3 and their regulator STIM1 was essential for MSCs proliferation/viability as gene silencing of Orai1/Orai3/TRPC1/STIM1 significantly inhibited stem cell viability. Finally, MSCs isolated from aged mice that were subjected to higher Ca2+ levels, were able to rescue age-induced loss of MSCs function. Together these results suggest that Ca2+ entry is essential for preventing the loss of aged stem cell function and supplementing Ca2+ not only restored their proliferative potential but, allowed them to develop into younger stem cell lineages that could be critical for regenerative medicine.

Tonsil-derived mesenchymal stem cells incorporated in reactive oxygen species-releasing hydrogel promote bone formation by increasing the translocation of cell surface GRP78

Controlling the senescence of mesenchymal stem cells (MSCs) is essential for improving the efficacy of MSC-based therapies. Here, a model of MSC senescence was established by replicative subculture in tonsil-derived MSCs (TMSCs) using senescence-associated β-galactosidase, telomere-length related genes, stemness, and mitochondrial metabolism. Using transcriptomic and proteomic analyses, we identified glucose-regulated protein 78 (GRP78) as a unique MSC senescence marker. With increasing cell passage number, GRP78 gradually translocated from the cell surface and cytosol to the (peri)nuclear region of TMSCs. A gelatin-based hydrogel releasing a sustained, low level of reactive oxygen species (ROS-hydrogel) was used to improve TMSC quiescence and self-renewal. TMSCs expressing cell surface-specific GRP78 (csGRP78+), collected by magnetic sorting, showed better stem cell function and higher mitochondrial metabolism than unsorted cells. Implantation of csGRP78+ cells embedded in ROS-hydrogel in rats with calvarial defects resulted in increased bone regeneration. Thus, csGRP78 is a promising biomarker of senescent TMSCs, and the combined use of csGRP78+ cells and ROS-hydrogel improved the regenerative capacity of TMSCs by regulating GRP78 translocation.

Abstract P506: Sirt6 Overexpression Inhibits Senescence And Inflammation In Human Mesenchymal Stem Cells

Background: Mesenchymal Stem Cells (MSCs) offer regenerative and therapeutic potential in an injured tissue in diabetic conditions. But the functional efficiency of MSCs has been shown to decrease with diabetes and aging. This reduces the potential of cell-based therapy in diabetic patients. Recent studies have established the role of sirtuin family proteins in metabolic disease, inflammation, longevity, and DNA repair. However, their potential to be used as a therapeutic target in cardiomyopathy and heart failure remain unexplored. Objective: To investigate the role of Sirtuin 6 (SIRT6) in mesenchymal stem cells senescence and cardiac regeneration. Methods and Results: We performed genomics and proteomics in the human control and diabetic heart tissues collected from the heart transplant. Human MSCs were treated with high glucose and mouse MSCs were derived from the bone marrow of diabetic db/db mice for this study. We found that SIRT6 expression is reduced in the myocardium of diabetic patients compared to non-diabetic controls. The SIRT6 expression decreased in high glucose-treated human MSCs compared to mannitol-treated control MSCs as well as in db/db mice MSCs compared to control mice MSCs. These high glucose-treated human MSCs and db/db mice MSCs showed increased expression of senescence and inflammation-related markers like that in diabetic human myocardium. Furthermore, we used small interfering RNA (SIRT6-siRNA) in human MSCs to knock down the SIRT6 gene and validated our findings. Indeed, the knockdown of SIRT6 promoted senescence and inflammation in those MSCs. Finally, we used adeno-associated viruses (AAV-SIRT6) to overexpress SIRT6 in MSCs treated with high glucose and performed proteomic analysis. SIRT6 overexpression in high glucose-treated MSCs reduced the expression of senescence and inflammation related genes and proteins. Conclusion: Our results highlight the importance of SIRT6 in diabetic myocardium and its role in balancing senescence and inflammation in MSCs. The validation of such in vitro studies in a diabetic mouse model ( db/db ) along with transplantation of SIRT6 overexpressed db/db MSCs into the myocardium of diabetic mice could open doors to successfully use MSC therapy in diabetic patients in the future.