Bone marrow mesenchymal stem cells (BMSCs) are stem cells that exist in bone marrow tissue and have osteogenic differentiation potential. Insulin growth factor-1 (IGF-1) plays a key role in the proliferation and osteogenic differentiation of BMSCs. However, the specific mechanism of IGF-1 in cell proliferation and osteogenic differentiation remains unclear. In the present study, BMSCs were transfected with lentivirus carrying the siRNA-Wnt3a gene, and the Wnt3a level in BMSCs was revealed to be reduced by western blotting, real-time quantitative polymerase chain reaction and immunofluorescence detection. Then, BMSCs were treated with 80 ng/ml IGF-1 in complete medium for 5 days. CCK-8 and cell cycle assays revealed that cell proliferation was significantly decreased in the siRNA-Wnt3a group than in the control group. The protein and mRNA levels of β-catenin and cyclin D1 were significantly downregulated in the siRNA-Wnt3a group compared with the control group. In addition, BMSCs were treated with IGF-1 in osteogenic differentiation medium for 7 and 21 days, and alkaline phosphatase staining and Alizarin Red staining demonstrated significantly reduced osteogenic differentiation ability in the siRNA-Wnt3a group compared with the control group. Furthermore, the protein and mRNA levels of β-catenin, RUNX2, and OPN were downregulated compared with the control group. Our findings revealed that IGF-1 promoted the proliferation and differentiation of BMSCs at least partially through the Wnt/β-catenin pathway. These findings provided new insight into the clinical treatment of bone disease.

Connective tissue growth factor (CTGF) is expressed at high levels in blood vessels, where it functions as a regulator of a number of physiological processes, such as cell proliferation, angiogenesis and wound healing. In addition, CTGF has been reported to be involved in various pathological processes, such as tumor development and tissue fibrosis. However, one of the main roles of CTGF is to promote the differentiation of fibroblasts into myofibroblasts, a process that is involved in disease progression. Therefore, the present study aimed to investigate the possible mechanism by which pathological changes in the microvasculature can direct the activation of fibroblasts into myofibroblasts in the context of hypoxia/reoxygenation (H/R). Human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts were used in the present study. The expression levels of CTGF were determined by western blot analysis and reverse transcription-semi-quantitative PCR. To analyze the paracrine effect of HUVECs on fibroblasts, HUVECs were infected with CTGF-expressing adenovirus and then the culture supernatant of HUVECs was collected to treat fibroblasts. The formation of α-smooth muscle actin (α-SMA) stress fibers in fibroblasts were observed by immunofluorescence staining. It was found that H/R significantly increased CTGF expression in HUVECs. CTGF was also able to directly induce the differentiation of fibroblasts into myofibroblasts. In addition, the culture supernatant from CTGF-overexpressing HUVECs stimulated the formation of α-SMA stress fibers in fibroblasts, which was inhibited by treatment with a functional blocking antibody against integrin αVβ3 and to a lesser degree by a blocking antibody against α6 integrin. The mechanism of CTGF upregulation by H/R in HUVECs was then evaluated, where it was found that the CTGF protein was more stable in the H/R group compared with that in the normoxic control group. These findings suggest that CTGF expressed and secreted by vascular endothelial cells under ischemia/reperfusion conditions can exert a paracrine influence on neighboring fibroblasts, which may in turn promote myofibroblast-associated diseases. This association may hold potential as a therapeutic target.

Joint cartilage damage affects 10-12% of the world's population. Medical treatments improve the short-term quality of life of affected individuals but lack a long-term effect due to injury progression into fibrocartilage. The use of mesenchymal stem cells (MSCs) is one of the most promising strategies for tissue regeneration due to their ability to be isolated, expanded and differentiated into metabolically active chondrocytes to achieve long-term restoration. For this purpose, human adipose-derived MSCs (Ad-MSCs) were isolated from lipectomy and grown in xeno-free conditions. To establish the best differentiation potential towards a stable chondrocyte phenotype, isolated Ad-MSCs were sequentially exposed to five differentiation schemes of growth factors in previously designed three-dimensional biphasic scaffolds with incorporation of a decellularized cartilage matrix as a bioactive ingredient, silk fibroin and bone matrix, to generate a system capable of being loaded with pre-differentiated Ad-MSCs, to be used as a clinical implant in cartilage lesions for tissue regeneration. Chondrogenic and osteogenic markers were analyzed by reverse transcription-quantitative PCR and cartilage matrix generation by histology techniques at different time points over 40 days. All groups had an increased expression of chondrogenic markers; however, the use of fibroblast growth factor 2 (10 ng/ml) followed by a combination of insulin-like growth factor 1 (100 ng/ml)/TGFβ1 (10 ng/ml) and a final step of exposure to TGFβ1 alone (10 ng/ml) resulted in the most optimal chondrogenic signature towards chondrocyte differentiation and the lowest levels of osteogenic expression, while maintaining stable collagen matrix deposition until day 33. This encourages their possible use in osteochondral lesions, with appropriate properties for use in clinical patients.

This study aimed to investigate the effects of vascular endothelial growth factor (VEGF) secreted by MCF-7 breast cancer cells on the differentiation, maturation and function of dendritic cells (DCs). Small interfering RNAs (siRNAs) directed against the VEGF gene were designed and transfected into MCF-7 breast cancer cells at an optimal concentration (100 nmol/l) using cationic liposome transfection reagent, whereas the control group was transfected with only transfection reagent. Western blot analysis and ELISA were used to determine VEGF protein expression and VEGF concentration, respectively. Mononuclear cells were cultured with the culture supernatants from primary MCF-7 cells (control group) and siRNA-treated MCF-7 cells (siRNA group). The DC phenotypes, including CD1a, CD80, CD83, CD86 and HLA-DR, were evaluated by flow cytometry. The MTT assay was used to assess the cytotoxicity of DC-mediated tumor-specific cytotoxic T lymphocytes (CTLs) against MCF-7 cells in the two different culture supernatants. The VEGF-targeted constructed siRNA inhibited VEGF expression in MCF-7 cells. Cultivation with the culture supernatants from MCF-7 cells treated with siRNA affected DC morphology. DCs in the siRNA group exhibited a significantly higher expression of CD86, CD80, CD83 and HLA-DR compared to the cells in the control group, whereas the expression of CD1a in the siRNA group was significantly lower compared to that in the control group. The cytotoxic activity of CTLs mediated by DCs was significantly altered by siRNA transfection. These results indicated that VEGF may play a significant role in tumor development, progression and immunosuppression.

Insulin-like growth factor 2 (IGF-2) is a growth factor that is involved in various functions of cells, including stem cells. The effects of IGF-2 on the cellular viability and osteogenic differentiation of stem cell spheroids were investigated in the present study. Stem cell spheroids were formed using concave microwells in the presence of IGF-2 at final concentrations of 0, 10 and 100 ng/ml. Cellular viability was measured qualitatively using a microscope and quantitatively using an assay kit based on water-soluble tetrazolium salt. The level of alkaline phosphatase activity, and an anthraquinone dye assay for calcium deposit evaluation, were used to assess osteogenic differentiation. A quantitative PCR analysis was conducted to evaluate the expression of Runx2 and Col1. Spheroid formation was noticed on day 1 in the microwells, and the spheroidal shape was maintained up to day 7. The cell viability assay values for IGF-2 at 0, 10 and 100 ng/ml at day 1 were 0.193±0.002, 0.191±0.002 and 0.201±0.006, respectively (P>0.05). The absorbance values at 405 nm for the alkaline phosphatase activity assays on day 21 were 0.221±0.006, 0.375±0.010 and 0.280±0.015 for IGF-2 at 0, 10 and 100 ng/ml, respectively. There were significantly higher values for IGF-2 in the 10 and 100 ng/ml groups when compared with the control (P<0.05). Significantly higher Alizarin red staining was noted for IGF-2 in the 10 ng/ml group when compared with the unloaded control at day 21 (P<0.05). Quantitative PCR revealed that mRNA levels of Runx2 and Col1 were significantly higher at 100 ng/ml on day 7. Conclusively, the present study demonstrated that the application of IGF-2 increased alkaline phosphatase activity, Alizarin red staining, and Runx2 and Col1 expression of stem cell spheroids.

Fibroblast growth factors (FGFs) are growth factors that were initially identified as proteins that stimulate fibroblast proliferation. The aim of the present study was to examine the effects of FGF-4 on the morphology, cellular viability and osteogenic differentiation of stem cell spheroids. Stem cell spheroids were generated using concave microwells in the presence of FGF-4 at concentrations of 0, 50, 100 and 200 ng/ml. Cellular viability was qualitatively assessed by a fluorometric live/dead assay using a microscope and quantitatively determined by using Cell Counting Kit-8. Furthermore, alkaline phosphatase activity and calcium deposition were determined to assess osteogenic differentiation. Reverse transcription-quantitative PCR (RT-qPCR) was performed to evaluate the mRNA expression levels of Runt-related transcription factor 2 (RUNX2) and bone γ-carboxyglutamate protein (BGLAP). Spheroidal shapes were achieved in the microwells on day 1 and a significant increase in the spheroid diameter was observed in the 200 ng/ml FGF-4 group compared with the control group on day 1 (P<0.05). The results regarding viability using Cell Counting Kit-8 in the presence of FGF-4 at 50, 100 and 200 ng/ml at day 1 were 98.0±2.5, 106.2±17.6 and 99.5±6.0%, respectively, when normalized to the control group (P>0.05). Furthermore, the alkaline phosphatase activity was significantly elevated in the 200 ng/ml group, when compared with the control group. The RT-qPCR results demonstrated that the mRNA expression levels of RUNX2 and BGLAP were significantly increased at 200 ng/ml. Therefore, the present results suggested that the application of FGF-4 maintained cellular viability while enhancing the osteogenic differentiation of stem cell spheroids, at least partially by regulating RUNX2 and BGLAP expression levels.

The proliferative rate of chondrocytes affects bone elongation. Chondrocyte hypertrophy is required for endochondral bone formation as chondrocytes secrete factors required for osteoblast differentiation and maturation. Previous studies have demonstrated that the Indian hedgehog (Ihh) signaling pathway is a key regulator of skeletal development and homeostasis. The aim of the present study was to investigate the function of Ihh in chondrocyte proliferation and differentiation, as well as the underlying mechanisms. Ihh was knocked down in mouse chondrocyte cells using short hairpin RNA. Chondrocyte apoptosis and cell cycle arrest were assessed using flow cytometry and the results indicated that knockdown of Ihh significantly inhibited cell growth (P<0.05) and increased apoptosis (P<0.001) compared with negative control cells. Downregulation of Ihh also resulted in cell cycle arrest at G1 to S phase in chondrocytes. It was also observed that knockdown of Ihh decreased alkaline phosphatase activity and mineral deposition of chondrocytes. The inhibitory roles of Ihh downregulation on chondrocyte growth and differentiation may be associated with the transforming growth factor-β/mothers against decapentaplegic and osteoprotegerin/receptor activator of nuclear factor κB ligand signaling pathway. The results of the present study suggest that chondrocyte-derived Ihh is essential for maintaining bone growth plates and that manipulation of Ihh expression or its signaling components may be a novel therapeutic technique for the treatment of skeletal diseases, including achondroplasia.

Triptolide has been demonstrated to induce tumor cell apoptosis. However, the role of triptolide in breast cancer angiogenesis remains unclear. The present study aimed to investigate the function of triptolide in breast cancer and the molecular mechanisms underlying this. The results revealed that triptolide could significantly decrease the expression of vascular endothelial growth factor A (VEGFA) in Hs578T and MDAMB231 breast cancer cells. Furthermore, human umbilical vein endothelial cells were used to perform tube formation and bromodeoxyuridine incorporation assays, which demonstrated an antiangiogenic effect of triptolide. In addition, the effect of triptolide in vivo was examined in a xenograft mouse model, which determined that VEGFA, cluster of differentiation 31 and anti-proliferation marker protein Ki67 expression in tumor sections was decreased in the triptolide treatment group compared with the control group. Western bolt analysis was performed to investigate the phosphorylation of extracellular signal-related kinase (ERK)1/2 and RAC-α serine/threonine-protein kinase after triptolide treatment, and it's effect on hypoxia inducible factor (HIF)1-α expression. The results demonstrated that triptolide suppressed ERK1/2 activation and HIF1-α expression. Furthermore, overexpression of HIF1-α could partially abrogate the inhibitory effect of triptolide on VEGFA expression. These results suggest that triptolide inhibits breast cancer cell angiogenesis in vitro and in vivo through inhibiting the ERK1/2-HIF1-α-VEGFA axis.

Infantile hemangioma (IH), which is the most common tumor in infants, is characterized by rapid proliferation followed by spontaneous regression into fibro-fatty tissue in childhood. However, its specific mechanism has not been clarified. Our previous studies showed that insulin-like growth factor 2 (IGF-2) is increased in the proliferative phase of IH, which is deemed to form from hemangioma-derived stem cells (HemSC). However, it remains unclear whether IGF-2 can promote the adipogenic differentiation of HemSCs and the signaling mechanisms involved require further elucidation. In the present study, CCK-8 assay was used to detect the effect of different concentrations of IGF-2 on the proliferation of HemSCs. Immunohistochemistry was applied to observe the expression of IGF-2 and its receptors in cells. Oil red o-staining of adipogenesis was conducted after cells recevied no treatment or were induced with IGF-2 or IGF-2 plus OSI-906 for 10 days. Cells were cultured in EGM-2/FBS-10% alone or containing IGF-2, IGF-2 plus OSI-906 or IGF-2 plus LY294002 and the protein expression of C/EBPα, C/EBPβ, PPARγ, adiponectin, p-AKT and total AKT was determined using western blot analysis. In another experiment, cells were treated with 25, 50 or 100 μM propranolol, or vehicle. C/EBPα, C/EBPβ, PPARγ and IGF-2 were analyzed using western blot analysis or reverse transcription-quantitative polymerase chain reaction. Results indicated that IGF-2 significantly promoted the cell proliferation and lipid accumulation of HemSCs. The expression of phosphorylated AKT (p-AKT), C/EBPα, C/EBPβ, PPARγ and adiponectin was increased in IGF-2-treated HemSCs culture, whereas these changes were repressed by the inhibition of either the IGF-1 receptor (IGF-1R) or phosphoinositide 3-kinase (PI3K). Our previous research showed that propranolol accelerated adipogenesis in HemSCs and induced the upregulation of IGF-2. The results of the present study indicate that IGF-2 is able to accelerate adipogenesis, and the propranolol-induced promotion of dysregulated adipogenesis may be mediated by the IGF-2 via IGF-1R and PI3K pathways.

Diabetic cardiomyopathy (DCM) is one of the major causes of morbidity and mortality in diabetic patients. Recent studies have demonstrated an increased level of fibroblast growth factor 21 (FGF21) in the plasma of DCM patients, and FGF21 has been proven to be a cardiovascular protector of the heart. The present study aimed to further investigate the pathogenic role of FGF21 in DCM, hypothesizing that a lack of FGF21 may promote the progression of DCM by regulating the lipid metabolism, cardiac hypertrophy and cardiac fibrosis, thus deteriorating the cardiac dysfunction. A total of 44 mice were randomly assigned into the normal (n=6), DCM (n=6), normal + scrambled siRNA (n=6), DCM + scrambled siRNA (n=6), normal + FGF21 siRNA (n=10) and DCM + FGF21 siRNA (n=10) groups. Type 1 diabetes mellitus was induced to mice in the DCM groups by streptozotocin injection, while FGF21 expression was inhibited by FGF21 siRNA. Normal and DCM mice administrated with scrambled siRNA were respectively regarded as the controls for the normal + FGF21 siRNA and DCM + FGF21 siRNA groups. In the DCM group, FGF21 inhibition promoted cardiac hypertrophy and fibrosis, and the expression levels of their indicators, including atrial natriuretic factor, α-skeletal actin, collagen type I and III, and transforming growth factor-β, increased, leading to further decreased cardiac function. In addition, FGF21 inhibition in DCM mice elevated the quantity of lipid droplets and the concentration of heart triglycerides, plasma triglycerides and cholesterol levels, accompanied by downregulation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and upregulation of cluster of differentiation (CD)36. Thus, the results indicated that FGF21 inhibition exacerbates the cardiac dysfunction by aggravating the lipid accumulation through regulating the expression levels of PGC-1α and CD36. In conclusion, it is suggested that FGF21 may be a potentially useful agent in the treatment of DCM.

The aim of this study was to observe the effects of cluster of differentiation (CD) 151 on the expression of vascular endothelial growth factor (VEGF) in ischemic myocardium by the injection of a recombinant adeno-associated virus (rAAV) vector carrying the human CD151 gene. A rat acute myocardial infarction model was established, and rAAV-CD151 was injected into the ischemic myocardium. Four weeks later, the ischemic myocardium was removed in order to detect the expression of exogenous CD151 mRNA by reverse transcriptase polymerase chain reaction. In addition, the expression of CD151 and VEGF was detected by western blot analysis to evaluate the effect of CD151 overexpression on VEGF expression. Four weeks after injection of the vector, exogenous CD151 mRNA was expressed in the myocardial tissues of the CD151 group, whereas it was not detected in sham surgery, model control or rAAV-green fluorescent protein (GFP) gene-treated groups. The expression levels of CD151 protein were significantly higher in the CD151 group compared with those in the other three groups (P<0.05). The VEGF expression level in the CD151 group was higher compared with those in the control and GFP groups (P>0.05). These results indicate that rAAV-CD151 effectively transfects rat myocardial tissues, and may promote angiogenesis of the ischemic myocardium, improve left ventricular function and increase VEGF expression to improve ventricular function.

The aim of the present study was to evaluate the effects of anionic, cationic and neutral liposomes containing doxorubicin on the cellular viability and osteogenic differentiation of three-dimensional stem cell spheroids. Doxorubicin-loaded liposomes were prepared using the traditional thin-lipid-film-hydration method and were characterized using transmission electron microscopy and a zeta potential analyzer. The doxorubicin release profile from these liposomes was also analyzed in vitro. Three-dimensional cell spheroids were fabricated using silicon elastomer-based concave microwells. Qualitative results of cellular viability were observed using a confocal microscope and quantitative cellular viability was evaluated using a Cell-Counting Kit-8 (CCK-8) assay. Furthermore, the secretion of vascular endothelial growth factor was evaluated. Western blot analysis was performed to assess the expression of collagen I and glyceraldehyde 3-phosphate. Results indicated that the spheroids were well formed in silicon elastomer-based concave microwells on day 1. In general, the shapes of the cells in the in the doxorubicin-loaded anionic, cationic and neutral liposome groups were similar to the control group except for the 10 µg/ml groups on days 3, 5, and 7. No significant changes in cellular viability were noted with the addition of doxorubicin at day 1 but significant decreases in cellular viability were noted with application of doxorubicin at day 5. Notably, higher concentrations of doxorubicin reduced the secretion of vascular endothelial growth factor and stem cell marker expression. To conclude, the present study indicated that doxorubicin-loaded anionic liposomes produced the most sustained release profile and cationic liposomes produced the highest uptake of the stem cell spheroids. These findings suggested that higher concentrations of doxorubicin-loaded liposomes affected cellular viability, the secretion of vascular endothelial growth factor and stem cell marker expression.

Clobetasol propionate (Clo) is a potent topical glucocorticoid and a potential remyelinating agent that has been approved by the U.S. Food and Drug Administration. However, the effect of Clo on neural stem cells (NSCs) remains largely unknown. The aim of the present study was to investigate the effect of Clo on the differentiation of NSCs in vitro. NSCs were isolated from mouse embryonic brain tissues and expanded in vitro. The effect of Clo on NSC viability was examined using an MTT assay. Differentiating NSCs were treated with 5 or 10 µM Clo, or with DMSO control, and the degree of differentiation was examined following culture in stem cell differentiation induction medium for 7 days. The effect of Clo on NSC differentiation was assessed using immunocytochemistry and western blot analyses. The results revealed that Clo significantly increased NSC viability compared with the DMSO control group. Treatment with Clo also significantly increased the number of NSCs that differentiated into growth associated protein 43 positive neurons and corresponding axon lengths were also significantly increased. In addition, treatment with Clo significantly increased the number of myelin basic protein positive oligodendrocytes and decreased the number of glial fibrillary acidic protein positive astrocytes. Furthermore, inhibition of the sonic hedgehog and AMP-activated protein kinase signaling pathways inhibited Clo-induced NSC differentiation, and treatment with Clo upregulated the expression of several neurotrophic factors. In conclusion, the results of the current study suggest that Clo may have a potential therapeutic benefit in neurological disorders affecting oligodendrocytes and neurons.

The differentiation of fibroblasts to myofibroblasts is critical for the development of idiopathic pulmonary fibrosis (IPF). T-cell lymphoma invasion and metastasis 1 (TIAM1) is known to be associated with amyotrophic lateral sclerosis 1 and colorectal cancer; however, its role in IPF is unclear. The aim of the present study was to investigate the expression and roles of TIAM1 in lung fibroblasts during pulmonary fibrosis. It was demonstrated that TIAM1 expression was significantly increased in fibrotic lung tissue and lung fibroblasts from bleomycin (BLM)-treated mice compared with control mice (P<0.05). TIAM1 expression and differentiation were significantly upregulated in human lung fibroblasts challenged with transforming growth factor-β (TGF-β) compared with unchallenged cells (P<0.05). Furthermore, inhibition of the nuclear factor (NF)-κB signaling pathway significantly attenuated TGF-β-induced TIAM1 expression and decreased fibroblast differentiation in human lung fibroblasts (P<0.05). Similarly, overexpression of TIAM1 significantly inhibited TGF-β-induced fibroblast differentiation, as indicated by decreased expression of fibronectin and α-smooth muscle actin (SMA; P<0.05). The results of the present study also demonstrated that TIAM1 knockdown increased TGF-β-induced fibroblast differentiation (P<0.05). These findings suggest that TIAM1 expression is associated with lung fibroblast differentiation in pulmonary fibrosis via an NF-κB-dependent pathway, and that TIAM1 inhibits lung fibroblast differentiation in pulmonary fibrosis.

The regulation of the mesenchymal stem cell (MSC) programming mechanism promises great success in regenerative medicine. Tissue regeneration has been associated not only with the differentiation of MSCs, but also with the microenvironment of the stem cell niche that involves various cytokines and immune cells in the tissue regeneration site. In the present study, fibroblast growth factor 2 (FGF2), the principal growth factor for tooth development, dental pulp homeostasis and dentin repair, was reported to affect the expression of cytokines in human dental pulp-derived MSCs. FGF2 significantly inhibited the expression of chemokine C-C motif ligand 11 (CCL11) in a time- and dose-dependent manner in the SDP11 human dental pulp-derived MSC line. This inhibition was diminished following treatment with the AZD4547 FGF receptor (FGFR) inhibitor, indicating that FGF2 negatively regulated the expression of CCL11 in SDP11 cells. Furthermore, FGF2 activated the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinases (JNK) in SDP11 cells. The mechanism of the FGFR-downstream signaling pathway was then studied using the SB203580, U0126 and SP600125 inhibitors for p38 MAPK, ERK1/2, and JNK, respectively. Interestingly, only treatment with SP600125 blocked the FGF2-mediated suppression of CCL11. The present results suggested that FGF2 regulated the expression of cytokines and suppressed the expression of CCL11 via the JNK signaling pathway in human dental pulp-derived MSCs. The present findings could provide important insights into the association of FGF2 and CCL11 in dental tissue regeneration therapy.

Mesenchymal stem cells (MSCs) may be easily isolated from the bone marrow, and possess multi-lineage differentiation potential and various therapeutic applications. The differentiation of MSCs into osteoblasts is a complex process that is regulated by multiple internal and external factors. In the present study, the differentiation of MSCs isolated from rabbit bone marrow into osteoblasts using different osteoblast inductive media in the presence of dexamethasone, bone morphogenetic protein 2 (BMP-2), 1,25-dihydroxyvitamin D3, transforming growth factor β (TGFβ), platelet lysate and cyclooxygenase 2 (COX2), respectively. Alkaline phosphatase (ALP) activity, mineralization, collagen type (Ct) I and osteocalcin activities, and the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), BMP-2 and Ct II were measured during the differentiation process in MSCs treated with different inducers. Rabbit MSCs were successfully isolated and were observed to be predominantly circular in shape after culture for 24 h. Following subculture for 5 days, the cells demonstrated a spindle shape. ALP, Ct I and osteocalcin activities were higher in cells cultured in dexamethasone, BMP-2 and TGFβ compared with the activities in control cells. Following differentiation, the dexamethasone, BMP-2 and TGFβ groups demonstrated significantly enhanced mineralization of MSCs detected by Alizarin Red S staining. The mRNA and protein expression levels of VEGF, BMP-2 and Ct II were significantly increased in the same groups compared with the levels in the control group. In conclusion, rabbit MSCs were successfully isolated from bone marrow and differentiated into osteoblasts indicated by raised ALP, Ct I and osteocalcin activities, mineralization and expression of osteogenesis-inducing genes and proteins. The present study revealed that dexamethasone, BMP-2 and TGFβ have a positive effect on cell differentiation.

Local anesthetics (LAs) are commonly used to provide peri-operative pain control in the peripheral joints. In the field of regenerative medicine, adipose-derived mesenchymal stem cells (ADMSCs) are gaining attention as a cellular source for repair and regeneration in degenerative diseases. However, previous studies have demonstrated that the commonly used drugs lidocaine, ropivacaine, bupivacaine and mepivacaine may be toxic to human chondrocytes, which has raised concerns over whether they exert similar negative effects on ADMSCs during early chondrogenic differentiation. In the present in vitro study, the cytotoxicity of different LAs to ADMSCs was determined during early chondrogenic differentiation. At concentrations similar to those after physiological dilution once injected into the degenerative tissues, LAs (1% lidocaine, 0.5% bupivacaine, 0.5% ropivacaine or 2% mepivacaine) and PBS (control group) were incubated with rabbit ADMSCs (rADMSCs) for 60 min. Following further culture for 3 or 7 days, the cell viability, apoptosis and morphological alterations of chondrogenic differentiation were measured by determining the mitochondrial activity, by flow cytometric analysis, Safranine Fast Green double staining and reverse transcription-quantitative polymerase chain reaction of chondrogenesis-associated genes. The results indicated that the mitochondrial activity in rADMSC was decreased and the apoptotic rate was increased, following treatment with LAs (P<0.05). Lidocaine (1%) was less cytotoxic to rADMSCs during early chondrogenesis compared with other LAs. The expression levels of chondrogenesis-associated markers, including collagen I, collagen III and sex-determining region Y box 9 were all decreased at day 3 following exposure to LAs compared with the control group (P<0.05). The expression levels of these chondrogenesis-associated genes began to increase on day 7 following exposure but remained lower compared with the control group (P<0.05). Of note, 2% mepivacaine and 1% lidocaine exhibited a less pronounced negative effect on chondrogenesis-associated gene expression compared with other LAs. Therefore, the present study concluded that LAs are cytotoxic to rADMSCs during early chondrogenesis. Attention should be paid to the different types of LA selected in conjunction with ADMSC injection therapy.

Neural stem and progenitor cells (NSPCs) are important pluripotent stem cells, which have potential applications for cell replacement therapy. Ephrin receptors (Ephs) and angiogenic growth factor receptors have a major impact on the proliferation and differentiation of NSPCs. Potential interactions between EphA4 and vascular endothelial growth factor (VEGF) receptor (VEGFR) 2, and their roles in NSPC differentiation in vitro remain unknown. In the present study, mouse embryonic NSPCs were treated with ephrin-A1 or VEGF165 alone as well as with combination treatment (ephrin-A1 + VEGF165). Immunoprecipitation and immunoblot assays demonstrated that wild-type EphA4, but not the EphA4 kinase-dead mutant, interacted with VEGFR2 when overexpressed in 293T cells. This interaction was inhibited by dominant-negative EphA4. The percentage of β-tubulin III (Tuj1)+, but not glial fibrillary acid protein (GFAP)+ cells, was increased in the ephrin-A1 + VEGF165 combination group as compared to the VEGF165 alone group in mouse embryonic NSPCs. VEGF165-induced neuronal differentiation was potentiated by ephrin-A1 in NSPCs in vitro and ephrin-A1- or VEGF165-stimulated EphA4 and VEGFR2 interactions may mediate the signaling pathway.

Induced pluripotent stem cells (iPSCs) reprogrammed by somatic cells may be used as a potentially novel treatment regimen in stem cell regenerative medicine, particularly in the central nervous system (CNS). In the present study, iPSCs were generated using mouse embryonic fibroblasts by ectopic overexpression of Sox-2, Oct-3/4, Klf-4 and c-Myc, and cultured under the same conditions as that used for embryonic stem cells. The neuronal differentiation capacity of mouse iPSCs was examined, and the involvement of the formation of embryoid bodies was assessed. The results suggested that after 15 days of neuronal inducement, Nestin, Vimentin and Glast protein expression levels were significantly increased in the mouse iPSC-derived cells. Additionally, Bmi1, which is selectively expressed in differentiated postnatal adult stem cells. such as hematopoietic stem cells and neural stem cells, was required for establishment of the neuronal differentiation of mouse iPSCs. In order to assess the effects of Bmi1 in neuronal differentiation, Bmi1 expression levels were inhibited with the small molecule PTC-209. The results showed that inhibition of Bmi1 expression reduced the expression of neuronal markers, such as Nestin, compared with the controls. These results suggested that mouse iPSCs can be induced to achieve neuronal differentiation. More interestingly, Bmi1 was required during the neuronal differentiation of mouse iPSCs.

Orthotopic liver or hepatocyte transplantation is effective for the treatment of acute liver injury and end-stage chronic liver disease. However, both of these therapies are hampered by the extreme shortage of organ donors. The clinical application of cell therapy through the substitution of hepatocytes with mesenchymal stem cells (MSCs) that have been differentiated into hepatocyte-like cells (HLCs) for liver disease treatment is expected to overcome this shortage. Bone marrow and adipose tissue are two major sources of MSCs [bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AT-MSCs), respectively]. However, knowledge about the variability in the differentiation potential between BM-MSCs and AT-MSCs is lacking. In the present study, the hepatogenic differentiation potential of rhesus macaque BM-MSCs and AT-MSCs was compared with the evaluation of morphology, immunophenotyping profiles, differentiation potential, glycogen deposition, urea secretion and hepatocyte-specific gene expression. The results indicated that BM-MSCs and AT-MSCs shared similar characteristics in terms of primary morphology, surface markers and trilineage differentiation potential (adipogenesis, osteogenesis and chondrogenesis). Subsequently, the hepatogenic differentiation potential of BM-MSCs and AT-MSCs was evaluated by morphology, glycogen accumulation, urea synthesis and expression of hepatocyte marker genes. The results indicated that rhesus BM-MSCs and AT-MSCs had hepatogenic differentiation ability. To the best of our knowledge, this is the first report to detect the hepatogenic differentiation potential of rhesus macaque BM-MSCs and AT-MSCs. The present study provides the basis for the selection of seed cells that can trans-differentiate into HLCs for cytotherapy of acute or chronic liver injuries in either clinical or veterinary practice.