Bone Marrow-derived Mesenchymal Stem Cells Research Articles

Effect of CD10-positive cells on osteogenic differentiation of human maxillary/mandibular bone marrow-derived mesenchymal stem cells

This study was aimed at investigating the effect of CD10-positive cells within the maxillary/mandibular bone marrow-derived mesenchymal stem cells (MBMSCs) on osteogenic differentiation of MBMSCs. CD10 expression in iliac bone marrow-derived MSCs (IBMSCs), MBMSCs, and gingival fibroblasts was measured using flow cytometry. The osteogenic potential of 19 MBMSC lines was evaluated, and based on it, they were classified into osteogenic-High and osteogenic-Low groups. The percentage of CD10-positive cells in each group was compared. Effect of coculturing gingival fibroblasts and CD10-positive cells on the osteogenic potential of MBMSCs was also assessed. Expression of tissue inhibitor of metalloprotease-1 (TIMP-1) in osteogenic-High and osteogenic-Low MBMSCs was measured using quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The molecular mechanisms underlying the regulation of osteogenic differentiation in MBMSCs were investigated. CD10 was not expressed in IBMSCs, but was highly expressed in fibroblasts. In MBMSCs, the CD10-positivity rate varied considerably between cells. MBMSCs with a high-CD10 positivity rate showed low osteogenic potential. Coculture with fibroblasts or CD10-positive cells reduced the osteogenic potential of MBMSCs. TIMP-1 was highly expressed in CD10-positive cells, and osteogenic-Low MBMSCs showed significantly higher TIMP-1 expression compared with osteogenic-High MBMSCs. β-catenin signaling was suppressed in osteogenic-Low MBMSCs. This study revealed that TIMP-1 secreted from CD10-positive cells may be involved in the suppression of the osteogenic potential of MBMSCs by contamination with CD10-positive cells. This finding provides important insights for developing bone regeneration therapies using MBMSCs.

Priming human bone marrow-derived mesenchymal stromal cells with signaling modifiers boosts their functionality: Potential application in regenerative therapies.

Mesenchymal stromal cells (MSCs) isolated from tissues such as bone marrow, cord, cord blood, etc., are frequently used as feeder layers to expand hematopoietic stem/ progenitor cells (HSCs/HSPCs) in vitro. They are also co-infused with the HSCs to improve the efficacy of transplantation. However, the MSCs sourced from non-hematopoietic tissues could have suboptimal hematopoiesis-supportive ability. Likewise, the functionality of the MSCs is known to decline after continuous in vitro culture - an unavoidable manipulation to get clinically relevant cell numbers. Hence, it may be necessary to boost the hematopoiesis-supportive ability of the long-term cultured MSCs so that they can, in turn, be used to prime the HSCs before their clinical applications. Here, I show that priming human bone marrow-derived MSCs (BMSCs) with appropriately selected signaling modifiers and integrin-activating bioactive peptides boosts their hematopoiesis-supportive ability, as seen by the formation of a significantly higher number of colonies from the bone marrow-derived mononuclear cells (MNCs) and extensive proliferation of CD34+ HSCS briefly interacted with them. Priming the BMSCs with signaling modifiers is a cost-effective and time-efficient process as synthesizing these small molecule compounds is relatively inexpensive - an advantage in clinical settings. The approach of briefly interacting the donor HSCs/HSPCs with the primed BMSCs just before their infusion into the recipients' bodies could save the cost of long-term ex vivo expansion of HSCs. This concept could also find applications in other regenerative medicine protocols after identifying suitable pharmacological modulators that have the desired effects on the target cells.

Mannose-modified exosomes loaded with MiR-23b-3p target alveolar macrophages to alleviate acute lung injury in Sepsis.

The anti-inflammatory role of miR-23b-3p (miR-23b) is known in autoimmune diseases like multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis. However, its role in sepsis-related acute lung injury (ALI) and its effect on macrophages in ALI remain unexplored. This investigation aimed to evaluate miR-23b's therapeutic potential in macrophages in the context of ALI. The study found reduced miR-23b expression in macrophages within ALI tissue. Intratracheal delivery of miR-23b mimics alleviated ALI by partially inhibiting M1 macrophage activation through the Lpar1-NF-κB pathway. Effective delivery systems are crucial for prolonging miR-23b activity in the lungs, reducing dosage, and minimizing side effects by specifically targeting macrophages. However, current vector systems for nucleic acid delivery, including viral, lipid-based, polymer-based, and peptide-based vectors, face limitations due to eliciting immune responses. Exosomes have garnered significant attention as a leading gene delivery system due to the safety, effectivity and low immunogenicity. We further isolated exosomes from bone marrow-derived mesenchymal stem cells, modified exosomes with mannosylated ligands to enhance the targeted delivery of miR-23b to macrophage. This approach represents a promising novel therapeutic strategy for treating sepsis-induced ALI.

Anti-arthritic Effects of Undifferentiated and Chondrogenic Differentiated MSCs in MIA-induced Osteoarthritis in Wistar Rats: Involvement of Oxidative Stress and Immune Modulation.

Osteoarthritis (OA) is a degenerative joint disease that can affect the many tissues of the joint. There are no officially recognized disease-modifying therapies for clinical use at this time probably due to a lack of complete comprehension of the pathogenesis of the disease. In recent years, emerging regenerative therapy and treatments with stem cells both undifferentiated and differentiated cells have gained much attention as they can efficiently promote tissue repair and regeneration. To determine how bone marrow-derived mesenchymal stem cells (BM-MSCs) and chondrogenic differentiated MSCs (CD-MSCs) can treat OA in rats, OA was induced in Wistar rats by injecting three doses of 100 μL physiological saline containing 1 mg of MIA into rat ankle joint of the right hind leg for three consecutive days. Following the induction, the osteoarthritic rats were injected weekly with BM-MSCs or CD-MSCs at a dose of 1x106 cells/rat/dose for three weeks. In addition to morphological and histological investigations of the ankle, spectrophotometric, ELISA, and Western blot analyses were applied to detect various immunological and molecular parameters in serum and ankle. The results of the study showed that in osteoarthritic rats, BM-MSCs and CD-MSCs significantly reduced right hind paw circumference, total leucocyte count (TLC), differential leukocyte count (DLC) of neutrophils, monocytes, lymphocytes, and eosinophils, serum rheumatoid factor (RF), prostaglandin E2 (PGE2) and interleukin (IL-) 1β levels, while they elevated serum IL-10 level. Additionally, BM-MSCs and CD-MSCs markedly reduced lipid peroxides (LPO) levels while they elevated superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities. The monocyte chemoattractant protein-1 (MCP-1) level was significantly downregulated in ankle joint articular tissues by treatment with BM-MSCs or CD-MSCs while nuclear factor erythroid 2-related factor 2 (Nrf2) was upregulated; CD-MSCs treatment was more effective. According to these findings, it can be inferred that BM-MSCs and CD-MSCs have anti-arthritic potential in MIA-induced OA; CD-MSCs therapy is more effective than MSCs. The ameliorative anti-arthritic effects may be mediated by suppressing inflammation and oxidative stress through the downregulation of MCP-1 and upregulation of Nrf2. Based on the obtained results, BM-MSCs and CD-MSCs therapies are promising new options that can be associated with other clinical treatments to improve cartilage regeneration and joint healing. However, more preclinical and clinical research is required to assess the benefits and safety of treating osteoarthritic patients with BM-MSCs and CD-MSCs.

Bone marrow mesenchymal stem cells derived cytokines associated with AKT/IAPs signaling ameliorate Alzheimer’s disease development

BackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative condition affecting around 50 million people worldwide. Bone marrow-derived mesenchymal stem cells (BMMSCs) have emerged as a promising source for cellular therapy due to their ability to differentiate into multiple cell types and their paracrine effects. However, the direct injection of BMMSCs can lead to potential unpredictable impairments, prompting a renewed interest in their paracrine effects for AD treatment. The specific mechanism and central role of cytokines in this process have not been fully elucidated.MethodsMouse BMMSCs were isolated, validated, and then transplanted intracerebrally into APP/PS1 female mice. The behavioral tests, including open-field test, novel object recognition test, and Morris water maze were performed, followed by β-amyloidosis plaque and neuron apoptosis analyses. Then the tissue RNA sequencing and mBMMSC cytokine analysis were performed. A cytokine antibody array for BMMSCs and the brain slice models were performed with AD model tissues were used to elucidate the molecular mechanisms. Finally, APP/PS1 mice were administrated with cytokine mixture for cognitive recovery.ResultsOur results demonstrated that BMMSCs significantly improved cognitive function, reduced beta-amyloid plaque deposition, and decreased apoptotic neurons through the activation of the AKT signaling pathway. Using a cytokine antibody array, we identified three highly expressed AKT pathway regulated neuroprotective factors in BMMSCs: IGF1, VEGF, and Periostin2. These cytokines were found to upregulate inhibitors of apoptosis family proteins (IAPs) and suppress Caspase-3 activity in brain slices induced with beta amyloidosis (Aβ), okadaic acid (OA), and lipopolysaccharide (LPS). When injection of this cytokine mixture to APP/PS1 mice also resulted in a mitigation of cognitive impairment.ConclusionsThese findings suggest that the secretory factors IGF1, VEGF, and Periostin2 derived from BMMSCs play a crucial role in neuroprotection by modulating the AKT/IAPs pathway to restore neuronal function. These cytokine sets could be a potential therapeutic strategy for AD and lay the groundwork for promising clinical applications.

Analysis of the therapeutic role of stem cells versus selenium nanoparticles in lipopolysaccharide-induced acute lung injury in adult male albino rat: A histological, immunohistochemical and histochemical study

Pharmacological research using Lipopolysaccharide (LPS) is a reliable model of acute lung injury. This study aims to compare the therapeutic efficacy of selenium nanoparticles (SeNPs) and bone-marrow-derived mesenchymal stem cells (BMSCs) against lung damage generated by lipopolysaccharides. Fifty adult male albino rats were employed and divided into 5 groups (control group, shame control group, LPS-treated group, SeNPs-treated group, and BMSCs-treated group). Histological and immunohistochemical staining for tumor necrosis factor-alpha (TNFα), Interleukin 6 (IL-6), and Caspase 3 were carried out and followed by histomorphometric analysis. Histochemical measurements of the oxidative-antioxidative markers, superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) were performed in addition to western blotting analysis to detect the antioxidative activity of Nrf2. LPS-treated group showed distorted pulmonary architecture with the upsurge in the caspase-3, TNF-α, IL-6, and MDA along with a significant decrease in GPx, SOD, and Nrf2 in the lung homogenates. These findings were relatively improved after adding SeNPs and stem cells. On comparing BMSCs and SeNPs treated group, there was a significant decrease in caspase-3, TNF-α, and IL-6 with BMSCs treatment relative to SeNPs. Treatment with selenium nanoparticles and BMSCs has improved pulmonary changes through their antioxidant and anti-inflammatory role. The level of pulmonary regeneration exerted by BMSCs is better than selenium nanoparticles so the BMSCs may be given preference for a particular course of treatment.

Unveiling the signal valve specifically tuning the TGF-β1 suppression of osteogenesis: mediation through a SMAD1-SMAD2 complex

BackgroundTGF-β1 is the most abundant cytokine in bone, in which it serves as a vital factor to interdict adipogenesis and osteogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, how TGF-β1 concurrently manipulates differentiation into these two distinct lineages remains elusive.MethodsTreatments with ligands or inhibitors followed by biochemical characterization, reporter assay, quantitative PCR and induced differentiation were applied to MSC line or primary BM-MSCs for signaling dissection. In vivo adipogenesis and ex vivo culture of bone explants were used to verify the functions of different SMAD complexes. Ingenuity Pathway Analysis, and analysis of transcriptomic datasets from human BM-MSCs in combination with hierarchical clustering and STRING assay were used to decipher the interplaying co-repressors. Mouse models of chronic and acute bone loss followed by biochemical assays and micro-computed tomography demonstrated the bone effects when functionally blocking the critical co-repressor HDAC1.ResultsDistinct from the TGF-β1 inhibition on adipogenesis through canonical SMAD2/3 signaling, we clarified that TGF-β1 suppresses osteogenesis by inducing the formation of previously unidentified mixed SMADs mainly composed of SMAD1 and SMAD2, in which SMAD2 recruits more TGF-β1-induced co-repressors including HDAC1, TGIF1 and ATF3, whereas SMAD1 allows directing the whole transcriptional suppression complex to the cis-elements of osteogenic genes. Depletion of the cross-activation to the mixed SMADs dismantled specifically the TGF-β1 suppression on osteogenesis without affecting its inhibition on adipogenesis. Such phenomena can be reproduced via knockdown of co-repressors such as Hdac1 or addition of HDAC1 inhibitors in TGF-β1-treated MSCs. In either the chronic or the acute bone loss model, we demonstrated that the TGF-β signaling was augmented in the bone niche during osteolysis, whereas administration of HDAC1 inhibitors significantly improved bone quality.ConclusionThis study identifies a new signal valve through which TGF-β1 can inhibit osteogenesis specifically. Functional interruption of this valve can tilt the seesaw balance of BM-MSC differentiation towards osteogenesis, highlighting the interplaying co-repressors, such as HDAC1, as promising therapeutic targets to combat diverse degenerative orthopedic diseases.

P0038 Perianal Fistula Closure after Heterologous Mesenchymal Stem Cell Injections is Associated with Protein Levels indicating Lower Tissue Immune Infiltrate, Specific Proteins Involved in the Control of Apoptosis, and Probable Higher Tissues Remodeling and Proteasome Activity

Abstract Background Perianal fistulae affects 30-34% of patients with Crohn’s Disease (CD)[1]. Scarce evidences are available on the perianal fistula pathophysiology. Bone marrow-derived Mesenchymal Stem Cells (bmMSCs) local injections have been found safe and efficient for 50% of CD patients included in a previous prospective monocentric study[2]. The present study aims at identifying potential proteins associated with fistulae closure following bmMSCs local injection, using the biological material collected during fistula tract curettage Methods In the prospective interventional uncontrolled study carried out at CHU of Liège (2019 - 2021), 16 CD have been treated and 10 curettage samples have been analyzed by label free proteomics using mass spectrometry technology[3]. Differential analysis comparing responders (R) with perianal fistula closure at W48 (n=5) with non-responders (NR), with absence of closure at any visit including W48, (n=5), identified specific proteomic profile changes, which were also evaluated by Gene Ontology analyses to identify biological processes, cell types and pathways associated with fistula closure. Results total of 1883 proteins were identified and the differential analysis revealed 48 proteins differentially distributed in R versus NR profiles. Only 18 were more abundant in R, among which the product of the STK4, PLP2, CYB5B and FLG genes, showing the strongest significant increases in R and therefore potential associations with fistula closure after bmMSCs injections. The fistula tract material of patients showing no closure (NR) reveal higher number of proteins, together with NR>R relative abundances, associated with inflammatory and immune infiltrates, with chemokines, bacterial invasion. NR profiles show as well lower numbers and relative levels of proteins involved in wound healing and in matrix remodeling than in R (figures 1 and 2). Among the proteins, differentially distributed in R versus NR, specific pro-apoptotic (R>NR) and anti-apoptotic proteins (NR>R) could be highlighted, in addition to specific proteins acting upstream pathways involved in morphogenesis, carcinogenesis and fibrosis (HIPPO, MAPK, JAK-STAT pathways and TGF signaling). Conclusion Our data provide the first descriptive picture of proteins and likely process/pathways associated with fistula closure after bmMSCs treatment. While independent confirmations are required these preliminary results suggest that the apoptotic process, cell infiltrate degree and proteins involved in tissue remodelling and wound healing, already impacted in fistulae tracts before the bmMSCs injections might be important factors associated with fistula closure outcome in this trial.

IL-1R1 and IL-18 Signals Regulate Mesenchymal Stromal Cells in an Aged Murine Model of Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are age-related diseases characterized by bone marrow (BM) dysfunction and an increased risk of developing acute leukemia. While there is growing evidence highlighting the crucial role of the BM microenvironment (BMME) in MDS, the specific influence of inflammation on BMME changes, as well as the potential benefits of targeting cytokines therapeutically, remain to be elucidated. We previously found interleukin-1 (IL-1) to be a driver of aging phenotypes of BMME and hematopoietic stem and progenitor cells (HSPCs). In the current study, BM samples from patients with MDS demonstrated upregulated levels of IL-1 family cytokines including IL-18. Utilizing highly purified primary BM-derived mesenchymal stromal cells (MSCs), both interleukin-1b (IL-1b) and IL-18 were found to exert direct effects on MSCs, thus influencing their ability to support HSPCs as well as erythroid progenitors. This confirms the significant involvement of both these IL-1 family cytokines in regulating the BM niche. Furthermore, targeting IL-1 receptor type I (IL-1R1) mitigated these aging phenotypes in elderly mice. We subsequently employed an age-appropriate murine model of MDS by transplanting NUP98-HOXD13 transgenic mice (NHD13Tg) cells into aged wild-type mice. Treatment with inhibitors targeting interleukin-1 receptor-associated kinase 4 (IRAK4) and NLR family pyrin domain containing 3 (NLRP3) reversed the proliferation of dysfunctional MSCs and enhanced their functionality. Additionally, IRAK4 inhibition selectively suppressed MDS clonal cells while sparing non-MDS cells in the BM. These findings suggest that targeting IL-1 signaling holds promise for MDS treatment by addressing the underlying myeloid malignancy and restoring the altered BMME via BM-MSCs.

Lymphatic platelet thrombosis limits bone repair by precluding lymphatic transporting DAMPs

In the musculoskeletal system, lymphatic vessels (LVs), which are interdigitated with blood vessels, travel and form an extensive transport network. Blood vessels in bone regulate osteogenesis and hematopoiesis, however, whether LVs in bone affect fracture healing is unclear. Here, we investigate the lymphatic draining function at the tibial fracture sites using near-infrared indocyanine green lymphatic imaging (NIR-ICG) and discover that lymphatic drainage insufficiency (LDI) starts on day one and persists for up to two weeks following the fracture in male mice. Sufficient lymphatic drainage facilitates fracture healing in male mice. Furthermore, we identify that lymphatic platelet thrombosis (LPT) blocks the draining lymphoid sinus and LVs, causes LDI, and inhibits fracture healing in male mice, which can be rescued by a blood thinner. Moreover, unblocked lymphatic drainage decreases neutrophils and increases M2-type macrophages of the hematoma niche to support osteoblast (OB) survival and bone marrow-derived mesenchymal stem cell (BMSC) proliferation via transporting damage-associated molecular patterns (DAMPs) in male rats. Lymphatic platelet thrombolysis also benefits senile fracture healing in female mice. These findings demonstrate that LPT limits bone regeneration by impeding lymphatic transporting DAMPs. Together, these findings represent a way forward in the treatment of bone repair.

Rap1 Guanosine Triphosphate Hydrolase (GTPase) Regulates Shear Stress-Mediated Adhesion of Mesenchymal Stromal Cells.

Intravenously transplanted mesenchymal stromal cells (MSCs) have been shown to interact with endothelial cells and to migrate to tissues. However, intracellular signals regulating MSC migration are still incompletely understood. Here, we analyzed the role of Rap1 GTPase in the migration of human bone marrow-derived MSCs in vitro and in short-term homing in mice in vivo. MSCs expressed both Rap1A and Rap1B mRNAs, which were downregulated after treatment with siRNA against Rap1A and/or B. In a flow chamber model with pre-established human umbilical vein endothelial cells (HUVECs), Rap1A/B downregulated MSCs interacted for longer distances before arrest, indicating adhesion defects. CXCL12-induced adhesion of MSCs on immobilized Vascular Cell Adhesion Molecule (VCAM)-1 was also decreased after the downregulation of Rap1A, Rap1B, or both, as was CXCL12-induced transwell migration. In a competitive murine short-term homing model with i.v. co-injection of Rap1A+B siRNA-treated and control MSCs that were labeled with PKH 26 and PKH 67 fluorescent dyes, the Rap1A+B siRNA-treated MSCs were detected at increased frequencies in blood, liver, and spleen compared to control MSCs. Thus, Rap1 GTPase modulates the adhesion and migration of MSCs in vitro and may increase the bio-availability of i.v.-transplanted MSCs in tissues in a murine model.

Youthful Stem Cell Microenvironments: Rejuvenating Aged Bone Repair Through Mitochondrial Homeostasis Remodeling.

Extracellularmatrix (ECM) derived from mesenchymal stem cells regulates antioxidant properties and bone metabolism by providing a favorable extracellular microenvironment. However, its functional role and molecular mechanism in mitochondrial function regulation and aged bone regeneration remain insufficiently elucidated. This proteomic analysis has revealed a greater abundance of proteins supporting mitochondrial function in the young ECM (Y-ECM) secreted by young bone marrow-derived mesenchymal stem cells (BMMSCs) compared to the aged ECM (A-ECM). Further studies demonstrate that Y-ECM significantly rejuvenates mitochondrial energy metabolism in adult BMMSCs (A-BMMSCs) through the promotion of mitochondrial respiratory functions and amelioration of oxidative stress. A-BMMSCs cultured on Y-ECM exhibited enhanced multi-lineage differentiation potentials in vitro and ectopic bone formation in vivo. Mechanistically, silencing of silent information regulator type 3 (SIRT3) gene abolished the protective impact of Y-ECM on A-BMMSCs. Notably, a novel composite biomaterial combining hyaluronic acid methacrylate hydrogel microspheres with Y-ECM is developed, which yielded substantial improvements in the healing of bone defects in an aged rat model. Collectively, these findings underscore the pivotal role of Y-ECM in maintaining mitochondrial redox homeostasis and present a promising therapeutic strategy for the repair of aged bone defects.

FOXG1 promotes osteogenesis of bone marrow-derived mesenchymal stem cells by activating autophagy through regulating USP14

The osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is key for bone formation, and its imbalance leads to osteoporosis. Forkhead Box Protein G1 (FOXG1) is associated with osteogenesis, however, the effect of FOXG1 on osteogenesis of BMSCs and ovariectomy (OVX)-induced bone loss is unknown. In our study, FOXG1 expression in BMSCs increases after osteogenic induction. FOXG1 overexpression significantly increases osteoblast marker expression ALP activity, and calcium deposition, while the opposite results are observed in FOXG1 knockdown BMSCs, suggesting that FOXG1 promotes osteogenic differentiation. Additionally, autophagy promotes the differentiation process in BMSCs. We find that FOXG1 induces autophagy, and osteogenic differentiation is blocked via inhibiting FOXG1-caused autophagy, indicating that FOXG1 accelerates osteogenic differentiation via inducing autophagy. Eight-week-old female C57BL/6J mice are used in OVX models, FOXG1 overexpression decreases bone loss by increasing bone formation. Moreover, FOXG1 overexpression suppresses osteoclast differentiation. Mechanically, FOXG1 transcriptionally represses ubiquitin-specific protease14 (USP14) via binding to the USP14 promoter. USP14 overexpression prevents the promoting effect of FOXG1 on osteogenic differentiation in BMSCs. Therefore, our findings suggest that FOXG1 promotes BMSC osteogenic differentiation and inhibits osteoclast differentiation, eventually blocking OVX-induced bone loss, which may provide a promising approach for osteoporosis treatment.

Improved Mesenchymal Stem Cell Viability in High-Stiffness, Translational Cartilage Matrix Hydrogels.

Scaffolds made from cartilage extracellular matrix are promising materials for articular cartilage repair, attributed to their intrinsic bioactivity that may promote chondrogenesis. While several cartilage matrix-based scaffolds have supported chondrogenesis in vitro and/or invivo, it remains a challenge to balance the biological response (e.g., chondroinductivity) with structural (e.g., robust mechanical performance, >1 MPa in compressive stiffness) and translational (e.g., ease of surgical implantation) considerations. Few studies have evaluated encapsulated cell viability within high-stiffness (>1 MPa) hydrogels. We previously fabricated one formulation of a high-stiffness (>3 MPa) pentenoate-functionalized, solubilized, devitalized cartilage (PSDVC) hydrogel that possessed an injectable, paste-like precursor for easy surgical application. In the current study, the characterization of the PSDVC material was expanded by varying the degree of functionalization (i.e., 0.45-1.09 mmol/g) and amount of crosslinker, dithiothreitol (DTT), to improve the reproducibility of the high compressive moduli and evaluate the viability of encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs) in high-stiffness cartilage matrix hydrogels. Prior to crosslinking, specific formulations functionalized with 0.80 mmol/g or less of pentenoate groups retained a paste-like precursor rheology. After crosslinking, these formulations produced hydrogels with greater than 1 MPa compressive stiffness. However, hBMSCs encapsulated in PSDVC hydrogels with lower functionalization (i.e., 0.57 mmol/g, no crosslinker) had a higher stiffness (i.e., 1.4 MPa) but the lowest viability of encapsulated hBMSCs (i.e., 5%). The middle PSDVC functionalization (i.e., 0.70 mmol/g) with DTT (i.e., 0.50 mmol thiols/g) demonstrated high cell viability (77%), high mechanical performance (1.65 MPa, 31% failure strain), and translational features (i.e., paste-like precursor, 1.5 min crosslinking time). For future evaluations of PSDVC hydrogels in cartilage repair, a middle functionalization (i.e., 0.70-0.80 mmol/g) with the addition of a crosslinker (i.e., 0.50 mmol thiols/g) had a desirable balance of high mechanical performance (i.e., >1 MPa compressive stiffness), high viability, and paste-like precursor for surgical translation.

Therapeutic effects of intracerebral transplantation of human modified bone marrow-derived stromal cells (SB623) with voluntary and forced exercise in a rat model of ischemic stroke.

Ischemic stroke results in significant long-term disability and mortality worldwide. Although existing therapies, such as recombinant tissue plasminogen activator and mechanical thrombectomy, have shown promise, their application is limited by stringent conditions. Mesenchymal stem cell (MSC) transplantation, especially using SB623 cells (modified human bone marrow-derived MSCs), has emerged as a promising alternative, promoting neurogenesis and recovery. This study evaluated the effects of voluntary and forced exercise, alone and in combination with SB623 cell transplantation, on neurological and psychological outcomes in a rat model of ischemic stroke. Male Wistar rats that had undergone middle cerebral artery occlusion (MCAO) were divided into six groups: control, voluntary exercise (V-Ex), forced exercise (F-Ex), SB623 transplantation, SB623 + V-Ex, and SB623 + F-Ex. Voluntary exercise was facilitated using running wheels, while forced exercise was conducted on treadmills. Neurological recovery was assessed using the modified neurological severity score (mNSS). Psychological symptoms were evaluated through the open field test (OFT) and forced swim test (FST), and neurogenesis was assessed via BrdU labeling. Both exercise groups exhibited significant changes in body weight post-MCAO. Both exercises enhanced the treatment effect of SB623 transplantation. The forced exercise showed a stronger treatment effect on ischemic stroke than voluntary exercise alone, and the sole voluntary exercise improved depression-like behavior. The SB623 + F-Ex group demonstrated the greatest improvements in motor function, infarct area reduction, and neurogenesis. The SB623 + V-Ex group was most effective in alleviating depression-like behavior. Future research should optimize these exercise protocols and elucidate the underlying mechanisms to develop tailored rehabilitation strategies for stroke patients.

Editorial Expression of Concern: The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

Editorial Expression of Concern: The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

Interleukin-12 Inhibits Tumor Growth and Metastasis Promoted by Tumor-Associated Mesenchymal Stem Cells in Triple-Negative Breast Cancer.

The aim of this study was to understand the interactions between tumor-associated mesenchymal stem cells (TA-MSCs) and triple-negative breast cancer (TNBC) cells, which appear to be necessary for developing effective therapies. In this experimental study, MDA-MB-231 and 4T1 TNBC cells were co-cultured with bone marrow-derived MSCs, and TA-MSCs conditioned media (CM) were collected. TA-MSC CM-treated TNBC cells were subjected to migration and invasion assays. Epithelial-mesenchymal transition (EMT) marker expression was quantified by real-time polymerase chain reaction (RT-PCR). Cell proliferation was measured using trypan blue exclusion technique, while cell cycle distribution and apoptosis were assessed by flow cytometry. The effects of TA-MSCs on tumor volume, survival rate, and lung metastasis were evaluated by subcutaneous co-injection of MSCs with 4T1 cells in the right flanks of BALB/c mice (n=5 per group). Intratumoral interleukin-12 (IL-12) immunotherapy was performed using lentiviral particles as a rescue experiment. The TA-MSCs RNA-seq dataset (PRJEB27694) was analyzed to detect elevated metastasis-associated oncogenes, downloaded from the European Nucleotide Archive database. For validation of the RNA-seq data analysis, the expression levels of candidate oncogenes were evaluated in TA-MSCs, TNBC cells, and tumor tissue using RT-PCR. TA-MSCs enhanced migration, invasion, and EMT of TNBC cells in vitro without affecting cell proliferation or apoptosis. In vivo, TA-MSCs increased tumor growth and lung metastasis, while decreasing survival rates. IL-12 therapy elevated serum IL-12 and interferon-gamma (IFN-γ) expression, suppressed tumor volume and lung metastasis, and improved overall survival in the TA-MSC group. RNA-seq data analysis identified upregulated oncogenes in TA-MSCs, among which MMP3, CXCL2, CXCL5, and ICAM1 were selected as the most relevant to metastasis. These genes showed increased expression in TA-MSCs, TNBC cells, and tumor tissues. The findings of the present study revealed a complex interplay between TA-MSCs and TNBC cells that affects tumor growth and metastasis. Preclinical results indicate that intratumoral IL-12 immunotherapy shows promise in overcoming TA-MSC-promoted tumor growth and metastasis.

Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

AbstractMesenchymal stem cell‐derived extracellular vesicles (MSC EVs) are an attractive therapeutic option for regenerative medicine applications due to their inherently pro‐angiogenic and anti‐inflammatory properties. However, reproducible and cost‐effective production of highly potent therapeutic MSC EVs is challenging, limiting their translational potential. Here, we investigated whether the well‐characterized responsiveness of MSCs to their mechanical environment—specifically, substrate stiffness—could be exploited to generate EVs with increased therapeutic bioactivity without the need for biochemical priming or genetic manipulation. Using polydimethylsiloxane and bone marrow‐derived MSCs (BM‐MSCs), we show that decreasing the stiffness of MSC substrates to as low as 3 kPa significantly improves the pro‐angiogenic bioactivity of EVs as measured by tube formation and gap closure assays. We also demonstrate that lower substrate stiffness improves EV production and overall yield, important for clinical translation. Furthermore, we establish the mechanoresponsiveness of induced pluripotent stem cell‐derived MSC (iMSC) EVs and their comparability to BM‐MSC EVs, again using tube formation and gap closure assays. With this data, we confirm iMSCs' feasibility as an alternative, renewable cell source for EV production with reduced donor variability. Overall, these results suggest that utilizing substrate stiffness is a promising, simple, and a potentially scalable approach that does not require exogenous cargo or extraneous reagents to generate highly potent pro‐angiogenic MSC EVs.

Synergistic Effects of Hydrogen Peroxide Preconditioning and Valproic Acid on Hepatic Differentiation of Mesenchymal Stem Cells.

Ex vivo preconditioning increases the therapeutic potential of mesenchymal stem cells (MSCs) in terms of antioxidant activity, growth factor production, homing, differentiation, and immunomodulation. Therefore, it is considered an effective strategy to be used before transplantation and therapeutic application of MSCs. Histone deacetylase inhibitor (HDACi), valproic acid (VPA), has been reported to induce hepatic differentiation in MSCs. Although individual studies have shown that preconditioning and epigenetic modification enhance the survival and differentiation of MSCs, the combined effects of these therapies have not been fully explored. This study aims to investigate the combined effect of hydrogen peroxide (H2O2) preconditioning and HDACi (valproic acid) on the differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) into hepatic-like cells. MSCs were first preconditioned with H2O2 and then cultured with VPA. The migration and proliferation potential of the treated cells were evaluated using wound healing and colony-- forming unit assays. Furthermore, the expression of hepatic genes (FOXA2, CK8, CK18, TAT) and proteins (AFP, ALB, TAT) was evaluated in all treated groups. The combined therapy group exhibited enhanced cell migration and proliferation, as evidenced by wound healing and colony-forming unit assays. Additionally, the combined treatment group showed higher expression of FOXA2, CK8, and CK18 hepatic genes and TAT protein, suggesting an improved differentiation of stem cells into hepatocytes. In conclusion, the combination of H2O2 and VPA emerges as an important factor in promoting hepatocyte differentiation. However, further studies are required to optimize this protocol for future therapeutics.

Comparative neuroprotective effects of xenogeneic and allogeneic bone marrow‐derived mesenchymal stromal cells in a murine model of sepsis

Aims/Purpose: Sepsis causes systemic inflammation (SI) and induces acute and long‐term neurological dysfunctions. In the retina, SI causes the loss of retinal ganglion cells (RGCs). Advanced cell therapy with bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) is a promising avenue for RGCs neuroprotection in SI due to their immunomodulatory properties. Here we analyze the neuroprotective properties of BM‐MSCs in two transplantation settings: xenogeneic and allogeneic in a murine model of SI induced by lipopolysaccharide (LPS).Methods: 2‐month‐old C57BL/6J male mice were used in all groups. LPS was intraperitoneally injected (5mg/kg) at day 0. At day 1, BM‐MSCs isolated from BALB/C mice (allogenic) or human donors (xenogenic) were intravitreally administered in the left eye. As controls we used intact and LPS‐treated C57BL/6J animals. RGCs survival (Brn3a+) and microglial activation (Iba1+) were studied at 7 days in both retinas, the treated and the contralateral to the injection.Results: BM‐MSCs were found forming an epiretinal membrane in both transplant modalities. RGCs death in LPS controls reached 20% of the original population. The allogeneic transplant rescued all RGCs in both eyes. In contrast, xenogeneic transplant only protected to the contralateral eyes and microglia were more activated and reactive.Conclusions: These preclinical data show that allogeneic BM‐MSCs elicits a greater neuroprotection for sepsis induced RGCs degeneration in both eyes, the treated and contralateral. While the xenogeneic transplant does not neuroprotect RGCs in the treated eye, it does in the contralateral one, a neuroprotection that could be due to paracrine effects. These findings may be the basis to treat sepsis induced neurodegeneration.