Migration Of rMSCs Research Articles

Activation of BK Channel Contributes to PL-Induced Mesenchymal Stem Cell Migration.

Due to their capacity to proliferate, migrate, and differentiate, mesenchymal stem cells (MSCs) are considered to be good candidates for regenerative medicine applications. The mechanisms underlying proliferation and differentiation of MSCs have been studied. However, much less is known about the mechanisms regulating the migration of MSCs. Platelet lysate (PL), a supplement used to promote cell expansion, has been shown to promote MSCs migration; however, the underlying mechanism are unknown. Here, by using adipose-derived rat MSCs (rMSCs) and the scratch assay in the absence and presence of various BK channels modulators, we evaluated the role of BK channels in mediating the PL-stimulated migration of rMSCs. We found that 5% PL increased rMSCs migration, and this effect was blocked by the addition of the BK channel selective antagonist Iberiotoxin (IBTX). In the absence of PL, the BK channel agonist NS1619, stimulated rMSCs migration to similar level as 5% PL. Addition of both NS1619 and 5% PL resulted in an increase in rMSCs migration, that was higher than when either one was added individually. From whole-cell recordings, it was found that the addition of 5% PL increased the magnitude of BK current density. By using Western blot and flow cytometry, it was found that PL did not affect the expression of BK channels. Together, our results indicate that as shown in other cell types, activation of BK channels by themselves also promote rMSC migration, and show that activation of BK channels contribute to the observed PL-induced increase in migration of rMSC.

LncRNA-TINCR Functions as a Competitive Endogenous RNA to Regulate the Migration of Mesenchymal Stem Cells by Sponging miR-761.

Mounting evidences have indicated that terminal differentiation-induced lncRNA (TINCR) contributes to various cellular processes, such as proliferation, apoptosis, autophagy, migration, invasion, and metastasis. However, the function of TINCR in regulating migration of MSCs is largely unknown. In this study, the effects of TINCR on the migration of rat MSCs from the bone marrow were studied by Transwell assays and wound healing assays. Our results suggested that TINCR positively regulated migration of rMSCs. miR-761 mimics suppressed rMSC migration, whereas miR-761 inhibitor promoted migration. Target prediction analysis tools and dual-luciferase reporter gene assay identified Wnt2 as a direct target of miR-761. miR-761 could inhibit the expression of Wnt2. Further, the investigation about the function of TINCR in miR-761-induced migration of rMSCs was completed. These results demonstrated that TINCR took part in the regulation of miR-761-induced migration in rMSCs through the regulation of Wnt2 and its Wnt2 signaling pathway. Taken together, our results demonstrate that lncRNA-TINCR functions as a competitive endogenous RNA (ceRNA) to regulate the migration of rMSCs by sponging miR-761 which modulates the role of Wnt2. These findings provide evidence that lncRNA-TINCR has a chance to serve as a potential target for enhancing MSC homing through the miR-761/Wnt2 signaling pathway.

MiR-539-5p negatively regulates migration of rMSCs induced by Bushen Huoxue decoction through targeting Wnt5a.

Bone fractures are very common, and above 5% of the fractures are impaired, leading to nonunions and severe disablilities. The traditional Chinese medicine Bushen Huoxue decoction (BHD) has been used to treat fracture in China. Our previous report has found that BHD promotes migration of rat mesenchymal stem cells (rMSCs) by activating Wnt5a signaling pathway. However, whether and how miRNAs are involved in modulating rMSCs migration induced by BHD has not been explored. In the present study, miRNA microarray analysis and further validation by real-time quantitative RT-PCR revealed that miR-539-5p was down-regulated in BHD-induced rMSCs. Transfection of miR-539-5p mimics suppressed rMSCs migration while the miR-539-5p inhibitor promoted rMSCs migration. Our results suggested that miR-539-5p was a negative regulator of migration of rMSCs induced by BHD. Target prediction analysis tools and Dual-luciferase reporter gene assay identified Wnt5a as a direct target of miR-539-5p. MiR-539-5p inhibited the expression of the Wnt5a and its downstream signaling molecules including JNK, PKC and CaMKII, which played a critical role in regulating migration of rMSCs. Taken together, our results demonstrate that miR-539-5p negatively regulates migration of rMSCs induced by BHD through targeting Wnt5a. These findings provide evidence that miR-539-5p should be considered as an important candidate target for the development of preventive or therapeutic approaches against bone nonunions.

MiR-124 suppresses the chemotactic migration of rat mesenchymal stem cells toward HGF by downregulating Wnt/β-catenin signaling

Mesenchymal stem cells (MSCs) exhibit the potential to repair a wide variety of injured adult tissues. The migration capability of MSCs is an important determinant of the efficiency of MSC transplant therapy. MicroRNAs (miRNAs) are increasingly implicated in regulating the migration of MSCs. Herein, we show that the expression of miR-124 was downregulated in rat MSCs (rMSCs) treated with hepatocyte growth factor (HGF). Overexpression of miR-124 significantly reduced the chemotactic migration of rMSCs toward HGF, while inhibition of endogenous miR-124 promoted the chemotactic migration. A further study revealed that miR-124 directly targeted FZD4 and LRP6, which encode a receptor and co-receptor of the Wnt/β-catenin signaling pathway, respectively, thus reducing the activity of this signaling. Consistently, activation of the Wnt/β-catenin signaling pathway by LiCl and ΔN89β-catenin rescued the inhibitory effect of miR-124 on the chemotactic migration of rMSCs toward HGF, while inhibition of Wnt/β-catenin signaling by FH535 abrogated the enhanced chemotactic response achieved by the miR-124 inhibitor. Collectively, our study demonstrates that miR-124 downregulates Wnt/β-catenin signaling via targeting FZD4 and LRP6 and thus suppresses the chemotactic migration of rMSCs toward HGF.

Mechano growth factor E peptide promotes rat bone marrow-derived mesenchymal stem cell migration through CXCR4-ERK1/2

Mechano growth factor (MGF) is a splicing variant of insulin-like growth factor 1 (IGF-1). The unique C-terminal E domain of MGF (MGF-E) makes it distinct from the other variants of IGF-1. Our previous work demonstrated that MGF-25E induces the migration of rat bone marrow-derived mesenchymal stem cells (rMSCs) by altering their mechanical properties, which is accompanied by the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. However, the relationship between ERK1/2 activation and the change in mechanical properties has not been illustrated. In the present study, we determined that MGF-25E induced the migration of rMSCs by modulating CXCR4 to activate the ERK1/2 pathway. The analysis of the Young’s modulus and F-actin remodeling indicated that MGF-25E increased the stiffness and the F-actin polymerization of rMSCs through the activation of the CXCR4-ERK1/2 pathway. For the first time, this study clarified the signaling pathway that regulates the mechanical properties of rMSCs and is responsible for MGF-25E-promoted migration.

Research on MMP-9 to promote mesenchymal stem cells migration through the blood-brain barrier

Objective To explore the role of MMP-9 in blood-brain barrier (BBB) disruption and to promote rMSCs migration.Methods To investigate the effect of MMP-9 on the permeability of BBB,an in vitro model of BBB was established and performed Transwell experiments.To observe the pathologic changes of the cerebral cortex,rat brains from Sham treated group,HIBD group,MMP-9 treated group and TIMP-1 (intervention) treated group at different time points (0.5,1,3,7,14 d) were prepared and stained with Hematoxylin-Eosin and quantified the brain water content.Permeability of BBB examined by EB values measurement.MMP-9 protein level of rat cortex was detected by Western Blot.The number of Brdu labeled rMSCs in the rat cerebral cortex of each group was quantified by Immunohistochemistry (IHC).Results Transwell experiments results showed that migration of rMSCs increased remarkably in hypoxic condition compared to that of normal control (P＜0.01).The number of rMSCs migrated in the MMP-9 treated group was much more than that of negative control group and TIMP-1 group (P＜0.01).The results of pathology showed that compared to HIBD group,brain water content and the permeability of the BBB were increased in MMP-9 treated group but reduced in TIMP-1 treated one.The expression of MMP-9 protein in MMP-9 treated group was reached the peak at 3 d point,which was higher than that of HIBD group and TIMP-1 group (P＜0.05,P＜0.01).The quantity of Brdu labeled rMSCs crossed BBB in MMP-9 treated group was extremely higher than that of HIBD group (P＜0.05,P＜0.01).Conclusions The expression level of MMp-9 protein was improved after hypoxic-ischemic.MMP-9 could facilitate the migration of rMSCs through vitro BBB and control the opening of BBB,which indicate that MMP-9 may facilitate the migration of rMSCs through BBB into brain. Key words: Mesenchymal stem cells; Matrix metalloproteinases-9; Hypoxia-ischemia; Blood-brain barrier

Conditioned Media from Mesenchymal Stem Cells Enhanced Bone Regeneration in Rat Calvarial Bone Defects

Tissue engineering has recently become available as a treatment procedure for bone augmentation. However, this procedure has several problems, such as high capital investment and expensive cell culture, complicated safety and quality management issues regarding cell handling, and patient problems with the invasive procedure of cell collection. Moreover, it was reported that stem cells secrete many growth factors and chemokines during their cultivation, which could affect cellular characteristics and behavior. This study investigated the effect of stem-cell-cultured conditioned media on bone regeneration. Cultured conditioned media from human bone marrow-derived mesenchymal stem cells (MSC-CM) enhanced the migration, proliferation, and expression of osteogenic marker genes, such as osteocalcin and Runx2, of rat MSCs (rMSCs) in vitro. MSC-CM includes cytokines such as insulin-like growth factor-1 and vascular endothelial growth factor. In vivo, a prepared bone defect of a rat calvarial model was implanted in five different rat groups using one of the following graft materials: human MSCs/agarose (MSCs), MSC-CM/agarose (MSC-CM), Dulbecco's modified Eagle's medium without serum [DMEM(-)]/agarose [DMEM(-)], PBS/agarose (PBS), and defect only (Defect). After 4 and 8 weeks, implant sections were evaluated using microcomputed tomography (micro-CT) and histological analysis. Micro-CT analysis indicated that the MSC-CM group had a greater area of newly regenerated bone compared with the other groups (p<0.05) and histological analysis at 8 weeks indicated that the newly regenerated bone bridge almost covered the defect. Interestingly, the effects of MSC-CM were stronger than those of the MSC group. In vivo imaging and immunohistochemical staining of transgenic rats expressing green fluorescent protein also showed that migration of rMSCs to the bone defect in the MSC-CM group was greater than in the other groups. These results demonstrated that MSC-CM can regenerate bone through mobilization of endogenous stem cells. The use of stem-cell-cultured conditioned media for bone regeneration is a unique concept that utilizes paracrine factors of stem cells without cell transplantation.

Research of HIF-1 Mediated SDF-1/CXCR4 Axis on the Directional Migration and Neural Differentiation of Mesenchymal Stem Cells

AbstractAbstract 4699 Introduction:Mesenchymal stem cells (MSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. At present, the mechanisms of targeted therapy of MSCs are known to relate with the hypoxia-inducible factor-1 (HIF-1) and its regulated biological axis stromal cell-derived factor-1/chemokine receptor 4 (SDF-1/CXCR4). SDF-1 and CXCR4 have a wide distribution in various cells and tissues, which plays an important role in the development of immune system, circulatory system and central nervous system. We thus inferred that SDF-1/CXCR4 may participate in the differentiation as well as the migration of stem cells. Our research aims to explore the effect of HIF-1α and its mediated SDF-1/CXCR4 axis on the directional migration and neural differentiation of MSCs, which may lead to a break in the efficiency and target distribution of MSCs therapy. Method:(1) Influence of hypoxia, CXCR4 antagonist (AMD3100) and SDF-1α on the proliferation of rat Mesenchymal Stem Cells (rMSCs): rMSCs were isolated from bone marrow of rats, and expanded in vitro. The growth feature of rMSCs exposed to hypoxia (PO2=1%) or normoxia was identified by growth curve, while the effect of AMD3100 (5ug/ml) and SDF-1α (10ng/ml and 100ng/ml) on the proliferation ability of rMSCs was detected by cell counting kit-8. (2) Effect of hypoxia on the expression of HIF-1α, CXCR4 and SDF-1α: Firstly, we used RT-PCR, western blotting and flow cytometry to detect the expression of HIF-1α and CXCR4 mRNA and protein levels in rMSCs which treated with hypoxia for 0h, 6h, 12h, 24h, 48h and 72h. Secondly, HIF-1α, SDF-1α mRNA and protein level in the hippocampus of rats which suffered hypoxia- ischemia for 1d, 3d, 5d, 7d, 14d and 21d were also detected by the same assays. (3) Research of HIF-1α and SDF-1/CXCR4 axis on the migration of rMSCs: We first detected the change of CXCR4 mRNA and protein levels in rMSCs treated with AMD3100 (5ug/ml) and SDF-1α (10ng/ml) by RT-PCR, western blotting and flow cytometry, and then studied SDF-1/CXCR4 axis on the migration of rMSCs using Transwell assay. (4) Effect of HIF-1α and SDF-1/CXCR4 axis on the differentiation of rMSCs: protein level of NSE and GFAP as well as positive rate of neural-induced rMSCs which have been pretreated with AMD3100 (5ug/ml) were detected by western blotting and immunocytochemistry. Results:Persistent hypoxia promoted the proliferation of rMSCs, while AMD3100 and SDF-1α at the concentration mentioned above had no effect. Compared to normal control, the protein expression of HIF-1α in rMSCs increased in hypoxic condition while the mRNA of HIF-1α did not change. Furthermore, the mRNA and protein level of CXCR4 both increased in rMSCs exposed to hypoxia for 6h and 12h, and the results confirmed by flow cytometry. We found HIF-1α mRNA was stably expressed in hippocampus, and increased significantly in hypoxia-ischemia brain damaged (HIBD) rats in a time dependent manner, which reached the peak on 7d. As expected, SDF-1α mRNA in hippocampus of HIBD rats was higher than that of normal control group, which reached the peak on 7d (P<0.01) and stably expressed till 21d, while the protein level is mainly in concordance. Moreover, CXCR4 mRNA was extremely up-regulated in rMSCs treated with SDF-1α (10ng/ml), however, in 5 ug/ml AMD3100 treated rMSCs, which decreased markedly (P<0.01), and the results were confirmed by western blotting and flow cytometry assays (P<0.05). Transwell assay manifested that SDF-1α had obvious chemotaxis to rMSCs. Protein level and positive cell number of NSE and GFAP were extremely down-regulated in rMSCs which pretreated with 5ug/ml AMD3100. Conclusion:Increased expression of HIF-1α led to the up-regulation of SDF-1/CXCR4 axis, and rMSCs displayed chemotaxis migration ascribed to the receptor-ligand interactions of SDF-1α and CXCR4, suggesting that HIF-1 and its mediated SDF-1/CXCR4 axis are of great significant on the directional migration of rMSCs. We also showed that CXCR4 antagonisation reduced the neural differentiation capabilities of rMSCs, thus suggested that SDF-1/CXCR4 axis may deeply involve in the neural differentiation of rMSCs. Disclosures:No relevant conflicts of interest to declare.

Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury.

Mesenchymal stem cells (MSCs), cultured ex vivo, recently were shown to be able to migrate into sites of brain injuries when transplanted systemically or locally, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. In this study, we examined the role of some chemokines and their receptors in the trafficking of rat MSCs (rMSCs) in a rat model of left hypoglossal nerve injury. rMSCs transplanted into the lateral ventricles of the rat brain migrated to the avulsed hypoglossal nucleus, where the expression of chemokines, stromal-cell-derived factor 1 (SDF-1), and fractalkine was observed to be increased. This increase temporally paralleled the migration of rMSCs into the avulsed nucleus at 1 and 2 weeks after operation. It has been found that rMSCs express CXCR4 and CX3CR1, the respective receptors for SDF-1 and fractalkine, and other chemokine receptors, CCR2 and CCR5. Furthermore, in vitro analysis revealed that recombinant human SDF-1 alpha (rhSDF-1alpha) and recombinant rat fractalkine (rrfractalkine) induced the migration of rMSCs in a G-protein-dependent manner. Intracerebral injection of rhSDF-1alpha has also been shown to stimulate the homing of transplanted rMSCs to the site of injection in the brain. These data suggest that the interactions of fractalkine-CX3CR1 and SDF-1-CXCR4 could partially mediate the trafficking of transplanted rMSCs. This study provides an important insight into the understanding of the mechanisms governing the trafficking of transplanted rMSCs and also significantly expands the potential role of MSCs in cell therapy for brain injuries and diseases.