Signaling In Mesenchymal Stem Cells Research Articles

Effect of DNA methylation on the osteogenic differentiation of mesenchymal stem cells: concise review.

Mesenchymal stem cells (MSCs) have promising potential for bone tissue engineering in bone healing and regeneration. They are regarded as such due to their capacity for self-renewal, multiple differentiation, and their ability to modulate the immune response. However, changes in the molecular pathways and transcription factors of MSCs in osteogenesis can lead to bone defects and metabolic bone diseases. DNA methylation is an epigenetic process that plays an important role in the osteogenic differentiation of MSCs by regulating gene expression. An increasing number of studies have demonstrated the significance of DNA methyltransferases (DNMTs), Ten-eleven translocation family proteins (TETs), and MSCs signaling pathways about osteogenic differentiation in MSCs. This review focuses on the progress of research in these areas.

Characteristic Differences between Freeze-dried Tenogenic Secretomes and Freeze-dried Crude Secretomes of Mesenchymal Stem Cells under Hypoxic Conditions

ABSTRACT Introduction: Tendons that are crucial for movement are struggling with self-healing. Regenerative medicine has been using mesenchymal stem cells (MSCs) and their secretomes, and it offers promise. These secretomes, rich in growth factors, aid tissue healing. Tenogenic secretomes target tendon-specific healing, while crude ones provide a broader view of MSC signaling. The study aims to enhance targeted interventions in tendon regeneration and improve the quality of life for those with tendon-related issues. Methods: This laboratory study compares freeze-dried tenogenic and crude secretomes of MSCs under hypoxic conditions for regenerative potential. Stem cells from adipose tissue undergo isolation, culture, and multiplication then treated in hypoxic conditions. Characterization, Interleukin-6 (IL-6) and Interleukin-10 (IL-10) analysis with enzyme-linked immunosorbent assay (ELISA) follow. The results, including organoleptic, pH, and scanning electron microscope assessments, were presented descriptively. Statistical analysis will enhance understanding. Results: Physical characteristics (odor, color, and pH) are similar in both groups. Tenogenic secretome has a more concave surface, potentially aiding cell migration compared to the crude secretome. There were significant differences in cytokine levels (P = 0.001). IL-6 levels expressed from tenogenic secretome were 10.2 ± 2.432 pg/mL compared to the crude one which 18.3 ± 3.175 pg/mL. Levels of IL-10 expressed from tenogenic and crude secretomes were 309.2 ± 50.973 pg/mL and 200.1 ± 32.030, respectively. Conclusion: The tenogenic secretome and crude secretome exhibit no differences in physical characteristics but differ in surface morphology. The concave surface of the tenogenic secretome facilitates cell migration more than the crude secretome. Under hypoxic conditions, the tenogenic secretome shows lower pro-inflammatory (IL-6) and higher anti-inflammatory (IL-10) effects than the crude secretome.

Vascular Cell Adhesion Molecule-1 Regulates Bone Marrow Mesenchymal Stem Cell Maintenance and Niche Function

Bone marrow (BM) mesenchymal stem cells (MSCs) are rare non-hematopoietic perivascular stromal cells characterized by their unique ability to self-renew and differentiate into bone, cartilage, and fat, ensuring proper skeletal development and maintenance. Different subsets of BM MSCs associate with arteriolar and sinusoidal vessels to form specialized niches that regulate hematopoietic stem cell (HSC) function by secreting high levels of niche factors such as CXC-chemokine ligand 12 (CXCL12), stem cell factor (SCF), and Vascular Cell Adhesion Molecule-1 (VCAM1). VCAM1 is classically expressed on endothelial and stromal cells where it acts as an adhesion molecule that preferentially binds to α4β1 integrin on HSCs and progenitor cells (HSPCs). Deletion of Vcam1 in endothelial and hematopoietic cells induces HSPC mobilization into the peripheral blood without affecting endothelial cells homeostasis. Recent work from our lab further demonstrated that VCAM1 expression on HSCs and leukemic stem cells confers innate immune tolerance ( Nat Cell Biol 2022). While the contribution of HSC and endothelial-derived VCAM1 to homeostasis has been well studied, the role of MSC-derived VCAM1 on HSC and MSC maintenance and multilineage potency remains unknown. Our supporting data suggest that MSCs are the BM's main source of Vcam1 (p<0.0001) and that VCAM1 is critical for the maintenance, survival, and function of MSCs. Using a mouse model in which Vcam1 can be conditionally deleted in BM MSCs by a tamoxifen-inducible N-Cadherin-Cre ER line (referred to as Vcam1 N-CadCreER), we found that deletion of Vcam1 significantly reduces the number (4-fold, p=0.0030), frequency (4-fold, p=0.0042), and viability (2-fold, p=0.0039) of BM MSCs. Previous findings in breast cancer revealed that VCAM1 promotes PI3K/Akt survival signaling via Akt phosphorylation. Accordingly, Vcam1 deletion in N-Cadherin-Cre ER MSCs, demonstrated a significant reduction in phospho-Akt (Ser473) (p<0.05), supporting the role of VCAM1 in mediating PI3K/Akt survival signaling in MSCs. Intriguingly, the remaining viable VCAM1-null MSCs isolated from Vcam1 N-CadCreER mice exhibited reduced clonogenic potential (determined by CFU-F assays; 3-fold, p=0.0001). Furthermore, micro-CT analysis of 6-month-old Vcam1 N-CadCreER mice presented an altered bone phenotype compared to the control as evidenced by their significantly increased bone thickness and volume (p<0.05), yet significantly decreased tissue mineral density and medullary cavity area (p<0.05). Bulk RNA sequencing (RNAseq) of sorted viable MSCs revealed distinct grouping between Control and Vcam1 N-CadCreER MSCs. Gene ontology analysis demonstrated that deletion of Vcam1 caused an upregulation of pathways associated with inflammation and cytokine release while pathways associated with ossification, extracellular matrix organization, and skeletal development were all downregulated. Surprisingly, both bulk RNAseq and qPCR analysis confirmed a significant reduction in the expression of HSC niche factors ( Vcam1 Cxcl12 and Scf) following Vcam1 deletion, indicative of reduced niche activity. As expected, Vcam1 deletion in N-Cadherin-Cre ER MSCs, caused significant mobilization of HSCs into the blood (p<0.0001) and BM homing defects (p<0.05), however the number and repopulation capacity of BM HSCs remained unaltered, as seen by BM transplantation experiments. Surprisingly, imaging analysis of whole-mount bone marrow sternum of phenotypic HSCs revealed no differences in HSC niche localization between Vcam1-null and Control mice. Accordingly, ELISA analysis of BM extracellular fluid revealed no differences in overall CXCL12 and SCF levels suggesting that there may be some local or systemic niche compensation at play. Single-cell (sc)RNAseq of pooled BM and compact bone sorted non-hematopoietic stromal cells (DAPI -Lineage -CD45 -Ter119 -CD19 -CD71 -)from Vcam1 N-CadCreERand Control mice further confirmed the downregulation of HSC niche factors in MSCs, and overall remodeling of the BM stromal cell composition following Vcam1 deletion. Interestingly, our scRNAseq analysis revealed increased expression of Cxcl12 and Scf in specific vascular and stromal cell populations, supporting the notion of HSC niche compensation. Overall, these findings demonstrate that MSC-derived Vcam1 is critical for BM MSC survival and their HSC niche function.

Comparative computational analysis to distinguish mesenchymal stem cells from fibroblasts.

Mesenchymal stem cells (MSCs) are considered to be the most promising stem cell type for cell-based therapies in regenerative medicine. Based on their potential to home to diseased body sites following a therapeutically application, these cells could (i) differentiate then into organ-specific cell types to locally restore injured cells or, most prominently, (ii) foster tissue regeneration including immune modulations more indirectly by secretion of protective growth factors and cytokines. As tissue-resident stem cells of mesenchymal origin, these cells are morphologically and even molecularly- at least concerning the classical marker genes- indistinguishable from similar lineage cells, particularly fibroblasts. Here we used microarray-based gene expression and global DNA methylation analyses as well as accompanying computational tools in order to specify differences between MSCs and fibroblasts, to further unravel potential identity genes and to highlight MSC signaling pathways with regard to their trophic and immunosuppressive action. We identified 1352 differentially expressed genes, of which in the MSCs there is a strong signature for e.g., KRAS signaling, known to play essential role in stemness maintenance, regulation of coagulation and complement being decisive for resolving inflammatory processes, as well as of wound healing particularly important for their regenerative capacity. Genes upregulated in fibroblasts addressed predominately transcription and biosynthetic processes and mapped morphological features of the tissue. Concerning the cellular identity, we specified the already known HOX code for MSCs, established a potential HOX code for fibroblasts, and linked certain HOX genes to functional cell-type-specific properties. Accompanied methylation profiles revealed numerous regions, especially in HOX genes, being differentially methylated, which might provide additional biomarker potential. Conclusively, transcriptomic together with epigenetic signatures can be successfully be used for the definition (cellular identity) of MSCs versus fibroblasts as well as for the determination of the superior functional properties of MSCs, such as their immunomodulatory potential.

The inhibition of pancreatic cancer progression by K-Ras-overexpressing mesenchymal stem cell-derived secretomes

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival. To explore an uncharted function of K-Ras proto-oncogene, K-Ras was activated in mesenchymal stem cells (MSCs) and the effects of MSC conditioned medium (CM) on PDAC were examined. Overexpression of K-Ras elevated PI3K signaling in MSCs, and K-Ras/PI3K-activated MSC-derived CM reduced the proliferation and migration of tumor cells, as well as the growth of ex vivo freshly isolated human PDAC cultures. CM’s anti-tumor capability was additive with Gemcitabine, a commonly used chemotherapeutic drug in the treatment of PDAC. The systemic administration of CM in a mouse model suppressed the colonization of PDAC in the lung. MSC CM was enriched with Moesin (MSN), which acted as an extracellular tumor-suppressing protein by interacting with CD44. Tumor-suppressive CM was also generated by PKA-activated peripheral blood mononuclear cells. Collectively, this study demonstrated that MSC CM can be engineered to act as a tumor-suppressive agent by activating K-Ras and PI3K, and the MSN-CD44 regulatory axis is in part responsible for this potential unconventional option in the treatment of PDAC.

Glucocorticoid-induced activation of NOX/ROS/NF-κB signaling in MSCs contributes to the development of GONFH.

This study aimed to investigate the pathogenic factors of glucocorticoids (GCs)-induced osteonecrosis of the femoral head (GONFH) and its underlying pathogenesis in vivo and in vitro. Radiographical (µCT) scanning, histopathological, immunohistochemical, reactive oxygen species (ROS) and tunel staining were conducted on GONFH patients and rats. ROS, tunel, flow cytometry, alkaline phosphatase, Oil red O staining, reverse transcription‑quantitative PCR and western blotting were applied to elucidate the exact pathogenesis mechanism. Clinical and animal studies demonstrated increased levels of ROS, aggravated oxidative stress (OS) microenvironment, augmented apoptosis and imbalance in osteogenic/lipogenic in the GONFH group compared to the control group. The fate of mesenchymal stem cells (MSCs) directed by GCs is a crucial factor in determining GONFH. In vitro studies further revealed that GCs promote excessive ROS production through the expression of NOX family proteins, leading to a deterioration of the OS microenvironment in MSCs, ultimately resulting in apoptosis and imbalance in osteogenic/lipogenic differentiation. Furthermore, our results confirmed that the NOX inhibitor-diphenyleneiodonium chloride and the NF-κB inhibitor-BAY 11-7082 ameliorated apoptosis and osteogenic/lipogenic differentiation imbalance of MSCs induced by an excess of GCs. We demonstrated for the first time that the aggravation of the OS microenvironment in MSCs caused by high doses of GCs leading to apoptosis and differentiation imbalance is a crucial factor in the pathogenesis of GONFH, mediated through activating the NOX/ROS/NF-κB signaling pathway.

Cell circuits between leukemic cells and mesenchymal stem cells block lymphopoiesis by activating lymphotoxin beta receptor signaling.

Acute lymphoblastic and myeloblastic leukemias (ALL and AML) have been known to modify the bone marrow microenvironment and disrupt non-malignant hematopoiesis. However, the molecular mechanisms driving these alterations remain poorly defined. Using mouse models of ALL and AML, here we show that leukemic cells turn off lymphopoiesis and erythropoiesis shortly after colonizing the bone marrow. ALL and AML cells express lymphotoxin α1β2 and activate lymphotoxin beta receptor (LTβR) signaling in mesenchymal stem cells (MSCs), which turns off IL7 production and prevents non-malignant lymphopoiesis. We show that the DNA damage response pathway and CXCR4 signaling promote lymphotoxin α1β2 expression in leukemic cells. Genetic or pharmacological disruption of LTβR signaling in MSCs restores lymphopoiesis but not erythropoiesis, reduces leukemic cell growth, and significantly extends the survival of transplant recipients. Similarly, CXCR4 blocking also prevents leukemia-induced IL7 downregulation and inhibits leukemia growth. These studies demonstrate that acute leukemias exploit physiological mechanisms governing hematopoietic output as a strategy for gaining competitive advantage.

MACF1 overexpression in BMSCs alleviates senile osteoporosis in mice through TCF4/miR-335-5p signaling pathway.

The decreased osteogenic differentiation ability of mesenchymal stem cells (MSCs) is one of the important reasons for SOP. Inhibition of Wnt signaling in MSCs is closely related to SOP. Microtubule actin crosslinking factor 1 (MACF1) is an important regulator in Wnt/β-catenin signal transduction. However, whether the specific expression of MACF1 in MSC regulates SOP and its mechanism remains unclear. We established MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-KI) mice, naturally aged male mice, and ovariectomized female mice models. Micro-CT, H&E staining, double calcein labeling, and the three-point bending test were used to explore the effects of MACF1 on bone formation and bone microstructure in the SOP mice model. Bioinformatics analysis, ChIP-PCR, qPCR, and ALP staining were used to explore the effects and mechanisms of MACF1 on MSCs' osteogenic differentiation. Microarray analysis revealed that the expression of MACF1 and positive regulators of the Wnt pathway (such as TCF4, β-catenin, Dvl) was decreased in human MSCs (hMSCs) isolated from aged osteoporotic than non-osteoporotic patients. The ALP activity and osteogenesis marker genes (Alp, Runx2, and Bglap) expression in mouse MSCs was downregulated during aging. Furthermore, Micro-CT analysis of the femur from 2-month-old MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-cKI) mice showed no significant trabecular bone changes compared to wild-type littermate controls, whereas 18- and 21-month-old MACF1 c-KI animals displayed increased bone mineral densities (BMD), improved bone microstructure, and increased maximum compression stress. In addition, the ovariectomy (OVX)-induced osteoporosis model of MACF1 c-KI mice had significantly higher trabecular volume and number, and increased bone formation rate than that in control mice. Mechanistically, ChIP-PCR showed that TCF4 could bind to the promoter region of the host gene miR-335-5p. Moreover, MACF1 could regulate the expression of miR-335-5p by TCF4 during the osteogenic differentiation of MSCs. These data indicate that MACF1 positively regulates MSCs osteogenesis and bone formation through the TCF4/miR-335-5p signaling pathway in SOP, suggesting that targeting MACF1 may be a novel therapeutic approach against SOP. MACF1, an important switch in the Wnt signaling pathway, can alleviate SOP through the TCF4/miR-335-5p signaling pathway in mice model. It might act as a therapeutic target for the treatment of SOP to improve bone function.

Protective effect of hydroxysafflor yellow A on MSCs against senescence induced by d-galactose

ObjectiveTo examine the protective effects of hydroxysafflor yellow A (HSYA) against the senescence of mesenchymal stem cells (MSCs) induced by d-galactose (d-gal) in vitro, and investigate the potential mechanism involved. MethodsGrouping experiment, Normal control (NC) group: conventional culture with complete medium; Senescence group: MSCs were cultured for 48 h with complete medium containing 10 g/L d-gal; HSYA group: on the basis of senescence induction, HSYA with the suitable concentration was used to protect MSCs. The key experimental indices associated with oxidative stress, inflammatory response, cell senescence, proliferation and apoptosis were measured through chemical colorimetry, β-galactosidase staining, EdU incorporation and flow cytometry, respectively. The relative quantity (RQ) of proteins related closely to cell proliferation, apoptosis, and NF-κB signaling were measured by Western blotting. ResultsAs compared with Senescence group, treatment with HSYA (120 mg/L) effectively ameliorated the adverse situation of MSCs. Oxidation stress and inflammation along with d-Gal induction was dramatically alleviated in MSCs; The β-Gal-positive staining indicated that MSC senescence was significantly mitigated; The proliferative capability of MSCs was significantly increased by up-regulating PCNA and inhibiting p16 expression; The anti-apoptotic effect on MSCs was exerted by down-regulating the RQ of cleaved Caspase-3 and Bax; The activity of NF-κB signaling in MSCs was notably suppressed through inhibiting phosphorylation of IKKβ and p65. ConclusionHSYA (120 mg/L) significantly delayed the d-Gal-induced senescence process in MSCs through attenuating inflammatory reaction and oxidative stress, and suppressing the activity of NF-κB signaling.

Inhibiting PI3K-AKT-mTOR Signaling in Multiple Myeloma-Associated Mesenchymal Stem Cells Impedes the Proliferation of Multiple Myeloma Cells.

The microenvironment of cancer cells is receiving increasing attention as an important factor influencing the progression and prognosis of tumor diseases. In multiple myeloma (MM), a hematological cancer of plasma cells, mesenchymal stem cells (MSCs) represent an integral part of the bone marrow niche and tumor microenvironment. It has been described that MM cells alter MSCs in a way that MM-associated MSCs promote the proliferation and survival of MM cells. Yet, our understanding of the molecular mechanisms governing the interaction between MM cells and MSCs and whether this can be targeted for therapeutic interventions is limited. To identify potential molecular targets, we examined MSCs by RNA sequencing and Western blot analysis. We report that MSCs from MM patients with active disease (MM-Act-MSCs) show a distinct gene expression profile as compared with MSCs from patients with other (non-) malignant diseases (CTR-MSCs). Of note, we detected a significant enrichment of the PI3K–AKT–mTOR hallmark gene set in MM-Act-MSCs and further confirmed the increased levels of related proteins in these MSCs. Pictilisib, a pan-PI3K inhibitor, selectively reduced the proliferation of MM-Act-MSCs as compared with CTR-MSCs. Furthermore, pictilisib treatment impaired the MM-promoting function of MM-Act-MSCs. Our data thus provide a deeper insight into the molecular signature and function of MSCs associated with MM and show that targeting PI3K–AKT–mTOR signaling in MSCs may represent an additional therapeutic pathway in the treatment of MM patients.

Autonomous and intercellular chemokine signaling elicited from mesenchymal stem cells regulates migration of undifferentiated gastric cancer cells.

Accumulating evidence demonstrates that bone marrow (BM)-derived mesenchymal stem cells (MSCs) play critical roles in regulating progression of various types of cancer. We have previously shown that Wnt5a-Ror2 signaling in MSCs induces expression of CXCL16, and that CXCL16 secreted from MSCs then binds to its cognate receptor CXCR6 on the surface of an undifferentiated gastric cancer cell line MKN45 cells, eventually leading to proliferation and migration of MKN45 cells. However, it remains unclear about a possible involvement of another (other) cytokine(s) in regulating progression of gastric cancer. Here, we show that CXCL16-CXCR6 signaling is also activated in MSCs through cell-autonomous machinery, leading to upregulated expression of CCL5. We further show that CCR1 and CCR3, receptors of CCL5, are expressed on the surface of MKN45 cells, and that CCL5 secreted from MSCs promotes migration of MKN45 cells presumably via its binding to CCR1/CCR3. These data indicate that cell-autonomous CXCL16-CXCR6 signaling activated in MSCs upregulates expression of CCL5, and that subsequent activation of CCL5-CCR1/3 signaling in MKN45 cells through intercellular machinery can promote migration of MKN45 cells. Collectively, these findings postulate the presence of orchestrated chemokine signaling emanated from MSCs to regulate progression of undifferentiated gastric cancer cells.

Exosome‑derived lncRNA‑Ankrd26 promotes dental pulp restoration by regulating miR‑150‑TLR4 signaling.

At present, retaining the biological function of dental pulp is an urgent requirement in the treatment of pulp disease; it has been recognized that application of dental pulp stem cells (DPSCs) in regenerating dental pulp and dentin complexes is expected to become a safe and effective treatment of pulp disease; meanwhile the role of DPSC‑derived exosomes in dental pulp regeneration and repair is gaining attention. However, the underlying mechanism of DPSCs in dental pulp regeneration and repair is still unclear. In the present study, a variety of invitro biological experiments and an animal model, as well as next‑generation sequencing and bioinformatics analysis, demonstrated that DPSCs promoted migration and osteoblastic differentiation of mesenchymal stem cells (MSCs) via exosomes; this was induced by DPSC‑derived exosomal long non‑coding (lnc)RNA‑ankyrin repeat domain (Ankrd)26. Mechanistically, the effect of exosomal lncRNA‑Ankrd26 on migration and osteoblastic differentiation of MSCs was dependent on microRNA (miR)‑150/Toll‑like receptor (TLR)4 signaling; this was regulated by lncRNA‑Ankrd26. The present study demonstrated that exosomes‑derived lncRNA‑Ankrd26 from DPSCs promoted dental pulp restoration via regulating miR‑150‑TLR4 signaling in MSCs; these findings help to understand the mechanism of dental pulp repair, identify therapeutic targets in the development of pulpitis and develop clinical treatments.

Indoleamine 2, 3 Dioxygenase 1 Impairs Chondrogenic Differentiation of Mesenchymal Stem Cells in the Joint of Osteoarthritis Mice Model.

Osteoarthritis (OA) is a serious joint inflammation that leads to cartilage degeneration and joint dysfunction. Mesenchymal stem cells (MSCs) are used as a cell-based therapy that showed promising results in promoting cartilage repair. However, recent studies and clinical trials explored unsatisfied outcomes because of slow chondrogenic differentiation and increased calcification without clear reasons. Here, we report that the overexpression of indoleamine 2,3 dioxygenase 1 (IDO1) in the synovial fluid of OA patients impairs chondrogenic differentiation of MSCs in the joint of the OA mice model. The effect of MSCs mixed with IDO1 inhibitor on the cartilage regeneration was tested compared to MSCs mixed with IDO1 in the OA animal model. Further, the mechanism exploring the effect of IDO1 on chondrogenic differentiation was investigated. Subsequently, miRNA transcriptome sequencing was performed for MSCs cocultured with IDO1, and then TargetScan was used to verify the target of miR-122-5p in the SF-MSCs. Interestingly, we found that MSCs mixed with IDO1 inhibitor showed a significant performance to promote cartilage regeneration in the OA animal model, while MSCs mixed with IDO1 failed to stimulate cartilage regeneration. Importantly, the overexpression of IDO1 showed significant inhibition to Sox9 and Collagen type II (COL2A1) through activating the expression of β-catenin, since inhibiting of IDO1 significantly promoted chondrogenic signaling of MSCs (Sox9, COL2A1, Aggrecan). Further, miRNA transcriptome sequencing of SF-MSCs that treated with IDO1 showed significant downregulation of miR-122-5p which perfectly targets Wnt1. The expression of Wnt1 was noticed high when IDO1 was overexpressed. In summary, our results suggest that IDO1 overexpression in the synovial fluid of OA patients impairs chondrogenic differentiation of MSCs and cartilage regeneration through downregulation of miR-122-5p that activates the Wnt1/β-catenin pathway.

Mesenchymal Stem Cells Resist Mechanical Confinement through the Activation of the Cortex during Cell Division.

The mechanical properties of the natural extracellular matrix (ECM) change extensively, but these specific properties provide a relatively stable environment for resident cells. Although the effect of matrix stiffness on cell functions has been widely studied, the molecular mechanism was still not fully understood. Matrix stiffening is a common phenomenon in tissue damaging processes. To explore the effect of the increase in local matrix stiffness on cell behaviors, a three-dimensional (3D) cell culture system with a tunable modulus but constant other physical parameters was constructed by the alginate hydrogel with different molecular weights and cross-linking degrees. By using this culture system, the transcriptome response of mesenchymal stem cells (MSCs) to matrix stiffness was explored. Furthermore, a finite element model was developed to simulate the interaction between cells and the matrix. Results revealed that the increased matrix stiffness promoted the proliferation-related signaling of MSCs, and this process depended on the increased cortex tension caused by the activation of RAS and myosin II.

Osteogenic Regulation by Graphene Nanoparticles in Human Mesenchymal Stem Cells

Background Degenerative bone diseases caused by trauma, cancer, or aging imposes a global health care burden every year. Treatment strategies include directing stem cell differentiation through osteogenesis, thereby creating new bone material for repairment. We previously identified that adult mesenchymal stem cells (MSCs) cultured on graphene nanoparticles spontaneously supports osteoblast development in vitro. However, the osteo-genetic signaling of MSCs in the presence of graphene is largely unknown. Therefore, this study sought to measure genetic expressions of MSCs cultured on graphene using focused PCR arrays. Methods MSCs were extracted from human adipose tissue (hAD-MSCs) and human bone marrow (hBM-MSCs) and expanded in growth media. For experimental conditions, hAD-MSCs and hBM-MSCs were seeded on a graphene surface and cultured for either 7 or 21 days. At each time point, cells were extracted for total RNA and reverse transcribed to cDNA before loading onto RT2 Profiler PCR Human Osteogenesis Array (Qiagen, Hilden, Germany). Gene expressions of both cell lines were compared either over time (day 7 as the control to day 21 as the treated group) or between both cell lines at each time point (hAD-MSCs to hBM-MSCs at day 7 or day 21). Results were analyzed using Qiagen Gene Globe software to determine the relative fold change in comparison to the control. Fold changes were considered statistically significant at p < 0.05. Results We examined expression of four transcriptional genes known to control stem cell fate: DLX5, RUNX2, SOX9, and SP7. In hAD-MSCs, RUNX2 was up-regulated while SOX9 and SP7 were down-regulated, suggesting RUNX2 to be a master regulator in hAD-MSCs. In contrast, all genes were down-regulated in hBM-MSCs. However, when hBM-MSCs were compared to hAD-MSCs, all genes were up-regulated at both time points. Interestingly, the fold changes at day 7 were more robust compared to that observed at day 21, suggesting transcriptional regulation of osteogenesis in hBM-MSCs occurs at an earlier time point. Secondly, we examined common markers of osteogenesis including ALPL, BGLAP, PHEX, and SPP1. In hAD-MSCs, ALPL, BGLAP, and PHEX were upregulated, while SPP1 was down-regulated.In hBM-MSCs, ALP, PHEX, and SPP1 were up-regulated, overall suggesting osteo-genetic signaling by graphene occurs in both cell types. Conclusion hAD-MSCs and hBM-MSCs cultured on graphene showed significant changes in osteogenic gene expressions. These cells were cultured in absence of any chemical inducers, suggesting graphene nanoparticles alone commits osteo-genetic signaling of MSCs. We also observed that osteogenesis of hBM-MSCs may occur at an earlier time point than hAD-MSCs. Lastly, we identified DLX5 as a novel osteogenic regulator supported by graphene. Overall, we show that MSCs isolated from two independent tissue sources spontaneously express osteogenic genes in the presence of graphene.

A low-temperature-printed hierarchical porous sponge-like scaffold that promotes cell-material interaction and modulates paracrine activity of MSCs for vascularized bone regeneration

Mesenchymal stem cells (MSCs) secrete paracrine trophic factors that are beneficial for tissue regeneration. In this study, a sponge-like scaffold with hierarchical and interconnected pores was developed using low-temperature deposition modeling (LDM) printing. Its effects on the cellular behavior, especially on the paracrine secretion patterns of MSCs, were comprehensively investigated. We found that compared with the scaffolds printed via the fused deposition modeling (FDM) technique, the LDM-printed sponges enhanced the adhesion, retention, survival, and ingrowth of MSCs and promoted cell-material interactions. Moreover, the paracrine functions of the cultured MSCs on the LDM-printed sponges were improved, with significant secretion of upregulated immunomodulatory, angiogenic, and osteogenic factors. MSCs on the LDM-printed sponges exert beneficial paracrine effects on multiple regenerative processes, including macrophage polarization, tube formation, and osteogenesis, verifying the enhanced immunomodulatory, angiogenic, and osteogenic potential. Further protein function assays indicated that focal adhesion kinase (FAK), downstream AKT, and yes-associated-protein (YAP) signaling might participate in the required mechanotransductive pathways, through which the hierarchical porous structures stimulated the paracrine effects of MSCs. In a rat distal femoral defect model, the MSC-laden LDM-printed sponges significantly promoted vascularized bone regeneration. The results of the present study demonstrate that the hierarchical porous biomimetic sponges prepared via LDM printing have potential applications in tissue engineering based on their cell-material interaction promotion and MSC paracrine function modulation effects. Furthermore, our findings suggest that the optimization of biomaterial properties to direct the paracrine signaling of MSCs would enhance tissue regeneration.

COX-2 promotes the osteogenic potential of BMP9 through TGF-β1/p38 signaling in mesenchymal stem cells

This study investigated the effects of transforming growth factor-β1 (TGF-β1) and cyclooxygenase-2 (COX-2) on bone morphogenetic protein 9 (BMP9) in mesenchymal stem cells (MSCs). We found that BMP9 increased mRNA levels of TGF-β1 and COX-2 in C3H10T1/2 cells. BMP9-induced osteogenic markers were enhanced by TGF-β1 and reduced by TGF-βRI-specific inhibitor LY364947. BMP9 increased level of p-Smad2/3, which were either enhanced or reduced by COX-2 and its inhibitor NS398. BMP9-induced osteogenic markers were decreased by NS398 and it was partially reversed by TGF-β1. COX-2 increased BMP9-induced osteogenic marker levels, which almost abolished by LY364947. BMP9-induced bone formation was enhanced by TGF-β1 but reduced by silencing TGF-β1 or COX-2. BMP9’s osteogenic ability was inhibited by silencing COX-2 but partially reversed by TGF-β1. TGF-β1 and COX-2 enhanced activation of p38 signaling, which was induced by BMP9 and reduced by LY364947. The ability of TGF-β1 to increase the BMP9-induced osteogenic markers was reduced by p38-specific inhibitor, while BMP9-induced TGF-β1 expression was reduced by NS398, but enhanced by COX-2. Furthermore, CREB interacted with Smad1/5/8 to regulate TGF-β1 expression in MSCs. These findings suggest that COX-2 overexpression leads to increase BMP9’s osteogenic ability, resulting from TGF-β1 upregulation which then activates p38 signaling in MSCs.

Primary Cilia as a Biomarker in Mesenchymal Stem Cells Senescence: Influencing Osteoblastic Differentiation Potency Associated with Hedgehog Signaling Regulation.

Bone tissue engineering-based therapy for bone lesions requires the expansion of seeding cells, such as autologous mesenchymal stem cells (MSCs). A major obstacle to this process is the loss of the phenotype and differentiation capacity of MSCs subjected to passage. Recent studies have suggested that primary cilia, primordial organelles that transduce multiple signals, particularly hedgehog signals, play a role in senescence. Therefore, we explored the relationships among senescence, primary cilia, and hedgehog signaling in MSCs. Ageing of MSCs by expansion in vitro was accompanied by increased cell doubling time. The osteogenic capacity of aged MSCs at passage 4 was compromised compared to that of primary cells. P4 MSCs exhibited reductions in the frequency and length of primary cilia associated with decreased intensity of Arl13b staining on cilia. Senescence also resulted in downregulation of the expression of hedgehog components and CDKN2A. Suppression of ciliogenesis reduced the gene expression of both Gli1, a key molecule in the hedgehog signaling pathway and ALP, a marker of osteoblastic differentiation. This study demonstrated that the senescence of MSCs induced the loss of osteoblastic differentiation potency and inactivated hedgehog signaling associated with attenuated ciliogenesis, indicating that primary cilia play a mediating role in and are biomarkers of MSC senescence; thus, future antisenescence strategies involving manipulation of primary cilia could be developed.

Impact of Alcohol on Bone Health, Homeostasis and Fracture repair.

Alcohol use continues to rise globally. We review the current literature on the effect of alcohol on bone health, homeostasis and fracture repair to highlight what has been learned in people and animal models of alcohol consumption. Recently, forkhead box O (FoxO) has been found to be upregulated and activated in mesenchymal stem cells (MSC) exposed to alcohol. FoxO has also been found to modulate Wnt/β-catenin signaling, which is necessary for MSC differentiation. Recent evidence suggests alcohol activates FoxO signaling, which may be dysregulating Wnt/β-catenin signaling in MSCs cultured in alcohol. This review highlights the negative health effects learned from people and chronic and episodic binge alcohol consumption animal models. Studies using chronic alcohol exposure or alcohol exposure then bone fracture repair model have explored several different cellular and molecular signaling pathways important for bone homeostasis and fracture repair, and offer potential for future experiments to explore additional signaling pathways that may be dysregulated by alcohol exposure.

Cytochrome P4501B1 in bone marrow is co-expressed with key markers of mesenchymal stem cells. BMS2 cell line models PAH disruption of bone marrow niche development functions

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that are metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by cytochrome P450 1B1 (CYP1B1). This metabolism occurs in bone marrow (BM) mesenchymal stem cells (MSC), which sustain hematopoietic stem and progenitor cells (HSPC). In BM, CYP1B1-mediated metabolism of 7, 12-dimethylbenz[a]anthracene (DMBA) suppresses HSPC colony formation within 6 h, whereas benzo(a)pyrene (BP) generates protective cytokines. MSC, enriched from adherent BM cells, yielded the bone marrow stromal, BMS2, cell line. These cells express elevated basal CYP1B1 that scarcely responds to Ah receptor (AhR) inducers. BMS2 cells exhibit extensive transcriptome overlap with leptin receptor positive mesenchymal stem cells (Lepr+ MSC) that control the hematopoietic niche. The overlap includes CYP1B1 and the expression of HSPC regulatory factors (Ebf3, Cxcl12, Kitl, Csf1 and Gas6). MSC are large, adherent fibroblasts that sequester small HSPC and macrophage in the BM niche (Graphic abstract). High basal CYP1B1 expression in BMS2 cells derives from interactions between the Ah-receptor enhancer and proximal promoter SP1 complexes, boosted by autocrine signaling. PAH effects on BMS2 cells model Lepr+MSC niche activity. CYP1B1 metabolizes DMBA to PAHDE, producing p53-mediated mRNA increases, long after the in vivo HSPC suppression. Faster, direct p53 effects, favored by stem cells, remain possible PAHDE targets. However, HSPC regulatory factors remained unresponsive. BP is less toxic in BMS2 cells, but, in BM, CYP1A1 metabolism stimulates macrophage cytokines (Il1b > Tnfa> Ifng) within 6 h. Although absent from BMS2 and Lepr+MSC, their receptors are highly expressed. The impact of this cytokine signaling in MSC remains to be determined.