Proliferation Of Bone Marrow Mesenchymal Stem Cells Research Articles

Synthesis and Evaluation of BMMSC-seeded BMP-6/nHAG/GMS Scaffolds for Bone Regeneration

Bioactive scaffolding materials and efficient osteoinductive factors are key factors for bone tissue engineering. The present study aimed to mimic the natural bone repair process using an osteoinductive bone morphogenetic protein (BMP)-6-loaded nano-hydroxyapatite (nHA)/gelatin (Gel)/gelatin microsphere (GMS) scaffold pre-seeded with bone marrow mesenchymal stem cells (BMMSCs). BMP-6-loaded GMSs were prepared by cross-linking and BMP-6/nHAG/GMS scaffolds were fabricated by a combination of blending and freeze-drying techniques. Scanning electron microscopy, confocal laser scanning microscopy, and CCK-8 assays were carried out to determine the biocompatibility of the composite scaffolds in vitro. Alkaline phosphatase (ALP) activity was measured to evaluate the osteoinductivity of the composite scaffolds. For in vivo examination, critical-sized calvarial bone defects in Sprague-Dawley rats were randomly implanted with BMMSC/nHAG/GMS and BMMSC/BMP-6/nHAG/GMS scaffolds, and compared with a control group with untreated empty defects. The BMP-6-loaded scaffolds showed cytocompatibility by favoring BMMSC attachment, proliferation, and osteogenic differentiation. In radiological and histological analyses, the BMMSC-seeded scaffolds, especially the BMMSC-seeded BMP-6/nHAG/GMS scaffolds, significantly accelerated new bone formation. It is concluded that the BMP-6/nHAG/GMS scaffold possesses excellent biocompatibility and good osteogenic induction activity in vitro and in vivo, and could be an ideal bioactive substitute for bone tissue engineering.

Preparation of bioactive denatured acellular dermal matrix from burn mice riched in mice bone marrow mesenchymal stem cells

Objective: To investigate the preparation of bioactive denatured acellular dermal matrix (DADM) from burn mice riched in mice bone marrow mesenchymal stem cells. Methods: Twelve BALB/c mice were collected and 20% total body surface area scalds (hereinafter referred to as burns) with deep partial thickness were inflicted on the back skin of each mouse. After removing epidermis, the burned skin were collected and divided into Triton X-100 group and elhylene diamine tetraacetic acid (EDTA) group according to the random number table, with 15 samples in each group. Samples in Triton X-100 group and EDTA group were respectively placed in mixture of 2.5 g/L Triton X-100 and 2.5 g/L trypsin solution and mixture of 0.2 g/L EDTA and 2.5 g/L trypsin solution for sustained vibration and elution for 24 hours to make mice DADM. The general appearance of DADM was observed. The structure and arrangement of collagen fibers of DADM were observed by scanning electron microscope and tissue structure of DADM were observed by fluorescence microscope. Bone marrow mesenchymal stem cells (BMSCs) from mice were transplanted in mice DADM in the two groups with concentration of 2×105 cells per well to prepare bioactive mice DADM. After cultured for 3 days, tissue structure of bioactive mice DADM was observed by hematoxylin and eosin staining, distribution and number of BMSCs of bioactive mice DADM were observed by immunofluorescence staining. Proliferation of BMSCs of bioactive mice DADM after cultured for 2 h, 1 d, 3 d, and 5 d was detected by cell count kit-8. Data were processed with analysis of variance for repeated measurement and t test. Results: (1) Mice DADM in the two groups were white in appearance with certain tenacity and elasticity. Mice DADM in the two groups maintained good three-dimensional porous network structure. Collagen fibers of mice DADM in EDTA group were with good continuity, and collagen fibers of mice DADM in Triton X-100 group were fractured in varying degrees. Mice DADM in the two groups were decellularized completely, and the collagen fibers were loose and arranged disorderly. The continuity of tissue structure of mice DADM in EDTA group was better than that of mice DADM in Triton X-100 group. (2) After cultured for 3 days, the BMSCs in bioactive mice DADM in the two groups were evenly distributed. The number of bioactive BMSCs in mice DADM in EDTA group was 37±7, which was significantly more than that of mice DADM in Triton X-100 group (25±8, t=0.128, P<0.05). The proliferation of bioactive BMSCs in mice DADM in Triton X-100 group and EDTA group was similar at 2 hours and on day 1 after cultured (t=1.292, 0.656, P>0.05). On 3, 5 days after cultured, the proliferation of bioactive BMSCs in mice DADM in EDTA group was significantly higher than that of mice DADM in Triton X-100 group (t=2.309, 14.128, P<0.05 or P<0.01). Conclusions: Mice DADM prepared by decellularization of EDTA has better three-dimensional porous network structure and good continuity of collagen fiber. The BMSCs in bioactive DADM from burn mice prepared by transplanting BMSCs are evenly distributed with large quantity and strong proliferative capacity, which has the potential to be good autologous dermal substitute.

Effect of Gefitinib inhibiting epidermal growth factor receptor signaling pathway on proliferation and osteogenic differentiation of endosteum-derived stem cells in rats

Objective To investigate the effect of gefitinib, an inhibitor of epidermal growth factor receptor (EGFR), on the proliferation and osteogenic differentiation of endosteum-derived stem cells (EDSCs) in rats. Methods Femoral fracture models were established in healthy male 4-week old SD rats. They were randomly divided into 2 groups. The experimental group was subjected to intragastric lavage with gefitinib, an EGFR signaling inhibitor (100 mg/kg·d) while the control group to intragastric lavage with an isodose of methyl cellulose. Bilateral femurs and tibias were harvested one week after lavage for separation of EDSCs and bone marrow mesenchymal stem cells (BMSCs) respectively using density gradient centrifugation. After proliferative cloning in vitro, expression of the cell surface antigens (CD29, CD34, CD44 and CD45) of the third passage cells was detected by flow cytometry (FCM). Proliferation of the cells was detected by BrdU, cell cycle was measured by FCM, and expression of the genes related to cell cycle inhibitory factors (p15, p16, p21 and p27) was determined by PCR. ALP staining was performed 14 days after osteogenesis induction. After 21 days of chondrogenic induction, von Kossa staining was conducted. qRT-PCR of the mRNA obtained was used to detect expression of osteogenic differentiation of related genes (osteocalcin, bsp, runx2 and osterix). Results CD29 and CD44 were positively expressed while CD34 and CD45 negatively expressed in EDSCs and BMSCs. After the EGFR signaling pathway was blocked by gefitinib, BrdU detection found that gefitinib inhibited BMSCs (11.15%) much more than EDSCs (0.25%). Cell cycle detection showed that the volume of EDSCs was increased in phases G0/G1 and S but decreased significantly in phase G2-M. ALP staining showed that the increase of EDSCs ALP+cells (53.31%) was significantly higher than that of BMSCs (25.04%). The increased expression percentages of the genes related to cell cycle inhibitors in EDSCs (103.9%, 58.0%, 117.3% and 105.1%, respectively) were significantly higher than those in BMSCs (39.3%, 38.4%, 24.5% and 83.4%, respectively)(P< 0.05). The increased expression percentages of the genes related to osteogenic differentiation in EDSCs (247.0%, 289.9%, 66.1% and 233.2%, respectively) were significantly higher than those in BMSCs (106.5%, 186.4%, 41.7% and 190.8%, respectively). All the above differences were statistically significant (P< 0.05). Conclusions Gefitinib, an EGFR inhibitor, can inhibit proliferation of EDSCs and BMSCs but promote their osteogenic differentiation. It inhibits proliferation of BMSCs more significantly as it promotes osteogenic differentiation of EDSCs. Key words: Receptor, epidermal growth factor; Bone marrow cells; Stem cells; Osteogenic differentiation

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.

Upregulation of UBAP2L in Bone Marrow Mesenchymal Stem Cells Promotes Functional Recovery in Rats with Spinal Cord Injury.

Post-translational modifications of cellular proteins with ubiquitin or ubiquitin-like proteins regulate many cellular processes, such as cell proliferation, differentiation, apoptosis, signal transduction, intercellular immune recognition, inflammatory response, stress response, and DNA repair. Nice4/UBAP2L is an important member in the family of ubiquitin-like proteins, and its biological function remains unknown. This study aimed to investigate the effect of UBAP2L on spinal cord injury (SCI). At first, rat bone marrow mesenchymal stem cells (BMSCs) were infected with adeno-associated virus to induce over-expression of Nice4. Subsequently, the infected BMSCs were transplanted into rats suffering from semi-sectioned SCI. The results showed that the over-expression of Nice4 significantly promoted the proliferation and differentiation of BMSCs. In addition, the transplantation of infected BMSCs into the injured area of SCI rats improved the function repair of SCI. Importantly, the immunohistochemical and hematoxylin-eosin staining and RT-PCR results showed that the number of neuronal cells, oligodendrocytes, and astrocytes was significantly increased in the injured area, along with significantly upregulated expression of cyclin D1 and p38 mitogen-activated protein kinase (MAPK). Meanwhile, the expression of caspase 3 protein was significantly down-regulated. In conclusion, the over-expression of Nice4 gene can promote the functional recovery in SCI rats by promoting cell proliferation and inhibiting apoptosis. The results of this study indicate an alternative option for the clinical treatment of SCI.

Klotho gene-modified BMSCs showed elevated antifibrotic effects by inhibiting the Wnt/β-catenin pathway in kidneys after acute injury.

Klotho is a protein primarily expressed in renal tubular epithelial cells. Studies have suggested that Klotho is an antiaging protein that reduces renal fibrosis after acute kidney injury (AKI) and inhibits stem cell senescence. Bone marrow mesenchymal stem cells (BMSCs) have consistent proliferation ability and multidirectional differentiation ability and have been used to treat tissue injury. Thus, we hypothesized that Klotho expressed in BMSCs could increase the renal protective effects of BMSCs. To verify the hypothesis, we isolated BMSCs from C57BL/6 mice, transfected them with Klotho-GFP-adenovirus and investigated the change in BMSC proliferation. We then transplanted Klotho-GFP-BMSCs into mice with AKI and investigated the therapeutic effect compared with that of sham-treated mice and GFP-BMSC-transplanted mice. Kidney fibrosis after ischemia/reperfusion injury (IRI) was relieved by BMSC transplantation, and the antifibrotic effect of BMSCs was significantly enhanced by overexpressing the Klotho gene. Mechanistic studies showed that Klotho increased pluripotency gene expression in BMSCs. Klotho produced by Klotho-GFP-BMSCs inhibited the Wnt/β-catenin pathway in renal tubular epithelial cells (TECs). Klotho-GFP-BMSCs showed increased proliferative ability and more potent immuno-regulation ability than did GFP-BMSCs. Our findings suggested that Klotho gene-modified BMSCs may be a better choice for cell therapy after AKI.

Low-level laser irradiation modulates the proliferation and the osteogenic differentiation of bone marrow mesenchymal stem cells under healthy and inflammatory condition.

The aim of this in vitro study was to evaluate the effects of low-level laser therapy (LLLT) at different energy intensities on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under healthy and inflammatory microenvironments. Human BMSCs and BMSCs from inflammatory conditions (i-BMSCs, BMSCs treated with tumor necrosis factor α; TNF-α) were subject to LLLT (Nd:YAG;1064nm) at different intensities. We designed one control group (without irradiation) and four testing groups (irradiation at 2, 4, 8, and 16J/cm2) for both BMSCs and i-BMSCs. Cell proliferation was measured using colony-forming unit fibroblast assay and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Osteogenic capacity of cells was determined by alkaline phosphatase (ALP) staining, ALP activity assay, Alizarin Red S staining and the mRNA transcript levels of genes runt-related transcription factor 2 (Runx2), ALP, and osteocalcin. Moreover, the effects of LLLT on secretion of TNF-α in BMSCs and i-BMSCs were measured by enzyme-linked immunosorbent assay. Our results demonstrated LLLT could significantly promote BMSC proliferation and osteogenesis at densities of 2 and 4J/cm2. LLLT at density of 8J/cm2 could promote the proliferation and osteogenesis of i-BMSCs. However, LLLT at 16J/cm2 significantly suppressed the proliferation and osteogenesis of BMSCs both in healthy and in inflammatory microenvironment. Moreover, we also found that the expression of TNF-α was obviously inhibited by LLLT at 4, 8, and 16J/cm2, in an inflammatory microenvironment. Considering these findings, LLLT could improve current in vitro methods of differentiating BMSCs under healthy and inflammatory microenvironments prior to transplantation.

Dorsal Root Ganglion Maintains Stemness of Bone Marrow Mesenchymal Stem Cells by Enhancing Autophagy through the AMPK/mTOR Pathway in a Coculture System.

Our previous studies found that sensory nerve tracts implanted in tissue-engineered bone (TEB) could result in better osteogenesis. To explore the mechanism of the sensory nerve promoting osteogenesis in TEB in vitro, a transwell coculture experiment was designed between dorsal root ganglion (DRG) cells and bone marrow mesenchymal stem cells (BMSCs). BMSC proliferation was determined by CCK8 assay, and osteo-, chondro-, and adipogenic differentiation were assessed by alizarin red, alcian blue, and oil red staining. We found that the proliferation and multipotent differentiation of BMSCs were all enhanced in the coculture group compared to the BMSCs group. Crystal violet staining showed that the clone-forming ability of BMSCs in the coculture group was also enhanced and mRNA levels of Sox2, Nanog, and Oct4 were significantly upregulated in the coculture group. Moreover, the autophagy level of BMSCs, regulating their stemness, was promoted in the coculture group, mediated by the AMPK/mTOR pathway. In addition, AMPK inhibitor compound C could significantly downregulate the protein expression of LC3 and the mRNA level of stemness genes in the coculture group. Finally, we found that the NK1 receptor antagonist, aprepitant, could partly block this effect, which indicated that substance P played an important role in the effect. Together, we conclude that DRG could maintain the stemness of BMSCs by enhancing autophagy through the AMPK/mTOR pathway in a transwell coculture system, which may help explain the better osteogenesis after implantation of the sensory nerve into TEB.

Effect of equiaxial tensile strain in early differentiation of mesenchymal stem cells into cartilage cells

To observe the effect of cyclic equiaxial tensile strain in the early differentiation of bone marrow mesenchymal stem cells(BMSCs) into cartilage in mouse under conditions of two-dimensional culture, and to investigate the mechanism of cyclic equiaxial tensile strain in early chondrogenic differentiation. Sixteen KM mouse aged 4 weeks were selected, male and female unlimited, with an average weight of 19.5 g (17 to 21 g). After extracting and isolating the BMSCs from KM mouse, then subculture the BMSCs to the 3rd generation. Seed the cells in the biological plate(BioFlex). According to experimental design, the cells were divided into 6 groups, blank group: ordinary culture medium was cultured for 8 days without isometric cyclic tensile strain stimulation. Control group: chondrogenic differentiation medium was used to culture for 8 days without isometric cyclic tensile strain stimulation. Experimental group: the experimental group was divided into 4 groups, all of which were cultured with chondrogenic differentiation medium for 8 days. During which isometric cyclic tensile strain stimulation was given for 1, 3, 5 and 7 days respectively. At the 8th day, all the cells were collected, the expression of the Sox9, Col-II and ROCK 1 signaling pathway-related molecules was analyzed by RT-PCR. Cells in each group were extracted, and the efficiency of cell proliferation in each group was detected by CCK-8. Glycosaminoglycan content in medium changed last was detected using ELISA. Pericellular matrix was observed by Safranin O staining and Alcian Blue staining. Normal measurement data using mean±standard deviation compared between the blank group and control group using paired t-test, compared between the experimental group and relative group using single factor analysis of variance. (1)After 8 days of culture, compared with the control group, the relative expression of Sox 9 and Col-II mRNA in the experimental group increased gradually with the increase of loading time(P<0.05), while the relative expression of ROCK1 mRNA decreased(P<0.05). Compared with the blank group, the relative expression of ROCK1 mRNA in experimental group and control group increased (P<0.05). (2)With the increase of loading time, the experimental group showed a trend of decreasing at first and then increasing, but compared with the blank group and the control group, the control group decreased significantly. (3)Glycosaminoglycan content in the medium changed last was detected by ELISA. The glycosaminoglycans in the experimental group increased gradually, and the content changes on 7 days loading group were statistically significant compared with other groups(P<0.05). (4)Safranin O and Alcilan staining showed that there was a tendency of cartilage differentiation in the experimental group, and the shape gradually increased with time, which was more obvious than that in the control group; The PCM, Col-II and GAG in the experimental group increased gradually with the increase of mechanical stimulation days, which were more obvious than those in the control group. Under conditions of two-dimensional culture, in the early differentiation of mesenchymal stem cells into cartilage, cyclic equiaxial tensile strain can promote the proliferation of BMSCs and the differentiation into chondrocytes. Moreover, cyclic equiaxial tensile strain may promote chondrogenic differentiation through inhibiting the Rho/ROCK 1 signaling pathway.

Geraniin promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) via activating β-catenin: a comparative study between BMSCs from normal and osteoporotic rats.

Abnormal osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) has been correlated with the pathogenesis of osteoporosis. Geraniin, a polyphenolic compound isolated from Phyllanthus amarus, is effective in preventing osteoporosis, but the mechanisms of action of geraniin and the impact of osteoporotic condition on drug action are not known. In this study we compared the proliferation and osteoblastic differentiation potential of BMSCs from normal rats with that from osteoporotic rats, and examined the responses of both BMSCs to geraniin in parallel. BMSCs of rats subjected to ovariectomy or sham operation were isolated and treated with geraniin. Cell proliferation was measured by CCK-8 assay. Osteoblastic differentiation was quantified by Alizarin Red S staining and alkaline phosphatase assay. Nuclear translocation of β-catenin was monitored by immunofluorescent staining. Expression of β-catenin was determined by Western blot and quantitative real-time polymerase chain reaction. Results showed that the proliferation and osteoblast formation of osteoporotic BMSCs decreased in comparison to that of normal BMSCs. Geraniin enhanced proliferation and osteoblastic differentiation of both BMSCs, but the responses of osteoporotic BMSCs to geraniin were less than those of normal BMSCs. Expression and nuclear accumulation of β-catenin in osteoporotic BMSCs were found to be diminished. Geraniin increased nuclear translocation and expression of β-catenin in both BMSCs. This study associated the osteogenic effect of geraniin to activation of Wnt/β-catenin signaling, and provided rationale for pharmacological investigation of geraniin in osteoporosis prevention and treatment.

Cajanine promotes osteogenic differentiation and proliferation of human bone marrow mesenchymal stem cells

Seed cells - mesenchymal stem cells (MSCs) - appear to be an attractive tool in the context of tissue engineering. Bone marrow represents the main source of MSCs for both experimental and clinical studies. However, the number limitation of bone marrow MSCs (BMSCs) and decreased function caused by proliferation make the search for adequate alternative sources of these cells for autologous and allogenic transplant necessary. This study was aimed to investigate the roles of cajanine isolated from the extracts of Cajanus cajan L. Millsp. in the proliferation and differentiation of BMSCs, and to discover the mechanism of proliferation of BMSCs promoted by cajanine. Bone marrow mesenchymal stem cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) and osteogenic differentiation was induced by adding dexamethasone, ascorbic acid and β-glycerophosphate supplements. Bone marrow MSCs were cultured in medium without cajanine or supplemented with cajanine. The information about the proliferation and osteogenic differentiation of BMSCs was collated. The osteogenic differentiation potential of BMSCs was also assessed at the 3rd passage by Von Kossa staining. To observe cell signal transduction changes of BMSCs after culturing them with cajanine for 24 h, the western blot analysis was performed to detect phosphorylated cell cycle proteins and activated cyclins. After osteogenic induction, the differentiation of BMSCs was accelerated by cajanine treatment. Osteogenesis markers were upregulated by cajanine treatment at both protein and mRNA levels. Cajanine obviously promoted the proliferation of BMSCs. After BMSCs were cultured with cajanine for 24 h, the cell cycle regulator proteins were phosphorylated or upregulated. Cajanine can promote the expansion efficiency of BMSCs, at the same time keeping their multi-differentiation potential. Cajanine can activate the cell cycle signal transduction pathway, thus inducing cells to enter the G1/S phase and accelerating cells entering the G2/M phase. This study can contribute to the development of cajanine-based drugs in tissue engineering.

The legacy effects of electromagnetic fields on bone marrow mesenchymal stem cell self-renewal and multiple differentiation potential

BackgroundThe effects of electromagnetic fields (EMF) on bone nonunion have been reported for many years. Many studies and randomized controlled trials have demonstrated that EMF exhibited benefits in curing delayed union and nonunion of long bone fractures. Most of them focused on the immediate effects, while the legacy effects of EMF remain poorly investigated.MethodsIn this study, rat bone marrow mesenchymal stem cells (BMSCs) were treated with EMF, and after a period of time the BMSC proliferation and differentiation were detected. Additionally, BMSC sheets with or without EMF treatment were transplanted into the rat tibia fracture nonunion models. The bone formation was evaluated after 2, 4, and 6 weeks.ResultsOur results showed that the proliferation capacity of BMSCs was heightened after EMF pretreatment. Over a period of time of EMF pretreatment, the capacities of osteogenic and chondrogenic differentiation were enhanced, while adipogenic differentiation was weakened. BMSC sheets pretreated with EMF could better promote the healing of tibia fracture in rats, compared to BMSC sheets alone. Furthermore, significantly higher values of radiographic grading scores were observed in the EMF group.ConclusionsEMF has lasting effects on the proliferation and differentiation of BMSCs, and together with cell sheet technology can provide a new method for the treatment of fracture nonunion.

Insulin-like growth factor binding protein 4 inhibits proliferation of bone marrow mesenchymal stem cells and enhances growth of neurospheres derived from the stem cells.

Insulin-like growth factor binding protein 4 (IGFBP-4) was reported to trigger cellular senescence and reduce cell growth of bone marrow mesenchymal stem cells (BMSCs), but its contribution to neurogenic differentiation of BMSCs remains unknown. In the present study, BMSCs were isolated from the femur and tibia of young rats to investigate effects of IGFBP-4 on BMSC proliferation and growth of neurospheres derived from BMSCs. Bone marrow mesenchymal stem cell proliferation was assessed using CCK-8 after treatment with IGFBP-4 or blockers of IGF-IR and β-catenin. Phosphorylation levels of Akt, Erk, and p38 in BMSCs were analysed by Western blotting. Bone marrow mesenchymal stem cells were induced into neural lineages in NeuroCult medium; the number and the size of BMSC-derived neurospheres were counted after treatment with IGFBP-4 or the blockers. It was shown that addition of IGFBP-4 inhibited BMSC proliferation and immunodepletion of IGFBP-4 increased the proliferation. The blockade of IGF-IR with AG1024 increased BMSC proliferation and reversed IGFBP-4-induced proliferation inhibition; however, blocking of β-catenin with FH535 did not. p-Erk was significantly decreased in IGFBP-4-treated BMSCs. IGFBP-4 promoted the growth of neurospheres derived from BMSCs, as manifested by the increases in the number and the size of the derived neurospheres. Both AG1024 and FH535 inhibited the formation of NeuroCult-induced neurospheres, but FH535 significantly inhibited the growth of neurospheres in NeuroCult medium with EGF, bFGF, and IGFBP-4. The data suggested that IGFBP-4 inhibits BMSC proliferation through IGF-IR pathway and promotes growth of BMSC-derived neurospheres via stabilizing β-catenin.

Highly dispersed lithium doped mesoporous silica nanospheres regulating adhesion, proliferation, morphology, ALP activity and osteogenesis related gene expressions of BMSCs.

Lithium (Li) doped mesoporous silica nanospheres (LMSNs) were synthesized by incorporation of 5 wt% Li into mesoporous silica nanospheres (MSNs) using sol-gel method. The results showed that LMSNs with a mean size of approximate 300 nm exhibited uniform and highly dispersed spherical morphology, which was similar to the morphology of MSNs. Moreover, the degradability of MSNs was significantly increased after the incorporation of Li, and LMSNs could release both silicon (Si) and Li ions in a sustained manner. Due to the release of Li ions, LMSNs showed higher stimulatory effects on the attachment and proliferation of bone marrow mesenchymal stem cells (BMSCs) than MSNs. In addition, LMSNs could also enhance the ALP activity of BMSCs as well as improving osteogenesis related genes (OPN, ALP, Runx2 and OCN) expression of BMSCs. In summary, LMSNs have shown the capability of being a carrier of biologically active ions, which exhibit great potential in bone repair/regeneration applications.

Adipose-derived and bone marrow mesenchymal stem cells: a donor-matched comparison

BackgroundAdipose-derived stem cells (ASCs) have been introduced as an alternative to bone marrow mesenchymal stem cells (BMSCs) for cell-based therapy. However, different studies comparing ASCs and BMSCs have shown conflicting results. In fact, harvesting ASCs and BMSCs from different individuals might influence the results, making comparison difficult. Therefore, this study aimed to characterize donor-matched ASCs and BMSCs in order to investigate proliferation, differentiation potential and possible effects of donor variation on these mesenchymal stem cells (MSCs).MethodsHuman bone marrow and adipose tissue samples were obtained from nine donors aged 8–14. ASCs and BMSCs were isolated and characterized based on expression of surface markers using flow cytometry. The proliferation up to 21 days was investigated. Multi-lineage differentiation was induced using osteogenic, chondrogenic and adipogenic differentiation media. Alkaline phosphatase (ALP) activity was monitored and collagen type I formation was evaluated by immunofluorescence staining. In vitro multi-potency was studied using tissue-specific stains and lineage-specific gene expression. In addition, the osteogenic lineage was evaluated at protein level.ResultsIsolated ASCs and BMSCs from all donors demonstrated morphologic and immunophenotypic characteristics of MSCs, with expression of MSCs markers and negative expression of hematopoietic markers. Unlike BMSCs, ASCs showed high expression of CD49d and low expression of Stro-1. In general, ASCs showed significantly higher proliferation and adipogenic capacity with more lipid vesicle formation and expression of the adipogenesis-related genes than BMSCs. In contrast, BMSCs showed significantly higher osteogenic and chondrogenic capacity compared to ASCs. BMSCs had earlier and higher ALP activity, calcium deposition, and expression of the osteogenesis- and chondrogenesis-related genes and the osteogenesis-related protein osteopontin. Proliferation and differentiation capacity of ASCs and BMSCs varied significantly among the donors.ConclusionsASCs and BMSCs showed tissue-specific differentiation abilities, but with significant variation between donors. The similarities and differences in the properties of ASCs and BMSCs should be taken into consideration when planning stem cell-based therapy.

(*)AG490 could suppress bone marrow mesenchymal stem cells migration, mineralization and bone defect healing via inhibiting Jak2-STAT3 pathway

Objective: To study the effect of Jak2-STAT3 pathway on cell proliferation, migration, mineralization and bone defect healing via simulating Jak2-STAT3 pathway inhibitor AG490 to bone marrow mesenchymal stem cells (BMSC) and bone defect mice models. Methods: The effect of AG490 on BMSC proliferation was measured by MTT (methyl thiazolyl tetrazolium) assay. Regulation of AG490 on BMSC migration was tested by scratch assay and transwell assay. The BMSC migration related gene, matrix metalloproteinase (MMP)-7, MMP-9 and CXC subfamily receptor 4 (CXCR4), regulated by AG490 was studied by real-time PCR. Western blotting was adopted to analyze the regulation of Jak2-STAT3 phosphorylation through the simulation of AG490. The alizarin red staining and alkaline phosphatase (ALP) activity assay were performed to measure the effect of AG490 on BMSC mineralization and osteogenic differentiation. Mice femur bone defect models were built to analyzed the effect of AG490 on bone remodeling. Results: AG490 significantly suppressed the migration rate of BMSC at 1 d and 2 d in the experiment group [(12.42±7.50) %, (41.8±2.6)%] compared with the control group [(55.5±9.9)%, (86.9±8.7)%] in scratch assay (P=0.000, P=0.000), the number of migrated BMSC in the experiment group (22.8± 5.9) was significantly suppressed compared with the control group (58.3±6.6) in Transwell assay (P=0.000). The expression of MMP-7, MMP-9 and CXCR4 were significantly downregulated in experiment group [(0.5± 0.1), (0.1±0.1) and (0.35±0.07)] compared with the control group [(1.1±0.1), (1.06±0.33), (1.08±0.13)] (P= 0.0003, P=0.000 and P=0.000). Also, the phosphorylation of Jak2-STAT3 was downregulated by AG490 in western blotting. After BMSCs were osteogenic induced for 14 days, the formation of mineralized nodule and the ALP activity of BMSC is significantly suppressed in experiment group (8.0±2.1) compared with the control group (35.7 ± 1.8) (P=0.0005). AG490 suppressed the bone defects healing,the expression level of phosphorylated Jak2 and phosphorylated STAT3, and the number of alkaline phosphatase positive cell at defect area in vivo is lower in experiment group than the control group. AG490 suppressed the relatively bone density at the defect area significantly (P=0.0004) at 5(th) week after the surgery. Conclusions: AG490 could suppress proliferation, migration and mineralization to of BMSCs regulate bone defect healing via inhibiting Jak2-STAT3 pathway.

Effect of double gene recombinant vector on the proliferation and osteogenic activity of rabbit stem cells induced by ethanol

Objective To investigate the effects of double gene recombinant vector on the proliferation and osteogenesis of alcohol-induced rabbit stem cells. Methods The rabbit bone marrow mesenchymal stem cells (BMSCs) at the third-generation were randomly divided into 6 groups: (1) normal group [bone marrow stem cells (BMSCs) without special treatment]; (2)model group (BMSCs without gene transfection); (3) unrelated sequence group (10 μl unrelated sequence transfected BMSCs); (4) siPPARγ group (10 μl siPPARγ gene vector transfected BMSCs); (5) exCGRP group (10 μl CGRP gene vector transfected BMSCs); (6) double gene group (10 μl double gene recombinant vector transfected BMSCs). The final mass concentration of 0.09 mol/L alcohol was added to all groups except the normal group. BMSCs proliferation was detected by methyl thiazol tetrazolium (MTT) method. Alkaline phosphatase (ALP) activity, laminin content, collagen type I content, and osteocalcin content in medium and ALP staining were detected at 14th day later. Results The proliferation in double gene group was obvious, and the proliferation curve was close to the normal group. The ALP activity, laminin content, collagen type I content and osteocalcin content in medium of the cells in the double gene group were the highest [(18.593±2.350) U/L, (24.422±3.110) ng/ml, (5.951±0.650) μg/L, (5.836±0.630) ng/ml respectively], which were significantly higher than those in model group [(6.528±0.830) U/L, (9.422±1.250) ng/ml, (1.733±0.230) μg/L, (2.016±0.240) ng/ml], unrelated sequence group [(7.011±0.850) U/L, (9.693±1.260) ng/ml, (1.663±0.220) μg/L, (1.913±0.230) ng/ml], obviously higher than those in siPPARγ group [(13.536±1.710) U/L, (17.103±2.230) ng/ml, (3.486±0.410) μg/L, (3.686±0.420) ng/ml], exCGRP group [(13.692±1.760) U/L, (17.185±2.240) ng/ml, (3.525±0.420) μg/L, (3.833±0.430) ng/ml], normal group [(15.056±1.910) U/L, (18.528±2.430) ng/ml, (4.035±0.460) μg/L, (4.012±0.460) ng/ml], and the difference was statistically significant (all P=0.000). The contents in model group and unrelated sequence group were significantly lower than those in normal group (all P=0.000). The contents in siPPARγ and exCGRP groups were slightly lower than those in normal group: for ALP activity (P=0.222, 0.274), for laminin (P=0.362, 0.390), for collagen type I (P=0.082, 0.105), and for osteocalcin (P=0.276, 0.543). The contents in double gene group were significantly higher than those in normal group: for ALP activity (P=0.031), for laminin (P=0.010), for collagen type I and osteocalcin (all P=0.001). The contents in double gene group were significantly higher than those in siPPARγ and exCGRP groups: for ALP activity (P=0.005, 0.006), for laminin (all P=0.003), for collagen type I and osteocalcin (all P=0.000). ALP staining showed that the cytoplasm in a large number of cells in double gene group was dyed dark blue and more than that in normal group. Conclusion The double gene recombinant vector can maintain the normal proliferation of rabbit stem cells induced by ethanol, and significantly promote osteogenic activity, which is superior to the single gene effect. Key words: Double gene recombinant vector; Ethanol; Stem cells; Proliferation; Osteogenic activity; Rabbit

Downregulation of MiR-199b-5p Inducing Differentiation of Bone-Marrow Mesenchymal Stem Cells (BMSCs) Toward Cardiomyocyte-Like Cells via HSF1/HSP70 Pathway.

BackgroundBone-marrow mesenchymal stem cells (BMSCs) are pluripotent stem cells with potent self-renewal and differentiation ability that are widely used in transplantation of cell therapy. But the mechanism on microRNA (miRNA) regulating stem cell differentiation is complicated and unclear. The aim of this study was to investigate whether miR-199b-5p is involved in differentiation of cardiomyocyte-like cells and identify potential signal pathways in BMSCs.Material/MethodsMouse BMSCs were treated with 5-azacytidine and transfected by miR-199b-5p mimic and inhibitor, respectively. qRT-PCR was used to detect the expression of miR-199b-5p in BMSCs, 5-azacytidine treated BMSCs, and neonatal murine cardiomyocytes. The expression of cardiac specific genes and the HSF1/HSP70 signal pathway were examined by qRT-PCR or western blotting. The proliferation and migration of BMSCs were evaluated by CCK-8 assay and wound-healing assay.ResultsThe expression of miR-199b-5p decreased gradually in the process of differentiation of BMSCs toward cardiomyocyte-like cells. The expression of cardiac specific genes and HSF1/HSP70 were increased in the miR-199b-5p inhibitor group; however, the miR-199b-5p mimic group presented an opposite result. Both the miR-199b-5p inhibitor group and the miR-199b-5p mimic group had no influence on BMSCs proliferation and migration. Using lentivirus vectors bearing HSF1 shRNA to silence HSF1 and HSP70, the anticipated elevated expression effect of cardiac specific genes induced by miR-199b-5p inhibitor was suppressed.ConclusionsDownregulation of miR-199b-5p induced differentiation of BMSCs toward cardiomyocyte-like cells partly via the HSF1/HSP70 signaling pathway, and had no influence on BMSCs proliferation and migration.

Effect of excess iron culturing on the proliferation and osteogenic differentiation of rat bone mesenchymal stem cells

Objective To investigate the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in the culture with excess iron. Methods Four-week old female Sprague-Dawley (SD) rats were used for BMSCs isolation from bone marrow. BMSCs were identified by flow cytometry and exposed to ammonium iron citrate (FAC) (50, 100 and 200 μmol/L) under osteogenic medium for 1 to 2 weeks. The proliferation and differentiation of BMSCs were analyzed by cell counting kit-8 (CCK-8), Alizarin Red S staining and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) tests. Results The proliferation activity of BMSCs decreased with the increase of FAC concentration. The formation of calcium nodules decreased with the increase of FAC concentration (P=0.000). The expression of osteogenic differentiation genes runt related transcription factor-2 (Runx2) decreased with the increase of FAC concentration (sham group: 1.000±0.229; 100 μmol/L FAC group: 0.198±0.044; 200 μmol/L FAC group: 0.149±0.009, P=0.001). The mRNA expression of ALP decreased with the increase of FAC concentration (sham group: 0.980±0.063; 50 μmol/L FAC group: 0.018±0.001; 100 μmol/L FAC group: 0.014±0.002; 200 μmol/L FAC group: 0.008±0.001, P=0.001). The mRNA expression of OPN decreased with the increase of FAC concentration (sham group: 0.999±0.001; 50 μmol/L FAC group: 0.170±0.072; 100 μmol/L FAC group: 0.142±0.008; 200 μmol/L FAC group: 0.117±0.028, P=0.003). Conclusion The ammonium iron citrate significantly inhibit the proliferation of rat BMSCs, and simultaneously suppress osteogenic differentiation of rat BMSCs dose-dependently. Key words: Bone marrow mesenchymal stem cells; Iron citrate; Osteogenic differentiation; Rat

Tenascin‑C promotes the migration of bone marrow stem cells via toll‑like receptor 4‑mediated signaling pathways: MAPK, AKT and Wnt.

There are currently limitations in stem cell therapy due to the low rate of homing and proliferation of cells following transplantation. The present study was designed to investigate the effects of Tenascin-C (TN-C) on bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanisms. BMSCs were obtained from C57BL/6 mice. The survival and proliferation of BMSCs was analyzed by Cell Counting Kit-8 assay, migration was evaluated using the Transwell method, and differentiation was assessed by immunocytochemistry and immunofluorescence. In addition, the levels of proteins were detected by western blotting. High concentrations of TN-C promoted the migration of BMSCs. H2O2 at concentrations of 60–90 µmol/ml induced cell death in BMSCs, and thus, it was used to simulate oxidative stress in the microenvironment of acute myocardial infarction (AMI). High concentrations of TN-C were able to protect BMSCs from cell death, and promoted the migration of BMSCs (P<0.05). However, TAK-242 [the inhibitor of Toll-like receptor 4, (TLR4)] reduced the promoting effect of TN-C (P<0.05). By contrast, TN-C had no effect on the proliferation and differentiation of BMSCs. TN-C reduced the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK), and increased the phosphorylation levels of Ser473 protein kinase B (AKT) and β-catenin, all of which were inhibited by TAK-242 (P<0.05). In the simulated AMI microenvironment, TN-C promoted the migration of BMSCs via TLR4-mediated signaling pathways, including MAPK, AKT and Wnt.