BMSCs promote alveolar epithelial cell autophagy to reduce pulmonary fibrosis by inhibiting core fucosylation modifications.

BACKGROUND: Idiopathic pulmonary fibrosis (PF) is a chronic progressive interstitial lung disease characterized by alveolar epithelial cell (AEC) injury and fibroblast activation. Inadequate autophagy in AECs may result from the activation of several signaling pathways following AEC injury, with glycoproteins serving as key receptor proteins. The core fucosylation (CF) modification in glycoproteins is crucial. Mesenchymal stem cells derived from bone marrow (BMSCs) have the ability to regenerate damaged tissue and treat PF. This study aimed to elucidate the relationship and mechanism of interaction between BMSCs, CF modification, and autophagy in PF. METHODS: C57BL/6 male mice, AEC-specific FUT8 conditional knockout (CKO) mice, and MLE12 cells were administered bleomycin (BLM), FUT8 siRNA, and mouse BMSCs, respectively. Experimental techniques including tissue staining, Western blotting, immunofluorescence, autophagic flux detection, and flow cytometry were used in this study. RESULTS: First, we found that autophagy was inhibited while FUT8 expression was elevated in PF mice and BLM-induced AEC injury models. Subsequently, CKO mice and MLE12 cells transfected with FUT8 siRNA were used to demonstrate that inhibition of CF modification induces autophagy in AECs and mitigates PF. Finally, mouse BMSCs were used to demonstrate that they alleviate the detrimental autophagy of AECs by inhibiting CF modification and decreasing PF. CONCLUSIONS: Suppression of CF modification enhanced the suppression of AEC autophagy and reduced PF in mice. Additionally, through the prevention of CF modification, BMSCs can assist AECs deficient in autophagy and partially alleviate PF.

DOT1L decelerates the development of osteoporosis by inhibiting SRSF1 transcriptional activity via microRNA-181-mediated KAT2B inhibition.

OBJECTIVE: Our study explored the function of DOT1L in osteoporosis (OP) via the microRNA (miR)-181/KAT2B/SRSF1 axis. METHODS: Osteoclast (OC) number was evaluated via TRAP staining, and serum CTXI, PINP, and ALP contents were tested by ELISA. Following identification of bone marrow mesenchymal stem cells (BMSCs), OC differentiation was induced by M-CSF and RANKL, followed by the detection of OC differentiation and the expression of bone resorption-related genes, DOT1L, miR-181, KAT2B, and SRSF1. RESULTS: Overexpressed DOT1L or miR-181 stimulated calcified nodule formation and increased alkaline phosphatase activity and osteogenic marker gene expression. KAT2B knockdown enhanced the osteogenic differentiation of BMSCs by reducing SRSF1 acetylation. The enhancement of OC differentiation induced by overexpressed SRSF1 was inhibited by simultaneous DOT1L or miR-181 overexpression. DOT1L suppressed OP development in vivo via the miR-181/KAT2B/SRSF1 axis. CONCLUSION: DOT1L overexpression slowed down bone loss and promoted bone formation via the miR-181/KAT2B/SRSF1 axis, thereby alleviating OP development.

Proteomic Analysis of the Supernatant from Bone Marrow Mesenchymal Stem Cells under High Glucose Conditions.

Diabetes mellitus hinders the process of bone regeneration by inhibiting the function of mesenchymal stem cells (MSCs) through elevated glucose levels, thereby impeding osteointegration. The stem cell niche (SCN) plays a crucial role in determining the fate of stem cells by integrating various signals. However, the precise mechanism by which high glucose levels affect the SCN and subsequently influence the function of MSCs remains unclear. In this study, we employed proteomic analysis to identify proteins with altered expression in the extracellular matrix (ECM), aiming to elucidate the underlying mechanism. Three cell supernatants were collected from bone marrow mesenchymal stem cells (BMSCs) or BMSCs stimulated with high glucose (BMSCs+Hg). A total of 590 differentially expressed proteins were identified, which were found to be associated with the ECM, including aging, autophagy, and osteogenic differentiation. The findings of our study indicate that elevated glucose levels exert an influence on the molecular aspects of the SCN, potentially contributing to a better comprehension of the underlying mechanism.

Long non-coding RNA H19 mediates osteogenic differentiation of bone marrow mesenchymal stem cells through the miR-29b-3p/DKK1 axis.

Single immobilization theory cannot fully account for the extensive bone loss observed after spinal cord injury (SCI). Bone marrow mesenchymal stem cells (BMSCs) are crucial in bone homeostasis because they possess self-renewal capabilities and various types of differentiation potential. This study aimed to explore the molecular mechanism of long non-coding RNA H19 in osteoporosis after SCI and provide new research directions for existing prevention strategies. We used small interfering RNA to knockdown H19 expression and regulated miR-29b-2p expression using miR-29b-3p mimetics and inhibitors. Western blotting, real-time fluorescence quantitative PCR, Alizarin red staining, alkaline phosphatase staining and double-luciferase reporter gene assays were used to assess gene expression, osteogenic ability and binding sites. lncRNA H19 was upregulated in BMSCs from the osteoporosis group, whereas miR-29b-3p was downregulated. We identified the binding sites between miR-29b-3p and lncRNAs H19 and DKK1. H19 knockdown promoted BMSCs' osteogenic differentiation, whereas miR-29b-3p inhibition attenuated this effect. We discovered potential binding sites for miR-29b-3p in lncRNAs H19 and DKK1. Our findings suggest that long non-coding RNA H19 mediates BMSCs' osteogenic differentiation in osteoporosis after SCI through the miR-29b-3p/DKK1 axis and by directly inhibiting the ß-catenin signalling pathway.

AMPK, a hub for the microenvironmental regulation of bone homeostasis and diseases.

AMP-activated protein kinase (AMPK), a crucial regulatory kinase, monitors energy levels, conserving ATP and boosting synthesis in low-nutrition, low-energy states. Its sensitivity links microenvironmental changes to cellular responses. As the primary support structure and endocrine organ, the maintenance, and repair of bones are closely associated with the microenvironment. While a series of studies have explored the effects of specific microenvironments on bone, there is lack of angles to comprehensively evaluate the interactions between microenvironment and bone cells, especially for bone marrow mesenchymal stem cells (BMMSCs) which mediate the differentiation of osteogenic lineage. It is noteworthy that accumulating evidence has indicated that AMPK may serve as a hub between BMMSCs and microenvironment factors, thus providing a new perspective for us to understand the biology and pathophysiology of stem cells and bone. In this review, we emphasize AMPK's pivotal role in bone microenvironment modulation via ATP, inflammation, reactive oxygen species (ROS), calcium, and glucose, particularly in BMMSCs. We further explore the use of AMPK-activating drugs in the context of osteoarthritis and osteoporosis. Moreover, building upon the foundation of AMPK, we elucidate a viewpoint that facilitates a comprehensive understanding of the dynamic relationship between the microenvironment and bone homeostasis, offering valuable insights for prospective investigations into stem cell biology and the treatment of bone diseases.

Mitochondria-specific antioxidant MitoTEMPO alleviates senescence of bone marrow mesenchymal stem cells in ovariectomized rats.

Senescence in bone marrow mesenchymal stem cells (BMSCs), triggered by excessive oxidative stress, plays a crucial role in the onset of postmenopausal osteoporosis. Recent studies underscore the importance of mitochondrial rehabilitation and quality control as key determinants in the modulation of oxidative stress and cellular senescence. MitoTEMPO, a mitochondria-targeted antioxidant, has been shown to mitigate the heightened levels of reactive oxygen species (ROS). In our research, we observed that BMSCs from ovariectomized (OVX) rats displayed premature senescence, which was attributed to combined mitochondrial and lysosomal dysfunction, a condition that worsens with extended estrogen deprivation. Treatment with MitoTEMPO effectively reversed these effects, reinstating lysosomal functionality and suppressing the mitochondrial unfolded protein response (UPRmt). Subsequent in vivo experiments corroborated these observations, revealing that MitoTEMPO administration in OVX rats curtailed trabecular bone loss and reduced the expression of p53, HSP60, and CLPP in the trabecular bone region of the proximal tibia. Overall, our findings suggest that MitoTEMPO holds promise as a therapeutic agent to counteract senescence in OVX-BMSCs, offering a potential strategy for treating postmenopausal osteoporosis.

PPARγ inhibition promotes osteogenic differentiation of bone marrow mesenchymal stem cells and fracture healing.

This study aimed to explore the effects of peroxisome proliferator-activated receptor γ (PPARγ) inhibition on fracture healing of nonunion and the underlying mechanisms. Bone marrow mesenchymal stem cells (BMSCs) were treated with PPARγ antagonist GW9662 (5 µM, 10 µM). Alkaline phosphatase (ALP) staining and Alizarin Red S was used to assess early stage of osteogenesis and osteogenic differentiation. GW9662 (1 mg/kg/day) were administered intraperitoneally into the rats with bone fracture. Bone healing processes in the rat femur fracture model were recorded and assessed by radiographic methods on Weeks 8, 14, and 20 postoperation. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative Week 20. GW9662 treatment increased ALP activity and Alp mRNA expression in rat BMSCs. Moreover, GW9662 administration increased matrix mineralization and mRNA and protein levels of Bmp2 and Runx2 in the BMSCs. In addition, GW9662 treatment improved radiographic score in the fracture rats and increased osteogenesis-related proteins, including type I collagen, osteopontin, and osteoglycin, in the bone tissues of the fracture sites. In conclusion, PPARγ inhibition promotes osteogenic differentiation of rat BMSCs, as well as improves the fracture healing of rats through Bmp2/Runx2 signaling pathway in the rat model of bone fracture.

A self-amplifying loop of TP53INP1 and P53 drives oxidative stress-induced apoptosis of bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cell (BMSC) transplantation is a promising regenerative therapy; however, the survival rate of BMSCs after transplantation is low. Oxidative stress is one of the main reasons for the high apoptosis rate of BMSCs after transplantation, so there is an urgent need to explore the mechanism of oxidative stress-induced apoptosis of BMSCs. Our previous transcriptome sequencing results suggested that the expression of P53-induced nuclear protein 1 (TP53INP1) and the tumor suppressor P53 (P53) was significantly upregulated during the process of oxidative stress-induced apoptosis of BMSCs. The present study further revealed the role and mechanism of TP53INP1 and P53 in oxidative stress-induced apoptosis in BMSCs. Overexpression of TP53INP1 induced apoptosis of BMSCs, knockdown of TP53INP1 alleviated oxidative stress apoptosis of BMSCs. Under oxidative stress conditions, P53 is regulated by TP53INP1, while P53 can positively regulate the expression of TP53INP1, so the two form a positive feedback loop. To clarify the mechanism of feedback loop formation. We found that TP53INP1 inhibited the ubiquitination and degradation of P53 by increasing the phosphorylation level of P53, leading to the accumulation of P53 protein. P53 can act on the promoter of the TP53INP1 gene and increase the expression of TP53INP1 through transcriptional activation. This is the first report on a positive feedback loop formed by TP53INP1 and P53 under oxidative stress. The present study clarified the formation mechanism of the positive feedback loop. The TP53INP1-P53 positive feedback loop may serve as a potential target for inhibiting oxidative stress-induced apoptosis in BMSCs.

MEG3 shuttled by exosomes released from human bone marrow mesenchymal stem cells promotes TP53 stability to regulate MCM5 transcription in keloid fibroblasts.

BACKGROUND: Despite the interest in mesenchymal stem cells (MSC), their potential to treat abnormal scarring, especially keloids, is yet to be described. The present study aimed to investigate the therapeutic potential of exosomes derived from human bone marrow MSCs (hBMSC-Exos) in alleviating keloid formation. METHODS: Exosomes were isolated from hBMSC, and keloid fibroblasts (KFs) were treated with hBMSC-Exos. Cell counting kit-8, wound healing, transwell invasion, immunofluorescence, and western blot assays were conducted to study the malignant phenotype of KFs. Mice were induced with keloids and treated with hBMSC-Exos. The effect of hBMSC-Exos on keloid formation in vivo was evaluated by hematoxylin and eosin staining, Masson staining, immunohistochemistry, and western blotting. The GSE182192 dataset was screened for differentially expressed long non-coding RNA during keloid formation. Next, maternally expressed gene 3 (MEG3) was knocked down in hBMSC to obtain hBMSC-Exossh-MEG3. The molecular mechanism of MEG3 was investigated by bioinformatic screening, and the relationship between MEG3 and TP53 or MCM5 was verified. RESULTS: hBMSC-Exos inhibited the malignant proliferation, migration, and invasion of KFs at same time as promoting their apoptosis, Moreover, hBMSC-Exos reduced the expression of fibrosis- and collagen-related proteins in the cells and the formation of keloids caused by KFs. The reduction in MEG3 enrichment in hBMSC-Exos weakened the inhibitory effect of hBMSC-Exos on KF activity. hBMSC-Exos delivered MEG3 to promote MCM5 transcription by TP53 in KFs. Overexpression of MCM5 in KFs reversed the effects of hBMSC-Exossh-MEG3, leading to reduced KF activity. CONCLUSIONS: hBMSC-Exos delivered MEG3 to promote the protein stability of TP53, thereby activating MCM5 and promoting KF activity.

Bioinformatics analysis and validation of RNA methylation-related genes in osteogenic and adipogenic differentiation of rat bone marrow mesenchymal stem cells.

BACKGROUND: The regulatory mechanisms of RNA methylation during the processes of osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) have yet to be fully understood. The objective of our study was to analyze and validate the contribution of RNA methylation regulators to the mechanisms underlying the osteogenic and adipogenic differentiation of rat BMSCs. METHODS: We downloaded the GSE186026 from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were screened using the DESeq2 package in R software (version 3.6.3). A total of 50 RNA methylation genes obtained from literature review and summary were intersected with the previous DEGs to obtain RNA methylation genes, which have different expressions (RM-DEGs). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were utilized to reveal the functional enrichment. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate RM-DEGs. Protein-protein interaction network (PPI) analysis and visual analysis were performed using STRING and Cytoscape. RM-DEGs regulatory network was constructed to analyze the top 10 hub genes. The relationship between RM-DEGs, some enriched GO and pathways was also been analyzed. The miRNAs and RM-DEGs regulatory networks were established by using miRWalk and TargetScan. RESULTS: As part of our research, we detected varying levels of expression for m6A regulators Mettl3 and Rbm15, as well as m7G regulators Mettl1 and Wdr4, in relation to osteogenic differentiation, along with m6A regulator Fmr1 in adipogenic differentiation. The protein-protein interaction (PPI) networks were constructed for 49 differentially expressed genes (DEGs) related to RNA methylation during the process of osteogenic differentiation, and 13 DEGs for adipogenic differentiation. Moreover, top10 hub genes were calculated. In osteogenic differentiation, Mettl3 regulated the Wnt pathway and Hippo pathway by regulating Smad3, Rbm15 regulated the Notch pathway by Notch1, Mettl1 regulated the PI3K-Akt pathway by Gnb4. In adipogenic differentiation, Fmr1 regulated the PI3K-Akt pathway by Egfr. M6A methylation sites of Smad3, Notch1 and Gnb4 were predicted, and the results showed that all three genes were possibly methylated by m6A, and more than 9 sites per gene were possibly methylated. Finally, we constructed the regulatory networks of Mettl3, Rbm15, Mettl1, and Wdr4 and 109 miRNAs in osteogenic differentiation, Fmr1 and 118 miRNAs in adipogenic differentiation. CONCLUSIONS: Mettl3(m6A), Rbm15(m6A), Wdr4 and Mettl1(m7G) were differentially expressed in osteogenic differentiation, while Fmr1(m6A) was differentially expressed in adipogenic differentiation. These findings offered potential candidates for further research on the involvement of RNA methylation in the osteogenic and adipogenic differentiation of BMSCs.

A Factor-Free Hydrogel with ROS Scavenging and Responsive Degradation for Enhanced Diabetic Bone Healing.

In view of the increased levels of reactive oxygen species (ROS) that disturb the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), the repair of diabetic bone defects remains a great challenge. Herein, a factor-free hydrogel is reported with ROS scavenging and responsive degradation properties for enhanced diabetic bone healing. These hydrogels contain ROS-cleavable thioketal (TK) linkers and ultraviolet (UV)-responsive norbornene (NB) groups conjugated with 8-arm PEG macromers, which are formed via UV crosslinking-mediated gelation. Upon reacting with high levels of ROS in the bone defect microenvironment, ROS-cleavable TK linkers are destroyed, allowing the responsive degradation of hydrogels, which promotes the migration of BMSCs. Moreover, ROS levels are reduced through hydrogel-mediated ROS scavenging to reverse BMSC differentiation from adipogenic to osteogenic phenotype. As such, a favorable microenvironment is created after simultaneous ROS scavenging and hydrogel degradation, leading to the effective repair of bone defects in diabetic mouse models, even without the addition of growth factors. Thus, this study presents a responsive hydrogel platform that regulates ROS scavenging and stromal degradation in bone engineering.

Hierarchically Assembled Nanofiber Scaffold Guides Long Bone Regeneration by Promoting Osteogenic/Chondrogenic Differentiation of Endogenous Mesenchymal Stem Cells.

Critical-sized segmental long bone defects represent a challenging clinical dilemma in the management of battlefield and trauma-related injuries. The residual bone marrow cavity of damaged long bones contains many bone marrow mesenchymal stem cells (BMSCs), which provide a substantial source of cells for bone repair. Thus, a three-dimensional (3D) vertically aligned nanofiber scaffold (VAS) is developed with long channels and large pore size. The pore of VAS toward the bone marrow cavity after transplantation, enables the scaffolds to recruit BMSCs from the bone marrow cavity to the defect area. In vivo, it is found that VAS can significantly shorten gap distance and promote new bone formation compared to the control and collagen groups after 4 and 8 weeks of implantation. The single-cell sequencing results discovered that the 3D nanotopography of VAS can promote BMSCs differentiation to chondrocytes and osteoblasts, and up-regulate related gene expression, resulting in enhancing the activities of bone regeneration, endochondral ossification, bone trabecula formation, bone mineralization, maturation, and remodeling. The Alcian blue and bone morphogenetic protein 2 (BMP-2) immunohistochemical staining verified significant cartilage formation and bone formation in the VAS group, corresponding to the single-cell sequencing results. The study can inspire the design of next-generation scaffolds for effective long-bone regeneration is expected by the authors.

Efficacy and safety of autologous or allogeneic mesenchymal stromal cells from adult adipose tissue expanded and combined with tricalcium phosphate biomaterial for the surgical treatment of atrophic nonunion of long bones: a phase II clinical trial.

BACKGROUND: Autologous bone grafting is the standard treatment for the surgical management of atrophic nonunion of long bones. Other solutions, such as bone marrow mesenchymal stem cells (BM-MSC) combined with phospho-calcium material, have also been used. Here we evaluate the safety and early efficacy of a novel procedure using autologous or allogenic adipose tissue mesenchymal stromal cells (AT-MSC) seeded in a patented tricalcium phosphate-based biomaterial for the treatment of bone regeneration in cases of atrophic nonunion. METHODS: This was a prospective, multicentric, open-label, phase 2 clinical trial of patients with atrophic nonunion of long bones. Biografts of autologous or allogenic AT-MSC combined with a phosphate substrate were manufactured prior to the surgical procedures. The primary efficacy was measured 6 months after surgery, but patients were followed for 12 months after surgery and a further year out of the scope of the study. All adverse events were recorded. This cohort was compared with a historical cohort of 14 cases treated by the same research team with autologous BM-MSC. RESULTS: A total of 12 patients with atrophic nonunion of long bones were included. The mean (SD) age was 41.2 (12.1) years and 66.7% were men. Bone healing was achieved in 10 of the 12 cases (83%) treated with the AT-MSC biografts, a percentage of healing similar (11 of the 14 cases, 79%) to that achieved in patients treated with autologous BM-MSC. Overall, two adverse events, in the same patient, were considered related to the procedure. CONCLUSIONS: The results of this study suggest that AT-MSC biografts are safe for the treatment of bone regeneration in cases of atrophic nonunion and reach high healing rates. TRIAL REGISTRATION: Study registered with EUDRA-CT (2013-000930-37) and ClinicalTrials.gov (NCT02483364).

Exosomes derived from Baicalin-pretreated bone mesenchymal stem cells improve Th17/Treg imbalance after hepatic ischemia-reperfusion via FGF21 and the JAK2/STAT3 pathway.

Baicalin is an active compound extracted from Scutellaria baicalensis with antioxidant and anti-inflammatory properties. Bone mesenchymal stem cells (BMSCs)-derived exosomes have shown promise for the treatment of hepatic ischemia-reperfusion (I/R) injury. This study aims to investigate the role of Baicalin-pretreated BMSCs-derived exosomes in hepatic I/R injury and its mechanisms. BMSCs were pretreated with or without Baicalin, and their exosomes (Ba-Exo and Exo) were collected and characterized. These exosomes were administered to mice via tail vein injection. Treatment with Exo and Ba-Exo significantly suppressed the elevation of ALT and AST induced by hepatic injury. Additionally, both Exo and Ba-Exo treatments resulted in a reduction in the liver weight-to-body weight ratio. RT-PCR results revealed a significant downregulation of pro-inflammatory cytokines with Exo and Ba-Exo treatment. Both Exo and Ba-Exo treatment improved the Th17/Treg cell imbalance induced by I/R and reduced hepatic injury. Additionally, exosomes were cocultured with normal liver cells, and the expression of fibroblast growth factor 21 (FGF21) in liver cells was elevated through Ba-Exo treatment. After treatment, the JAK2/STAT3 pathway was inhibited, and FOXO1 expression was upregulated. Finally, recombinant FGF21 was injected into mouse tail veins to assess its effects. Recombinant FGF21 injection further inhibited the JAK2/STAT3 pathway, increased FOXO1 expression, and improved the Th17/Treg cell imbalance. In conclusion, this study confirms the protective effects of Exo and Ba-Exo against hepatic I/R injury. Ba-Exo mitigates hepatic I/R injury, achieved through inducing FGF21 expression in liver cells, inhibiting the JAK2/STAT3 pathway, and activating FOXO1 expression. Therefore, baicalin pretreatment emerges as a promising strategy to enhance the therapeutic capability of BMSCs-derived exosomes for hepatic I/R.

Chondroprotective effects of bone marrow mesenchymal stem cell-derived exosomes in osteoarthritis.

Chondrocyte ferroptosis constitutes a major cause of the development of osteoarthritis (OA). Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have a protective role against ferroptosis in various diseases. Hence, we aimed to determine whether BMSC-Exos alleviated chondrocyte ferroptosis and its effect on OA, and to dissect out the possible mechanisms. An OA rat chondrocyte model was established by interleukin-1ß (IL-1ß) exposure, and treated with BMSC-Exos/ferroptosis inhibitor Ferrostatin-1. Cell viability/ferroptosis-related index levels [reactive oxygen species (ROS)/malondialdehyde (MDA)/glutathione (GSH)]/cell death/ACSL4 mRNA and protein levels and METTL3 levels were assessed by MTT/kits/immunohistochemical method and TUNEL staining/RT-qPCR and Western blot. METTL3/ACSL4 were overexpressed in rat chondrocytes to evaluate their role in BMSC-Exo-produced repression on chondrocyte ferroptosis. Bioinformatics website predicted the presence of m6A modification sites on ACSL4 mRNA, with the m6A level enriched on it assessed by MeRIP/RT-qPCR. ACSL4 mRNA stability was detected by actinomycin D assay. A surgical destabilized medial meniscus rat OA model was also established, followed by injection with BMSC-Exos to verify their function. IL-1ß stimulation in rat chondrocytes inhibited cell viability, elevated Fe2+/ROS/MDA levels, declined GSH levels and increased TUNEL positive cell number and ACSL4 level, which were neutralized by BMSC-Exos. BMSC-Exos limited chondrocyte ferroptosis by down-regulating METTL3, with the effect abrogated by METTL3 overexpression. METTL3 regulated the m6A modification of ACSL4 mRNA, increasing ACSL4 mRNA stability and ACSL4 expression. BMSC-Exos reduced chondrocyte ferroptosis and prevented OA progression via disruption of the METTL3-m6A-ACSL4 axis. BMSC-Exos might exert a chondroprotective effect by attenuating chondrocyte ferroptosis and alleviate OA progression.

Hydrogen Sulfide Attenuates Mesenchymal Stem Cell Aging Progress via the Calcineurin-NFAT Signaling Pathway.

Aging is a gradual process that is coupled with a decline in the regenerative capacity of stem cells and a subsequent reduction in tissue function and repair. Hydrogen sulfide (H2S) plays an important role in maintaining the function of stem cells. The present study aimed to investigate the role of H2S in mesenchymal stem cell aging and the underlying mechanism and to provide novel insights into stem cell therapies in elderly people. Bone marrow mesenchymal stem cells (BMMSCs) were isolated from young mice (2 months) and from old mice (12 months). Senescence-associated ß-galactosidase (SA-ß-Gal) activity, reactive oxygen species (ROS) production, ROS scavenging enzymes, and the expression of cell-cycle-related genes were compared between those young and old BMMSCs. The expression of H2S-producing enzymes and the production of H2S in BMMSCs were examined. In vitro osteogenic differentiation and cell senescence were analyzed in young and old BMMSCs before and after H2S treatment. The underlying mechanism was investigated using calcineurin and NFAT1 inhibitors or a Foxp3 siRNA. Bone volume/tissue volume (BV/TV) of femurs in mice was examined using micro-CT with or without systemic injection of an H2S donor. Here, we found that H2S levels in BMMSCs declined with age. When the generation of H2S was blocked with the CBS inhibitor hydroxylamine and the CSE inhibitor dl-propargylglycine, BMMSCs underwent senescence. The elevation of H2S levels rescued BMMSC function in vitro and prevented bone loss in vivo. Mechanistically, H2S represses cell aging via the calcineurin-NFAT1 signaling pathway.

Transcriptional Targets of TWIST1 in Human Mesenchymal Stem/Stromal Cells Mechanistically Link Stem/Progenitor and Paracrine Functions.

Despite extensive clinical testing, mesenchymal stem/stromal cell (MSC)-based therapies continue to underperform with respect to efficacy, which reflects the paucity of biomarkers that predict potency prior to patient administration. Previously, we reported that TWIST1 predicts inter-donor differences in MSC quality attributes that confer potency. To define the full spectrum of TWIST1 activity in MSCs, the present work employed integrated omics-based profiling to identify a high-confidence set of TWIST1 targets, which mapped to cellular processes related to ECM structure/organization, skeletal and circulatory system development, interferon gamma signaling, and inflammation. These targets are implicated in contributing to both stem/progenitor and paracrine activities of MSCs indicating these processes are linked mechanistically in a TWIST1-dependent manner. Targets implicated in extracellular matrix dynamics further implicate TWIST1 in modulating cellular responses to niche remodeling. Novel TWIST1-regulated genes identified herein may be prioritized for future mechanistic and functional studies.

Bone Marrow Mesenchymal Stem Cells-Derived miR-21-5p Protects Grafted Islets Against Apoptosis by Targeting PDCD4.

The apoptosis of grafted islets is an urgent problem due to the high rate of islet loss soon after transplantation. MicroRNA-21-5p (miR-21-5p) is an essential mediator of bone marrow mesenchymal stem cells-derived exosomes (BMSCs-Exo) during anti-apoptosis, but its effect and the underlying molecular mechanism in islet transplantation remain partially understood. Here, we found that miR-21-5p could be delivered to islet cells via BMSCs-Exo. Subsequently, we demonstrated that miR-21-5p overexpression reduced apoptosis in islets and INS-1 cells, whereas miR-21-5p inhibition enhanced apoptosis. A mechanistic analysis involving RNA sequencing and bioinformatic analysis was performed to determine the interaction between miR-21-5p and its target gene programmed cell death 4 (PDCD4), which was further verified by a dual luciferase assay. In vivo, the grafted islets overexpressing miR-21-5p showed a higher survival rate, better insulin secretion function, and a lower apoptosis rate. In conclusion, these results demonstrated that miR­21­5p from BMSCs-Exo protects against the apoptosis of grafted islets by inhibiting PDCD4 expression. Hence, miR-21-5p can be used as a cell-free therapeutic agent to minimize ß-cell apoptosis at the early stage of islet transplantation.

Targeted Deletion of Rictor in BMSCs Reduces the Biological Activity of K7M2 Cells and Mitigates OS-Induced Bone Destruction.

Bone marrow mesenchymal stem cells (BMSCs) are indispensable cells constituting the bone marrow microenvironment that are generally recognized as being involved in the development and progression of osteosarcoma (OS). To explore whether mTORC2 signaling inhibition in BMSCs suppressed OS growth and tumor-caused bone destruction, 3-month-old littermates genotyped Rictorflox/flox or Prx1-cre; Rictorflox/flox (with same gender) were injected with K7M2 cells in the proximal tibia. After 40 days, bone destruction was alleviated in Prx1-cre; Rictorflox/flox mice, as observed on X-ray and micro-CT. This was accompanied by decreased serum N-terminal propeptide of procollagen type I (PINP) levels and reduced tumor bone formation in vivo. Interactions between K7M2 and BMSCs were studied in vitro. Rictor-deficient BMSCs, which were cultured in tumor-conditioned medium (TCM), caused reduced bone proliferation and suppressed osteogenic differentiation. Moreover, compared with the control group, K7M2 cells cultured in BCM (culture medium extracted from Rictor-deficient BMSCs) displayed less proliferation, migration, and invasion, and attenuated osteogenic activity. Forty types of cytokines were then analyzed by mouse cytokine array and decreased levels CCL2/3/5 and interleukin-16 were detected in Rictor-deficient BMSCs. These results suggested that inhibition of mTORC2 (Rictor) signaling pathway in BMSCs exerted anti-OS effects through 2 mechanisms: (1) by suppressing the proliferation and osteogenic differentiation of BMSCs induced by OS to alleviate bone destruction; (2) by reducing the secretion of cytokines by BMSCs, which are closely related to OS cell growth, migration, invasion, and tumorigenic osteogenesis.

Iron overload and programmed bone marrow cell death: Potential mechanistic insights.

Iron overload has detrimental effects on bone marrow mesenchymal stem cells (BMMSCs), cells crucial for bone marrow homeostasis and hematopoiesis support. Excessive iron accumulation leads to the production of reactive oxygen species (ROS), resulting in cell death, cell cycle arrest, and disruption of vital cellular pathways. Although apoptosis has been extensively studied, other programmed cell death mechanisms including autophagy, necroptosis, and ferroptosis also play significant roles in iron overload-induced bone marrow cell death. Studies have highlighted the involvement of ROS production, DNA damage, MAPK pathways, and mitochondrial dysfunction in apoptosis. In addition, autophagy and ferroptosis are activated, as shown by the degradation of cellular components and lipid peroxidation, respectively. However, several compounds and antioxidants show promise in mitigating iron overload-induced cell death by modulating ROS levels, MAPK pathways, and mitochondrial integrity. Despite early indications, more comprehensive research and clinical studies are needed to better understand the interplay between these programmed cell death mechanisms and enable development of effective therapeutic strategies. This review article emphasizes the importance of studying multiple cell death pathways simultaneously and investigating potential rescuers to combat iron overload-induced bone marrow cell death.