Differentiation Of Bone Marrow Stromal Stem Cells Research Articles

Muscle-derived extracellular vesicles improve disuse-induced osteoporosis by rebalancing bone formation and bone resorption.

Osteoporosis is a highly prevalent skeletal bone disorder worldwide with characteristics of reduced bone mass and increased risk of osteoporotic fractures. It has been predicted to become a global challenge with the aging of the world population. However, the current therapy based on antiresorptive drugs and anabolic drugs has unwanted side effects. Although cell-based treatments have shown therapeutic effects for osteoporosis, there are still some limitations inhibiting the process of clinical application. In the present study, we developed EVs derived from skeletal muscle tissues (Mu-EVs) as a cell-free therapy to treat disuse-induced osteoporosis. Our results showed that Mu-EVs could be prepared easily and abundantly from skeletal muscle tissues, and that these Mu-EVs had typical features of extracellular vesicles. In vitro studies demonstrated that Mu-EVs from normal skeletal muscles could be phagocytized by bone marrow stromal/stem cells (BMSCs) and osteoclasts (OCs), and promoted osteogenic differentiation of BMSCs while inhibited OCs formation. Correspondingly, Mu-EVs from atrophic skeletal muscles attenuated the osteogenesis of BMSCs and strengthened the osteoclastogenesis of monocytes. In vivo experiments revealed that Mu-EVs could efficiently reverse disuse-induced osteoporosis by enhancing bone formation and suppressing bone resorption. Collectively, our results suggest that Mu-EVs may be a potential cell-free therapy for osteoporosis treatment. STATEMENT OF SIGNIFICANCE: Osteoporosis is a highly prevalent skeletal bone disorder worldwide and has become a global health concern with the aging of the world population. The current treatment for osteoporosis has unwanted side effects. Extracellular veiscles (EVs) from various cell sources are a promising candidate for osteoporosis treatment. In the present study, our team established protocols to isolate EVs from culture supernatant of skeletal muscles (Mu-EVs). Uptake of Mu-EVs by BMSCs and osteoclasts influences the balance of bone remodeling via promoting the osteogenic differentiation of BMSCs and inhibiting the osteoclasts formation of monocytes. In addition, exogenous Mu-EVs from normal skeletal muscles are proved to reverse the disuse-induced osteoporosis. We provide experimental evidence that Mu-EVs therapy is a potential cell-free platform for osteoporosis treatment towards clinical application.

Mg2+ -mediated autophagy-dependent polarization of macrophages mediates the osteogenesis of bone marrow stromal stem cells by interfering with macrophage-derived exosomes containing miR-381.

Magnesium ion (Mg2+ ) has received increased attention due to the roles it plays in promoting osteogenesis and preventing inflammation. This study was designed to investigate the mechanism by which Mg2+ influences the osteoblastic differentiation of bone marrow stromal stem cells (BMSCs). The polarization of Mø (macrophages) was measured after treatment with Mg2+ . Meanwhile, autophagy in Mø was measured by detecting LC3B expression. Mø-derived exosomes were isolated and cocultured with BMSCs; after which, osteogenic differentiation was evaluated by Alizarin Red staining and detection of alkaline phosphatase (ALP). Our results showed that Mg2+ could induce autophagy in macrophages and modulate the M1/M2 polarization of macrophages. Mg2+ -mediated macrophages could facilitate the osteogenic differentiation of BMSCs by regulating autophagy, and this facilitation by Mg2+ -mediated macrophages was closely related to macrophage-derived exosomes, and especially exosomes containing miR-381. However, miR-381 in macrophages did not influence autophagy or the polarization of Mg2+ -mediated macrophages. Furthermore, macrophage-derived exosomes containing miR-381 mainly determined the osteogenic differentiation of BMSCs. Mg2+ -mediated macrophages were shown to promote the osteogenic differentiation of BMSCs via autophagy through reducing miR-381 in macrophage-derived exosomes. In conclusion, our results suggest Mg2+ -mediated macrophage-derived exosomes containing miR-381 as novel vehicles for promoting the osteogenic differentiation of BMSCs.

Reciprocal effect of microRNA-224 on osteogenesis and adipogenesis in steroid-induced osteonecrosis of the femoral head.

The adverse effects of glucocorticoids (GCs) on bone marrow stromal stem cells (BMSCs) play an important role in steroid-induced osteonecrosis of the femoral head (ONFH). Our previous miRNA microarray analysis indicated that microRNA-224-5p (miR-224-5p) could be a potential regulator; however, the underlying mechanism remains unclear. In the present study, we demonstrated that miR-224-5p was upregulated in GC-treated BMSCs, and functional experiments revealed that miR-224-5p could suppress osteogenic but promote adipogenic differentiation of BMSCs. Smad4 was identified as a direct target gene of miR-224-5p, and the Smad4-Taz axis was confirmed as the regulatory pathway for adipo-osteogenic differentiation of BMSCs. Our in vivo experiments further confirmed that the miR-224-5p antagomir could alleviate the inhibitory effects of GCs and facilitate bone formation in steroid-induced ONFH models. Therefore, these findings provide insight into the function of miR-224-5p as a reciprocal regulator of the adipo-osteogenic differentiation of BMSCs, and it could serve as a novel therapeutic target for steroid-induced ONFH.

Lnc-RNA BLACAT1 regulates differentiation of bone marrow stromal stem cells by targeting miR-142-5p in osteoarthritis.

Osteogenic differentiation of bone marrow stromal stem cells (BMSCs) is beneficial to the treatment of osteoarthritis (OA). Lnc-RNA BLACAT1 involves in occurrence and development of various diseases. However, the role of Lnc-RNA BLACAT1 in BMSCs differentiation under inflammation remains unclear. Rat BMSCs were isolated and randomly divided into control group and inflammation group (addition of IL-6). The inflammation group was further divided into BLACAT1 siRNA group and BLACAT1 siRNA+miR-142-5p inhibitor group, followed by analysis of Lnc-RNA BLACAT1 expression by real time PCR, BMSCs proliferation, Caspase 3 activity, ALP activity, expression of Runx2, OC and PPARγ2 by real time PCR, and secretion of TNF-α and IL-1β by enzyme-linked immunosorbent assay (ELISA). The bioinformatics software and the Luciferase reporter system analyze the targeted relationship between BLACAT1 and miR-142-5p. In inflammation group, Lnc-BLACAT1 expression was increased, along with inhibited BMSCs proliferation, increased Caspase 3 activity, decreased ALP activity, and expression of Runx2 and OC, increased PPARγ2 expression and secretion of TNF-α and IL-1β. The difference was statistically significant compared with control group (p<0.05). MiR-142-5p is the target miRNA of Lnc-RNA BLACAT1. BLACAT1 siRNA down-regulated BLACAT1 expression, promoted cell proliferation, inhibited Caspase 3 activity, increased ALP activity and Runx2 and OC expression, decreased PPARγ2 expression and TNF-α and IL-1β secretion. Compared with inflammation group, the difference was statistically significant (p<0.05). Of note, BLACAT1 siRNA+miR-142-5p inhibitor group reversed the effect of siRNA-mediated knockdown of BLACAT1. Lnc-RNA BLACAT1 expression was increased in inflammatory BMSCs, and knockdown of BLACAT1 promoted proliferation and osteogenic differentiation of BMSCs targeting miR-142-5p.

Decrease of MiR-31 induced by TNF-α inhibitor activates SATB2/RUNX2 pathway and promotes osteogenic differentiation in ethanol-induced osteonecrosis.

Suppressed osteogenic differentiation is considered a main cause of ethanol-induced osteonecrosis. Tumor necrosis factor α (TNF-α) and miR-31 have been reported to be involved in the osteogenic induction. This study aimed to explore a possible molecular mechanism regulating osteogenic differentiation in ethanol-induced osteonecrosis bone marrow stromal stem cells (BMSCs). Alizarin red staining was used to examine the level of mineralization in osteogenic differentiation process. Alkaline phosphatase assay was applied to the validation of ALP level which was essential to bone mineralization. The level of osteogenesis markers was determined by western blot assay, whereas the fluctuations of messenger RNA levels were tested by quantitative real-time polymerase chain reaction. Microarray analysis was conducted to identify differentially expressed genes, because the possible target relationship was predicted and validated by miRBase and luciferase reporter assay, respectively. Colony forming unit of fibroblast assay was used to observe the proliferation of BMSCs. BMSCs from patients with ethanol-induced osteonecrosis exhibited weaker osteogenic differentiation and proliferation abilities. TNF-α inhibitor added in the osteogenic medium significantly enhanced the osteogenic differentiation ability and BMSCs proliferation ability. TNF-α by regulating miR-31 downregulated the expressions of RUNX2 and SATB2, two contributors of osteoblast differentiation, further suppressed osteogenic differentiation. On the contrary, TNF-α inhibitor could promote osteogenic differentiation in BMSCs from patients with ethanol-induced osteonecrosis. TNF-α inhibitor could downregulate miR-31 expressions, which directly promoted SATB2 and RUNX2 expressions and enhanced osteogenic differentiation of BMSCs from patients with ethanol-induced osteonecrosis.

Early Growth Response (EGR)-1 Expression Regulates Colony Forming Capacity and Hematopoietic Support Function in Human Primary Bone Marrow Stromal Stem Cells

Bone marrow stromal stem cells (BMSCs) are essential components of the hematopoietic environment. BMSCs play a key role in regulating hematopoiesis and, furthermore, as skeletal stem cells they give rise to osteoblasts, adipocytes, and chondrocytes. However, despite this important role of BMSCs in bone and bone marrow physiology, little is known about how proliferation, differentiation and hematopoietic support functions of BMSCs are regulated. We hypothesized that primary human BMSCs have a distinct transcriptional regulatory system, which control BMSC stem cell properties and biological functions. We have previously reported gene expression profiling of highly-enriched human primary BMSCs (Li et al, Stem Cell Reports, 3(6):965-74, 2014), which demonstrated a substantially higher expression of early growth response 1 (EGR1) in primary cells compared to the non-colony-forming cells and cultured stromal cells, respectively. EGR1 is a member of the immediate early response transcription factor family, which has a function in cell growth, development, and stress responses in many tissues. EGR1 has been previously reported to be important for hematopoietic stem cell (HSC) proliferation and localization (Min et al. Cell Stem Cell, 2(4):380-91, 2008), but its role in non-hematopoietic bone marrow cells has thus far not been investigated. Therefore, we aimed to study the possible role of EGR1 in stroma stem cell proliferation and hematopoietic supporting function.Our data demonstrate that the expression of EGR1 as measured by qPCR was 126 ± 9-fold higher in highly fibroblast colony-forming cells (CFU-F) enriched human primary linneg/CD45neg/CD271pos/CD140aneg bone marrow cells compared to the non-colony forming CD45neg/CD271neg cell population. Furthermore, EGR1 expression in CD271posCD140aneg cells was 3 ± 0.2 -fold higher than in the CD271posCD140apos cell population, which has only minimal CFU-F activity. EGR1 expression decreased dramatically during culture with a more than 30-fold difference between primary and passage one and six cells. Down-regulation of EGR1 expression by shRNA did not affect the multi-differentiation capacity (adipogenic, osteogenic) and surface marker expression profile of BMSCs in vitro compared to controls. However, colony-forming capacity and proliferation was considerably increased in EGR1 knockdown cells, i.e. shRNA- transduced stromal cells produced up to 1.8 ± 0.1-fold more CFU-F compared to controls, whereas CFU-F were virtually absent when assaying EGR1 overexpressing cells. Furthermore, population doubling times were decreased in EGR1 knockdown cells but significantly increased (2.4 ± 0.3-fold) in EGR1 overexpressing cells. These data indicate that EGR1 expression negatively regulates BMSC proliferation and colony-forming capacity. On the other hand, hematopoietic support function was decreased in EGR1 knockdown cells as measured by the production of transplantable CD34posCD90pos HSC in stroma co-culture experiments (4-day serum-free culture supplemented with SCF 25 ng/ml, TPO 25 ng/ml, and FLT3L 25 ng/ml, 1×104 CD34pos seeded cord blood cells). Here, the positive effect of the supporting stroma was neutralized by knockdown of EGR1. Numbers of CD34posCD90pos HSC produced in co-cultures with EGR1 knockdown stroma cells were as low as 1,053 ± 316 compared to 6,100 ± 840 in control co-cultures (scamble control). Without stroma, 840 ± 210 CD34posCD90pos cells were generated from 1×104 seeded CD34pos cells. Furthermore, expansion of CD34posCD90posCB cells was increased in co-cultures with EGR1 overexpressing cells, indicating that EGR1is a positive regulator of hematopoietic stroma support in human BMSC, and confirmatory in-vivo xenotransplantation studies are ongoing.In summary, EGR1 is highly expressed by primary BM-MSC compared with non-colony forming cells and is downregulated during culture. EGR1 expression negatively regulates BMSC proliferation and colony formation while positively regulating hematopoietic stroma support function. Our data thus indicate that EGR1 may act as an important BMSC regulator coordinating the specific functions of BMSC in their different biological contexts. DisclosuresNo relevant conflicts of interest to declare.

Effects of Nano-hydroxyapatite/Poly(DL-lactic-co-glycolic acid) Microsphere-Based Composite Scaffolds on Repair of Bone Defects: Evaluating the Role of Nano-hydroxyapatite Content.

The aim of the current study was to prepare microsphere-based composite scaffolds made of nano-hydroxyapatite (nHA)/poly (DL-lactic-co-glycolic acid) (PLGA) at different ratios and evaluate the effects of nHA on the characteristics of scaffolds for tissue engineering application. First, microsphere-based composite scaffolds made of two ratios of nHA/PLGA (nHA/PLGA = 20/80 and nHA/PLGA = 50/50) were prepared. Then, the effects of nHA on the wettability, mechanical strength, and degradation of scaffolds were investigated. Second, the biocompatibility and osteoinductivity were evaluated and compared by co-culture of scaffolds with bone marrow stromal stem cells (BMSCs). The results showed that the adhesion, proliferation, and osteogenic differentiation of BMSCs with nHA/PLGA (50/50) were better than those with nHA/PLGA (20/80). Finally, we implanted the scaffolds into femur bone defects in a rabbit model, then the capacity of guiding bone regeneration as well as the in vivo degradation were observed by micro-CT and histological examinations. After 4 weeks' implantation, there was no significant difference on the repair of bone defects. However, after 8 and 12 weeks' implantation, the nHA/PLGA (20/80) exhibited better bone formation than nHA/PLGA (50/50). These results suggested that a proper concentration of nHA in the nHA/PLGA composite should be taken into account when the composite scaffolds were prepared, which plays an important role in the biocompatibility, degradation rate and osteoconductivity.

Calcitonin gene-related peptide promotes the expression of osteoblastic genes and activates the WNT signal transduction pathway in bone marrow stromal stem cells.

Calcitonin gene-related peptide (CGRP) is known to induce osteoblastic differentiation and alkaline phosphatase activity in bone marrow stromal stem cells (BMSCs). However, it has remained elusive whether this effect is mediated by CGRP receptors directly or whether other signaling pathways are involved. The present study assessed the possible involvement of the Wnt/β-catenin signaling pathway in the activation of CGRP signaling during the differentiation of BMSCs. First, the differentiation of BMSCs was induced in vitro and the expression of CGRP receptors was examined by western blot analysis. The effects of exogenous CGRP and LiCl, a stimulator of the Wnt/β-catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/β-catenin signaling pathway was assessed using quantitative PCR and western blot analyses. The results revealed that CGRP receptors were expressed throughout the differentiation of BMSCs, at days 7 and 14. Incubation with CGRP and LiCl led to the upregulation of the expression of osteoblastic genes associated with the Wnt/β-catenin pathway, including the mRNA of c-myc, cyclin D1, Lef1, Tcf7 and β-catenin as well as β-catenin protein. However, the upregulation of these genes and β-catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled-related protein, an antagonist of the Wnt/β-catenin pathway. The results of the present study therefore suggested that the Wnt/β-catenin signaling pathway may be involved in CGRP- and LiCl-promoted osteoblastic differentiation of BMSCs.

Effect of fibroblast growth factor 9 on the osteogenic differentiation of bone marrow stromal stem cells and dental pulp stem cells.

The role of fibroblast growth factor 9 (FGF9) in bone formation may depend on gene dosage, developmental stage, cell type or interactions with other cytokines. In the present study bone marrow stromal stem cells (BMSCs) and dental pulp stem cells (DPSCs) were cultured and osteogenically induced in vitro, treated with exogenous FGF9 at varying concentrations. Alkaline phosphatase staining, alizarin red S staining, reverse transcription quantitative polymerase chain reaction and western blot analyses were performed in order to investigate the gene expression levels of osteogenic markers. The results of the present study demonstrated that FGF9 enhanced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) during osteogenic induction in BMSCs and DPSCs, which are derived from different tissues. FGF9 also inhibited the osteogenic differentiation of BMSCs and DPSCs through the activation of ERK1/2. These findings suggested that FGF9 may be an inhibitor of osteogenesis in mesenchymal stem cells in vitro and its application in vivo requires investigation in the future.

Dentin matrix proteins (DMPs) enhance differentiation of BMMSCs via ERK and P38 MAPK pathways

Dentin, the predominant mineralized tissue of the tooth, comprises an extracellular matrix of collagen and a heterogeneous mixture of non-collagenous components, many of which have cellular signaling properties. These properties may be important in signaling stem cell involvement in tissue regeneration following injury and the present study investigates their morphogenic effects on differentiation of Bone Marrow Stromal Stem Cells (BMMSCs) in vitro. Non-collagenous dentin matrix proteins (DMPs) were isolated from healthy human teeth and their effects on BMMSCs behavior examined during in vitro culture. In vitro, DMPs enhanced alkaline phosphatase activity and mineralization in BMMSCs cultures as well as increasing the expression of dentinogenic and osteogenic differentiation markers (including runt-related transcription factor 2, osterix, bone sialoprotein, dentin sialophosphoprotein and osteocalcin) at both transcript and protein levels, with 10 μg/mL DMPs being the optimal stimulatory concentration. Expression of phosphor-ERK/phosphor-P38 in BMMSCs was up-regulated by DMPs and, in the presence of the ERK1/2- and p38-specific inhibitors, the differentiation of BMMSCs was inhibited. These data indicate that DMPs promote the dentinogenic/osteogenic differentiation of BMMSCs via the ERK/p38 MAPK pathways.

A comparative study of effects of autologous versus allogeneic platelet-rich plasma on the proliferation and differentiation of bone marrow stromal stem cells in rabbits

Objective To investigate whether autologous platelet-rich plasma (PRP) can be replaced by allogeneic PRP to promote the in vitro proliferation and differentiation of bone marrow stromal stem cells (BMSCs) in rabbits and to find an optimal concentration at which allogeneic PRP can best promote the proliferation and differentiation of BMSCs.Methods Three New Zealand rabbits were used for conventional culture of BMSCs and preparation of PRP.Experiments were conducted in 4 groups with 4 kinds of culture medium:blank group (DMEM only),conventional group [DMEM containing 10％ fetal bovine serum (FBS)],autologous group (DMEM containing 10％ FBS plus autologous PRP extract) and allogeneic group (DMEM containing 10％ FBS plus allogeneic PRP extract).The allogeneic group was further divided into five subgroups according to the concentrations of PRP extract in the medium:10％,20％,30％,40％ and 50％ respectively.After the best concentration was determined in the allogeneic group,it was used in the autologous group to compare the effects of the 2 kinds of PRP.On day 4,8,12,16 and 20 after intervention,optical density (OD) value in MTT assay and alkaline phosphatase (ALP) activity were tested in all groups and the cell apoptosis rate at the best concentration of PRP extract was also assessed in each group.Results The effect of PRP on the proliferation and differentiation of BMSCs was negatively related to the concentration of PRP.The optimal concentration of PRP for promotion of the proliferation of BMSCs was 30％.There was no significant difference between the autologous and allogeneic PRP (30％) regarding their effects on proliferation and differentiation of BMSCs on day 4,8,12,16 or 20 (P ＞ 0.05).On day 20,the cell apoptosis rates in blank group,conventional group,autologous group (30％) and allogeneic group (30％)were respectively 38.7％ ± 2.0％,24.1％ ± 1.7％,9.7％ ±0.8％ and 10.3 ± l.2％,with no significant difference between autologous and allogeneic groups (P ＞ 0.05).Conclusions Autologous PRP could be replaced by allogeneic PRP in vitro culture of BMSCs; the optimal concentration of PRP for promotion of the proliferation of BMSCs is about 30％. Key words: Platelet-rich plasma; Cell proliferation; Cell differentiation; Alkaline phosphatase

Overexpression of β-NGF promotes differentiation of bone marrow mesenchymal stem cells into neurons through regulation of AKT and MAPK pathway

Bone marrow stromal stem cells (BMSCs) are fibroblastic in shape and capable of self-renewal and have the potential for multi-directional differentiation. Nerve growth factor (NGF), a homodimeric polypeptide, plays an important role in the nervous system by supporting the survival and growth of neural cells, regulating cell growth, promoting differentiation into neuron, and neuron migration. Adenoviral vectors are DNA viruses that contain 36 kb of double-stranded DNA allowing for transmission of the genes to the host nucleus but not inserting them into the host chromosome. The present study aimed to investigate the induction efficiency and differentiation of neural cells from BMSCs by β-NGF gene transfection with recombinant adenoviral vector (Ad-β-NGF) in vitro. The results of immunochemical assay confirmed the induced cells as neuron cells. Moreover, flow cytometric analysis, Annexin-V-FITC/PI, and BrdU assay revealed that chemical inducer β-mercaptoethanol (β-met) triggered apoptosis of BMSCs, as evidenced by inhibition of DNA fragmentation, nuclear condensation, translocation of phospholipid phosphatidylserine, and activation of caspase-3. Furthermore, the results of western blotting showed that β-met suppressed AKT signaling pathway and regulated the MAPKs during differentiation of BMSCs. In contrast, Ad-β-NGF effectively induced the differentiation of BMSCs without causing any cytopathic phenomenon and apoptotic cell death. Moreover, Ad-β-NGF recovered the expression level of phosphorylated AKT and MAPKs in cells exposed to chemical reagents. Taken together, these results suggest that β-NGF gene transfection promotes the differentiation of BMSCs into neurons through regulation of AKT and MAPKs signaling pathways.

A new multistep induction protocol for the transdifferentiation of bone marrow stromal stem cells into GABAergic neuron-like cells.

Bone marrow stromal stem cells (BMSC) are appropriate source of multipotent stem cells that are ideally suited for use in various cell-based therapies. It can be differentiated into neuronal-like cells under appropriate conditions. This study examined the effectiveness of co-stimulation of creatine and retinoic acid in increasing the differentiation of BMSC into GABAergic neuron-like cells (GNLC). BMSC isolated from the femurs and tibias of adult rats were cultured in DMEM/F12 medium supplemented with 10% FBS, pre-induced using β-mercaptoethanol β-ME) and induced using retinoic acid (RA) and creatine. Immunostaining of neurofilament 200 kDa, neurofilament 160 kDa, nestin, fibronectin, Gamma-amino butyric acid (GABA) and glutamic acid decarboxylase (GAD) 65/67 were used to evaluate the transdifferentiation of BMSC into GNLC and to evaluate the effectiveness of pre-induction and induction assays. The expression of genes that encode fibronectin, octamer-binding transcription factor 4 (Oct-4), GAD 65/67 and the vesicular GABA transporter was examined in BMSC and GNLC by using RT-PCR assays during transdifferentiation of BMSC into GLNC. Co-stimulation with RA and creatine during the induction stage doubled the rates of GABAergic differentiation compared with induction using creatine alone, resulting in a 71.6% yield for GLNC. RT-PCR showed no expression of Oct-4 and fibronectin after the induction stage. The results of this study showed that the application of β-ME, RA, and creatine induced the transdifferentiation of BMSC into GLNC.

Nicotine-induced chondrogenic differentiation of human bone marrow stromal cells in vitro

Nicotine has been reported that it has a dose-dependent effect on matrix mineralization by human bone marrow cells. However, there is no relevant research concerning on chondrogenic differentiation potential of bone marrow stromal stem cells (BMSCs) treated with nicotine in vitro. The aims of the study were to examine the effects of nicotine (0, 10(-7), 10(-6) and 10(-5)M) on the proliferation and chondrogenic differentiation of BMSCs from three healthy donors in vitro. BMSCs proliferation was analyzed by CCK8 assay and real-time polymerase chain reaction was used to assay the expression of type II collagen, aggrecan, type I collagen and type X collagen. The proteoglycan content was stained by Alcian blue, and the sulfated glycosaminoglycan (sGAG) content of BMSCs was quantified spectrofluorometrically using dimethylmethylene blue. The cell viability was not significantly impaired until up to a concentration of 10(-5)M nicotine. Nicotine promoted the proliferation and enhanced the expression of type II collagen at the level up to 10(-6)M (P<0.05). The expression of aggrecan was reduced at the concentration of 10(-5) M nicotine at day 14 (P<0.05), and there was no significant difference in aggrecan gene expression at 10(-7) and 10(-6) M nicotine levels compared to control group (n.s.). Also the fibroblastic and hypertrophic gene expressions were down-regulated in the chondrogenic medium with 10(-7)-10(-5) M nicotine (P<0.05). It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing chondrogenic differentiation capacity of BMSCs in cell-based cartilage tissue engineering. Also these results indicate that nicotine maybe a potentially useful drug for the treatment of Osteoarthritis.

NGF and BDNF expression drop off in neurally differentiated bone marrow stromal stem cells

Bone marrow stromal stem cells (BMSC) express two neurotrophins nerve growth factor (NGF) and brain derived growth factor (BDNF) constitutively and can be differentiated into neuronal-like cells and used to treat neural injuries and diseases. The neurotrophins are required for repair of neural tissues. However, it is not evident whether these cells supply the sufficient amounts of the functional growth factors following neuronal differentiation. This study investigates the expression of NGF, BDNF and their processing enzymes Prohormone convertases (PC) Furin, PC5 and PC6 by Real-time RT-PCR during neural differentiation of rat BMSC. The results showed that all inspected processing enzymes are expressed in the cells. The expression of NGF, BDNF and PC5 decreases following differentiation. In addition, BMSCs express Survivin, an anti-apoptotic gene; however, the differentiated cells reduce its expression similar to two neurotrophins, which could make them susceptible to apoptotic death.

Proprotein convertases 1 and 2 (PC1 and PC2) are expressed in neurally differentiated rat bone marrow stromal stem cells (BMSCs)

Neural-like cells derived from bone marrow stromal stem cells (BMSCs) have potential usefulness in cell therapy of degenerative or traumatic diseases of the central nervous system (CNS). The functional recovery mediated by these cells, however, depends on the secretion of neurotrophins (NTs) and their cognate receptors, as the main regulators of neural survival and death. The function of NTs is further modulated by proprotein convertase (PC) enzymes which function in converting proproteins (including proNTs) into their functional end products. Accordingly, failure in converting proprotein forms of NTs into their mature forms may lead to neuronal cell death. In the present study, we have investigated the expression profile of PCs before and during neural differentiation of rat BMSCs by RT-PCR. Our results show that major members of the PC family functioning in the constitutive secretory pathway (furin, PACE4 and PC7/LPC) are highly expressed in both undifferentiated and neurally differentiated BMSCs. In contrast, while PC1/PC3 and PC2 (specific to neural and endocrine cells) are absent in undifferentiated BMSCs, their expression is initiated upon the induction of differentiation. In conclusion, our results suggest that neurally differentiated BMSCs have acquired the functional machinery to process the precursor forms of proteins in both the constitutive and regulated pathways.

Defective osteogenesis of the stromal stem cells predisposes CD18-null mice to osteoporosis

Osteogenesis by the bone marrow stromal stem cells (BMSSCs) supports continuous bone formation and the homeostasis of the bone marrow microenvironment. The mechanism that controls the proliferation and differentiation of BMSSCs is not fully understood. Here, we report that CD18, a surface protein present primarily on hematopoietic cells, but not on differentiated mesenchymal cells, is expressed by the stromal stem cells and plays a critical role in the osteogenic process. Constitutive expression of CD18 on BMSSCs using a retroviral promoter significantly enhances bone formation in vivo, whereas genetic inactivation of CD18 in mice leads to defective osteogenesis due to decreased expression of the osteogenic master regulator Runx2/Cbfa1. The defective osteogenesis of the CD18-null BMSSCs can be restored by expressing full-length, but not cytoplasmic domain-truncated, CD18. Radiographic analyses with dual-energy x-ray absorptiometry and 3D microcomputed tomography show that mice lacking CD18 have decreased bone mineral density and exhibit certain features of osteoporosis. Altogether, this work demonstrates that CD18 functions critically in the osteogenesis of BMSSCs, and thus lack of CD18 expression in the leukocyte adhesion deficiency patients may predispose them to osteoporosis.