Ceramide kinase regulates the migration of bone marrow-derived mesenchymal stem cells

Endogenous bone marrow-derived mesenchymal stem cells (BM-MSCs) are mobilized into peripheral blood and injured tissues by various growth factors and cytokines that are expressed in the injured tissues, such as substance P (SP), stromal cell derived factor-1 (SDF-1), and transforming growth factor-beta (TGF-β). Extracellular bioactive lipid metabolites such as ceramide-1-phosphate and sphingosine-1-phosphate also modulate BM-MSC migration as SP, SDF-1, and TGF-β. However, the roles of intrinsic lipid kinases of BM-MSCs in the stem cell migration are unclear. Here, we demonstrated that ceramide kinase mediates the chemotactic migration of BM-MSCs in response to SP, SDF-1, or TGF-β. Furthermore, a specific inhibitor of ceramide kinase inhibited TGF-β-induced migration of BM-MSCs and N-cadherin that is necessary for BM-MSCs migration in response to TGF-β. Therefore, these results suggest that the intracellular ceramide kinase is required for the BM-MSCs migration and the roles of the intrinsic ceramide kinase in the migration are associated with N-cadherin regulation.     

Differentiation into neurons of rat bone marrow-derived mesenchymal stem cells

Purpose

It has been reported that mesenchymal stem cells (MSCs) can differentiate into neurons as an effect of adding extraneous factors, such as β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanisole. However, many of these compounds could harm MSCs and the human body, which restricts their application. We examined whether MSCs could differentiate into neuron-like cells under the influence of natural growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1, and neurotrophin 3 (NT-3).

Methods

MSCs were collected from rat bone marrow using the plastic adherent selection method, and induced in culture media to which was added different combinations of EGF, bFGF, IGF-1 and NT-3. The shape of the induced cells was observed daily and the differentiated cells were characterized by immunocytochemistry with neural-specific markers.

Result

With bFGF and NT-3 in the medium, the induced cells became slim, gradually developing protruding processes, with parts of them forming net- or ring-like structures. Cells with processes showed expression of microtubule-associated protein 2 (MAP2) and nestin (NES), which was enhanced when bFGF and NT-3 were added in combination. However, with IGF-1 added to the medium, there was no evidence of neurite-like processes or any net- or ring-like structures; the MSCs retained their round or slim shape.

Conclusion

Using natural cytokines in vitro, MSCs successfully differentiated into neuron-like cells. Our study confirms that bFGF and NT-3 exerts a neural-induction effect on the differentiation of MSCs, but that IGF has a rather negative effect on this process.

     

Cell-autonomous heparanase modulates self-renewal and migration in bone marrow-derived mesenchymal stem cells

Background

Stem cell-fate is highly regulated by stem cell niche, which is composed of a distinct microenvironment, including neighboring cells, signals and extracellular matrix. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells and are potentially applicable in wide variety of pathological conditions. However, the niche microenvironment for BM-MSCs maintenance has not been clearly characterized. Accumulating evidence indicated that heparan sulfate glycosaminoglycans (HS-GAGs) modulate the self-renewal and differentiation of BM-MSCs, while overexpression of heparanase (HPSE1) resulted in the change of histological profile of bone marrow. Here, we inhibited the enzymatic activity of cell-autonomous HPSE1 in BM-MSCs to clarify the physiological role of HPSE1 in BM-MSCs.

Results

Isolated mouse BM-MSCs express HPSE1 as indicated by the existence of its mRNA and protein, which includes latent form and enzymatically active HPSE1. During in vitro osteo-differentiations, although the expression levels of Hpse1 fluctuated, enzymatic inhibition did not affect osteogenic differentiation, which might due to increased expression level of matrix metalloproteinase 9 (Mmp9). However, cell proliferation and colony formation efficiency were decreased when HPSE1 was enzymatically inhibited. HPSE1 inhibition potentiated SDF-1/CXCR4 signaling axis and in turn augmented the migratory/anchoring behavior of BM-MSCs. We further demonstrated that inhibition of HPSE1 decreased the accumulation of acetylation marks on histone H4 lysine residues suggesting that HPSE1 also modulates the chromatin remodeling.

Conclusions

Our findings indicated cell-autonomous HPSE1 modulates clonogenicity, proliferative potential and migration of BM-MSCs and suggested the HS-GAGs may contribute to the niche microenvironment of BM-MSCs.     

Sonic hedgehog enhances the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells

MSCs (mesenchymal stem cells) may be promising seed cells for tissue regeneration because of their self-renewal and multi-differentiation potential. Shh (sonic hedgehog) is involved in the skeletal formation during embryo development and skeletal regeneration. However, how Shh regulates the biological characteristics of BM-MSCs (bone marrow-derived MSCs) is poorly understood. We have investigated the effect of rShh-N (recombinant N-terminal Shh) on the proliferation and osteogenic differentiation of rBM-MSCs (rat BM-MSCs) in vitro. rBM-MSCs were treated with rShh-N at concentrations up to 200 ng/ml. Proliferation and colony-forming ability of rBM-MSCs were increased in a dose-dependent manner. rShh-N increased the ratio of cells in S and G2/M phase, as well as the number of Ki-67+ cells. In addition, ALP (alkaline phosphatase) activity and matrix mineralization were enhanced by 200 ng/ml rShh-N. Real-time PCR showed that rShh-N (200 ng/ml) up-regulated the expression of genes encoding Cbfa-1 (core-binding factor α1), osteocalcin, ALP and collagen type I in rBM-MSCs. This information reveals some potential of rShh-N in the therapeutics of bone-related diseases.     

Loss of Lgr4 inhibits differentiation,migration and apoptosis,and promotes proliferation in bone mesenchymal stem cells

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine-rich repeat-containing G protein-coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β-geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4^+/– mice at E16.5 by β-gal staining, and Lgr4-deficiency promoted BMSC proliferation (16 ± 4 in wild-type [WT] and 28 ± 2 in Lgr4^−/−) using colony forming units-fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4^−/−) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4-regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4-inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs-delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm-derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy.     

Effects of mechanical stimulation on the proliferation of bone marrow-derived human mesenchymal stem cells

To support and enhance thein vitro growth and activity of mesenchymal stem cells (MSCs), the cell culture medium may be supplemented with various proteins and factors to mimic the physiological environment in which the cells optimally proliferate and differentiate. In this study, the effects of mechanical factors on cellular metabolic responses were investigated experimentally using a bioreactor. The effects of various chemical factors, such as growth factors, cytokines, and hormones, were also investigated. Based on previous reports demonstrating the important roles of mechanical factors in the growth and activity of MSCs, we sought to evaluate the effects of mechanical stimuli on the proliferation of bone marrow-derived MSCs using a cell training bioreactor that imposed cyclic mechanical stretch, with parameters of 240 min/day, 0.03 Hz, and 5–15% strain. The application of cyclic stretch (5–15% strain) to the MSCs enhanced their proliferation during the early stage (3 days), but not the late stage (14 days), of batch culture. Mechanical stretch did not increase the release of lactate dehydrogenase (LDH) from the MSCs during culture. Appropriate levels of mechanical stretch (5–10% strain) increased collagen synthesis, but did not alter MSC surface antigen expression. It is thought that the appropriate level of mechanical stretch was able to serve as a potent positive modulator of MSC proliferation during the initial stages of culture.     

A critical role of the KCa3.1 channel in mechanical stretch-induced proliferation of rat bone marrow-derived mesenchymal stem cells

Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca²⁺-activated K⁺ channel, K_Ca3.1, is critically engaged in MSC proliferation but its role in mechanical regulation of MSC proliferation remains unknown. Here, we examined the K_Ca3.1 channel expression and its role in rat bone marrow-derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up-regulated the K_Ca3.1 channel expression and pharmacological or genetic inhibition of the K_Ca3.1 channel strongly suppressed stretch-induced increase in cell proliferation and cell cycle progression. These results support that the K_Ca3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation.     

Lipocalin 2 decreases senescence of bone marrow-derived mesenchymal stem cells under sub-lethal doses of oxidative stress

The regenerative potential of mesenchymal stem cells (MSCs) is impaired by cellular senescence, a multi factorial process that has various functions. However, pathways and molecules involved in senescence have not been fully identified. Lipocalin 2 (Lcn2) has been the subject of intensive research, due to its contribution to many physiological and pathophysiological conditions. The implication of Lcn2 has been reported in many conditions where senescence also occurs. In the present study, we evaluated the role of Lcn2 in the occurrence of senescence in human bone marrow-derived mesenchymal stem cells (hB-MSCs) under oxidative conditions. When hB-MSCs were genetically engineered to over-express Lcn2 (MSC-Lcn2) and exposed to H₂O₂, the proliferation rate of the cells increased. However, the number of colonies and the number of cells that made up each colony in both MSC-V and MSC-Lcn2 cells decreased compared to those cultivated under normal conditions. Our results revealed that over-expression of recombinant Lcn2 in hB-MSCs decreases senescence induced by H₂O₂ treatment. Senescent cells were observed in aged hB-MSCs; however, no alteration in the expression level of Lcn2 was detected compared to earlier passages. Finally, a higher amount of Lcn2 protein was detected in the plasma of the elderly than in young people. Our findings suggest that Lcn2 might restore the health and regeneration potential of MSCs by decreasing senescence.     

In vitro proliferation and differentiation potential of bone marrow-derived mesenchymal stem cells from ovariectomized rats

Bone marrow-derived mesenchymal stem cells (BMMSCs) from the patients suffering from age-related osteoporosis were found to have numerous degeneration, such as decreased growth rate, impaired capacity of differentiating into local tissue, and repressed telomerase activity. However, it is not clear whether post-menopausal osteoporotic bone is either subject to such decline in cellular function. In the present study, bone marrow cells were harvested from ovariectomized (OVX) and Sham rats and cultured in vitro at 3 months post-surgery. MTT assay indicated that the proliferation potential of ^OVXBMMSCs was always higher than that of ^ShamBMMSCs, no matter cultured in basic, osteoblastic or adipogenic medium. Alkaline phosphatase activity assay, Alizarin red S staining, Oil red O staining and real-time RT-PCR analysis further demonstrated that bilateral ovariectomization positively influenced the osteoblastic and adipogenic differentiation potential of BMMSCs, this action may be partly mediated through up-regulation of osteoblastic special markers core binding factor a1, collagen type I and low-density lipoprotein receptor-related protein 5, as well as adipogenic special markers peroxisome proliferators activated receptor gamma, CCAAT/enhancer binding protein alpha and adipocyte lipid-binding protein 2. These results may hold great promise for using post-menopausal osteoporotic bone as an attractive autologous marrow source for tissue engineering and cell-based therapies.     

Mitophagy promotes the stemness of bone marrow-derived mesenchymal stem cells

Previous studies demonstrated that mitochondrial fission arguments the stemness of bone marrow-derived mesenchymal stem cells (BMSCs). Because mitophagy is critical in removing damaged or surplus mitochondrial fragments and maintaining mitochondrial integrity, the present study was undertaken to test the hypothesis that mitophagy is involved in mitochondrial fission-enhanced stemness of BMSCs. Primary cultures of rat BMSCs were treated with tyrphostin A9 (TA9, a potent inducer of mitochondrial fission) to increase mitochondrial fission, which was accompanied by enhanced mitophagy as defined by increased co-staining of MitoTracker Green for mitochondria and LysoTracker Deep Red for lysosomes, as well as the increased co-localization of autophagy markers (LC3B, P62) and mitochondrial marker (Tom20). A mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was used to promote mitophagy, which was confirmed by an increased co-localization of mitochondrial and lysosome biomarkers. The argumentation of mitophagy was associated with enhanced stemness of BMSCs as defined by increased expression of stemness markers Oct4 and Sox2, and enhanced induction of BMSCs to adipocytes or osteocytes. Conversely, transfection of BMSCs with siRNA targeting mitophagy-essential genes Pink1/Prkn led to diminished stemness of the stem cells, as defined by depressed stemness markers. Importantly, concomitant promotion of mitochondrial fission and inhibition of mitophagy suppressed the stemness of BMSCs. These results thus demonstrate that mitophagy is critically involved in mitochondrial fission promotion of the stemness of BMSCs.     

microRNA-16-5p-containing exosomes derived from bone marrow-derived mesenchymal stem cells inhibit proliferation,migration, and invasion,while promoting apoptosis of colorectal cancer cells by downregulating ITGA2

Colorectal cancer (CRC) is a form of cancer developing from either the colon or rectum. Nowadays, research supports the functionality of exosome expressing microRNAs (miRNAs) as potential biomarker for various cancers including CRC. This study was performed with the intent of investigating the roles of both bone marrow-derived mesenchymal stem cells (BMSCs) and exosomal miR-16-5p in CRC by regulating integrin α2 (ITGA2). A microarray-based analysis was conducted to screen the CRC-associated differentially expressed genes (DEGs) as well as potential regulatory miRNAs. Next, the role of miR-16-5p in terms of its progression in association with CRC was determined. Subsequently, CRC cells were exposed to exosomes secreted by BMSCs transfected with miR-16-5p, isolated and cocultured with CRC cells in an attempt to identify the role of exosomes. Effects of BMSCs-derived exosomes overexpressing miR-16-5p on biological functions of CRC cells and tumorigenicity were all subsequently detected. Effects of miR-16-5p treated with CRC cells in regard to CRC in vivo were also measured. ITGA2 was overexpressed, while miR-16-5p was poorly expressed in CRC cells and miR-16-5p targeted ITGA2. The in vitro experiments revealed that the BMSCs-derived exosomes overexpressing miR-16-5p inhibited proliferation, migration, and invasion, while simultaneously stimulating the apoptosis of the CRC cells via downregulation of ITGA2. Furthermore, the results of in vivo experiments confirmed that the BMSCs-derived exosomes overexpressing miR-16-5p repressed the tumor growth of CRC. Collectively, BMSCs-derived exosomes overexpressing miR-16-5p restricted the progression of CRC by downregulating ITGA2.     

Hypoxic culture enhances the expansion of rat bone marrow-derived mesenchymal stem cells via the regulatory pathways of cell division and apoptosis

This study aimed to examine the proliferative behavior and molecular mechanisms of rat bone marrow-derived MSCs (rBMSCs) cultured under three different oxygen concentrations. Passaged rBMSCs exhibited significantly greater proliferation rates at 1% O₂ and 5% O₂ than those at 18% O₂ and the cells exposed to 1% O₂ showed the highest proliferative potential, which was evidenced by the growth curves, colony-forming efficiencies, and CCK-8 absorbance values. The rBMSCs grown under hypoxic culture conditions (1% O₂ and 5% O₂) had the increased percentage of cells in S?+?G₂/M-phase and the decreased apoptotic index, compared with normoxia (18% O₂). It was revealed for the first time that there were more phosphohistone H3 (PHH3)-positive cells and higher expressions of proliferating cell nuclear antigen (PCNA) in the hypoxic cultures of rBMSCs than in the normoxic culture. Hypoxia upregulated the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic proteins Bax and the cleaved caspase-3 in cultured rBMSCs. The levels of hypoxia-inducible factor-1α (HIF-1α) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) were increased in the hypoxic-cultured rBMSCs. Nevertheless, no significant difference was observed in p53 level of rBMSCs between different oxygen concentrations. In conclusion, the hypoxia exerts a promoting effect on the in vitro expansion of rBMSCs via several signaling and molecular pathways involved in the control of cell cycle and apoptosis.     

Glycosaminoglycans mimetics potentiate the clonogenicity, proliferation, migration and differentiation properties of rat mesenchymal stem cells

Successful use of stem cell-based therapeutic products is conditioned by transplantation of optimized cells in permissive microenvironment. Mesenchymal stem cell (MSC) fates are tightly regulated by humoral factors, cellular interactions and extracellular matrix (ECM) components, such as glycosaminoglycans (GAG), which are complex polysaccharides with structural heterogeneity. During osteogenesis, a temporally controlled expression of particular GAG species is required to interact with specific growth promoting and differentiating factors to regulate their biological activities. As a comparative tool to study natural GAG, we used structurally and functionally related synthetic GAG mimetics. One of these compounds [OTR₄₁₂₀] was previously shown to stimulate bone repair in rat models. Here, we demonstrate that structurally distinct GAG mimetics stimulate differentially clonogenicity, proliferation, migration and osteogenic phenotype of MSC in vitro, according to their specific chemical signature, underlying the role of sulfate and acetyl groups in specific interactions with heparin binding factors (HBF). These effects are dependent on FGF-2 interactions since they are inhibited by a FGF receptor 1 signaling pathway blocker. These data suggest that the in vivo [OTR₄₁₂₀] bone regenerative effect could be due to its ability to induce MSC migration and osteogenic differentiation. To conclude, we provide evidences showing that GAG mimetics may have great interest for bone regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of MSC.     

Effect of bone sialoprotein on proliferation and osteodifferentiation of human bone marrow-derived mesenchymal stem cells in vitro

We performed this study to investigate the effects of recombinant human bone sialoprotein (BSP) on the proliferation and osteodifferentiation of human BMSCs(hBMSCs). The hBMSC cultures were divided into 4 groups: control group, 10⁻¹⁰ M BSP group (BSP group), osteogenic medium group (10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 mg/L ascorbic acid, OM group) and BSP + OM group (OM plus10⁻¹⁰ M BSP). Compared with the control group, cell growth of the other three groups slowed down, while fluorescence at the G₀/G₁ phase increased. After 28 days, in the OM group and the BSP + OM group, the proportion of STRO-1-positive cells decreased by 22.7% and 38.4% and ALP activity increased by 50% and 71.43%, respectively. CD271 mRNA expression decreased while Cbfa1, osteocalcin and osterix mRNA levels increased in the OM and BSP + OM groups, and the mRNA level change was greater in the BSP + OM group. After 28 days, the number of nodules in the BSP + OM group was 112.5% more than that in the OM group, but nodules did not formed in the control or BSP group. We conclude that BSP is capable of inhibiting hBMSCs proliferation and enhancing their osteogenic differentiation and mineralization in the presence of OM.     

BMP-2, VEGF and bFGF synergistically promote the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) were treated with bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) dose-dependently and time-dependently. Together they caused a strong synergistic effect on the osteogenic differentiation of MSCs, with lower concentrations of each factor being enough to show the synergistic promotion (50 ng BMP-2/ml, 1 ng VEGF/ml and 10 ng bFGF/ml). When both VEGF and bFGF were added in the early proliferating stage (the first 7 days) and BMP-2 was added in the late differentiation stage (the last 7 days), osteogenic differentiation of MSCs could be enhanced more effectively.