Differentiation Of Marrow Mesenchymal Stem Cells Research Articles

DLX2 activates Wnt1 transcription and mediates Wnt/β-catenin signal to promote osteogenic differentiation of hBMSCs.

The molocular mechanism underlying human bone marrow mesenchymal stem cells (hBMSCs) differentiation remains to be further elucidated. DLX2 has been confirmed to accelerate osteogenic differentiation which is one member of Distal-less family genes. However, how DLX2 regulates in osteogenic differentiation is still unclear. The hBMSCs were isolated and identified by the antigen CD29, CD4, CD90 through flow cytometry. DLX2 expression level, molecules related signaling pathways and transcriptional markers in osteogenesis were examined by western blot and real time-PCR. Osteogenic state was weighed by the ALP Detection Kit and Alizarin red S staining. The combination between DLX2 and WNT1 was detected by Chromatin immunoprecipitation (CHIP) assay. The results showed that in the process of osteoblast differentiation, DLX2 was up-regulated accompanied with osteogenic transcriptional factor. DLX2 elevated cellular alkaline phosphatase activity, accelerated BMSC mineralization along with up-regulation of osteogenic-related gene expression. Besides, DLX2 is a transcription factor of WNT1, which activated the Wnt/β-Catenin signaling pathway resulting in osteogenic differentiation. Whereas, the inhibitor of β-Catenin FH535 restrained enhanced osteogenic capability induced by DLX2. Taken together, these results suggest that by up-regulation of Wnt/β-Catenin signaling, DLX2 accelerated human osteogenic differentiation.

Effect of MicroRNA-126a-3p on Bone Marrow Mesenchymal Stem Cells Repairing Blood-brain Barrier and Nerve Injury after Intracerebral Hemorrhage

Objective: Intracerebral hemorrhage (ICH) is a disease that threatens human health due to its high morbidity and mortality. On behalf of finding the better methods in the treatment of ICH, researchers pay more attention to a new technology which is finding effective genes to modify stem cells. Methods: In this study, we isolated, cultured and identified bone marrow mesenchymal stem cells (MSCs) in vitro. Further, the MSCs (transfected with lentivirus expressing microRNA-126a-3p (miR-126)) were injected into the type Ⅶ collagenase-induced ICH rats to investigate the recovery effects of blood-brain barrier (BBB) and nerve damage in vivo. Results: The MSCs surface marker molecules (CD29: 98.5%; CD90: 96.5%) were highly expressed, and the blood cell surface molecule was negatively expressed (CD45: 2%). Meanwhile, it was verified that miR-126 facilitated the differentiation of MSCs into vascular endothelial cells, owing to the rise of markers (CD31 and VE-cadherin). The modified neurological severity score, modified limb placing test score, brain water content and evans blue content were reduced after transplanted miR-126-modified MSCs. It was found that miR-126 accelerated the differentiation of MSCs into vascular endothelial cells via immunohistochemical staining in vivo. HE staining indicated the area of edema was obviously decreased compared with that in ICH + vector-MSCs group. MiR-126-modified MSCs alleviated the cell apoptosis in brain tissues by TUNEL assay. In addition, the mRNA and protein expression of protease activated receptor-1 and matrix metalloproteinase-9 were diminished, whilst the expression of zonula occludens-1 (ZO-1) and claudin-5 were enhanced in ICH+miR-126-MSCs group. Immunofluorescence assay revealed that miR-126-modified MSCs decreased the disruption of tight junction (ZO-1 and claudin-5). Conclusions: All data illustrate that miR-126-modified MSCs repair BBB and nerve injury after ICH.

PKM2 suppresses osteogenesis and facilitates adipogenesis by regulating β-catenin signaling and mitochondrial fusion and fission.

Bone marrow mesenchymal stem cells (BMSCs) differentiation dysfunction is a common pathological phenotype of several prevalent metabolic and genetic bone diseases. Pyruvate kinase muscle isoenzyme 2 (PKM2) regulates the last step of glycolysis, and its role in BMSCs differentiation is still unknown. In this study, the influence of PKM2 on osteogenesis and adipogenesis was assessed in vitro and in vivo. We found that DASA-58 (the activator of PKM2) reduced the enzymatic activity of ALP, and inhibited the levels of osteogenic marker genes, especially RUNX2, which is a crucial transcription factor for osteogenesis. Besides, we provided evidence that C3k, an inhibitor of PKM2, caused increase in mitochondrial membrane potential and maintained low levels of ROS, and promoted mitochondrial fusion. Furthermore, after treatment with DASA-58, the level of active β-catenin gradually decreased, which also inhibited the transport of active β-catenin into the nucleus, but C3k obviously promoted its nuclear translocation. As for adipogenesis, PKM2 activation increased the expression of adipogenic related genes and decreased active-β-catenin expression, whereas treatment of C3k had the opposite effect. In addition, C3k significantly attenuated ovariectomy-induced trabecular bone loss in vivo. Our findings helped uncover the molecular mechanisms underlying PKM2 regulation of BMSCs differentiation.

Effect of Substance P on Bone Marrow Mesenchymal Stem Cells Differentiation in High Glucose Environment

Bone marrow mesenchymal stem cells (BMSCs) can be used to treat bone defects. The neuropeptide substance P (SP) plays an important role in a variety of life activities. However, the effect of SP on BMSCs differentiation in high glucose environment remains unclear. Rat BMSCs were isolated and divided into control group; high glucose group; and SP group. The secretion of SP was detected by ELISA; cell proliferation was detected by MTT assay; apoptosis activity was detected by Cas-pase3 activity kit. Real time PCR was performed to measure Bax and Bcl-2 expression. Alizarin red staining was to detect calcified nodule formation. Western blot was done to measure AMPK/mTOR signaling protein expression. In high glucose environment, SP secretion was significantly decreased, along with increased cell proliferation, Caspase3 activity and Bax expression. Meanwhile, Bcl-2 expression, ALP activity and calcified nodules formation was significantly decreased with reduced AMPK phosphorylation and increased mTOR expression (P < 0.05). SP addition in high glucose environment significantly promoted SP secretion and cell proliferation, decreased Caspase3 activity and Bax expression, increased Bcl-2 expression, ALP activity and calcification nodules formation with increased AMPK phosphorylation and decreased mTOR expression (P < 0.05). In high glucose environment, SP secretion is decreased in BMSCs. Up-regulation of SP in BMSCs cells in high glucose environment inhibit the apoptosis of BMSCs and promote cell proliferation and osteogenesis by regulating AMPK/mTOR signaling pathway.

LncRNA Urothelial Carcinoma-Associated 1 (UCA1) Regulates Rat Bone Marrow Mesenchymal Stem Cell Differentiation and Promotes Repair of Bone Defects

Bone marrow mesenchymal stem cells (BMSCs) have characteristics of self-renewal and multidirectional differentiation. LncRNA UCA1 regulates BMSCs differentiation. Whether LncRNA UCA1 plays a role in bone defects remains unclear. BMSCs were randomly divided into control group, radiation group (6Gy), radiation + UCA1 group followed by analysis of the expression of LncRNA UCA1, RUNX2 and OPN by real time PCR, BMSCs proliferation by MTT assay as well as ALP activity. Healthy Sprague-Dawley rats were randomly divided into control group; bone defect group; UCA1 group, in which UCA1-transfected BMSCs were infused into bone defect rats followed by analysis of bone mineral density, ALP activity as well as the formation of type II collagen by ELISA. LncRNA UCA1 expression was significantly decreased in BMSCs of irradiated group, with decreased BMSCs proliferation, reduced expression of RUNX2 and OPN as well as decreased ALP activity (P < 0.05). Transfection of UCA1 significantly up-regulated LncRNA UCA1 expression in BMSCs, promoted BMSCs proliferation, increased the expression of RUNX2 and OPN, and the activity of ALP (P < 0.05). In addition, UCA1 promoted bone mineral density, increased ALP activity and type II collagen formation in rats with bone defect. LncRNA UCA1 promotes osteogenic differentiation of BMSCs, and targeting it might be a novel approach to promote bone remodeling at the bone defect site.

Interleukin-15 Regulates Osteogenic/Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Under Oxidative Stress by Regulating PI3K/Akt/mTOR Signaling Pathway

The oxidative stress process can affect bone marrow mesenchymal stem cells (BMSCs) differentiation. Interleukin (IL-15) regulates fat differentiation of BMSCs. However, the role of IL-15 in osteogenic/adipogenic differentiation of BMSCs under oxidative stress remains unclear. Rat BMSCs were cultured and randomly divided into control group; oxidative stress group and IL-15 treatment group followed by analysis of IL-15 secretion by ELISA, expression of osteocalcin, type I collagen, RUNX2 and OPN mRNA as well as FABP4 and PPARγ 2 by Real time PCR, ALP activity, ROS content and SOD activity, and expression of PI3K/Akt/mTOR signaling proteins by Western blot. In oxidative stress group, IL-15 secretion was significantly decreased, osteocalcin, type I collagen, RUNX2 and OPN mRNA expression was reduced, along with deceased ALP activity and SOD activity, increased ROS content and FABP4 and PPARγ 2 protein expression as well as decreased expression of p-AKT and mTOR in comparison of control (P < 0 05). IL-15 treatment on oxidative stress BMSCs significantly increased IL-15 secretion and the expression of osteocalcin, type I collagen, RUNX2 and OPN mRNA, along with increased ALP activity and SOD activity, decreased FABP4 and PPAR 2 protein expression and ROS content as well as increased expression of p-AKT and mTOR (P < 0 05). IL-15 secretion was reduced in BMSCs under oxidative stress. Promoting IL-15 secretion can improve redox balance through PI3K/Akt/mTOR signaling pathway, promote osteogenic differentiation of BMSCs.

Bmi1 Induces Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Under Inflammatory Environment

Bmi1 is a polycomb histone that regulates stem cells, but the role and mechanism of Bmi1 in bone marrow mesenchymal stem cells (BMSCs) differentiation has not been elucidated. Rat BMSCs were cultured in vitro and randomly divided into control group and inflammation group (treated with LPS). Bmi1 and Bmi1 siRNA were transfected into inflammatory BMSCs, followed by analysis of Bmi1 expression by Real time PCR, cell proliferation by MTT assay, Caspase3 activity, ALP activity, expression of Runx2, OP and PPARγ 2 by Real time PCR, as well as secretion of TNF-α and IL-1β by ELISA. In inflammatory environment, Bmi1 expression was significantly decreased, cell proliferation was significantly inhibited, along with increased Caspase3 activity, decreased ALP activity and the expression of Runx2 and OP, increased PPAR 2 expression and secretion of TNF-α and IL-1β (P < 0 05). Transfection of Bmi1 siRNA into inflammatory BMSCs further significantly aggravated the above changes (P < 0 05). Bmi1 plasmid transfected into inflammatory BMSCs significantly promoted Bmi1 expression and cell proliferation, decreased Caspase3 activity, increased ALP activity and expression of Runx2 and OP, decreased PPAR γ2 expression and TNF-α and IL-1β secretion (P < 0 05). Bmi1 expression is reduced in BMSCs under inflammation. Up-regulation of Bmi1 can inhibit the secretion of inflammatory factors, regulate the proliferation and apoptosis of BMSCs, and promote the proliferation and osteogenic differentiation of BMSCs.

Cytoprotective effect of Fufang Lurong Jiangu capsule against hydrogen peroxide-induced oxidative stress in bone marrow stromal cell-derived osteoblasts through the Nrf2/HO-1 signaling pathway

ObjectiveOxidative stress is increasingly recognized as a risk factor associated with the development and progression of osteoporosis. Fufang Lurong Jiangu Capsule (FLJC) has a known anti-osteoporotic effect, but its pharmacological effect on osteoblasts is not clearly understood. This study was designed to investigate FLJC effects/mechanisms on in vitro hydrogen peroxide (H2O2)-induced oxidative damage of osteoblasts and on in vivo lipopolysaccharide (LPS)-induced mice bone loss. FLJC alleviates osteoporosis via unknown pharmacological mechanisms. MethodsChemical compositions of FLJC preparations were analyzed using high-performance liquid chromatographic fingerprinting. After rat bone marrow mesenchymal stem cell differentiation induction, resulting osteoblasts received various 48 h FLJC pretreatments before H2O2-based (200 μM) oxidative stress exposure. FLJC effects were measured on osteoblast cell viability, morphological changes, levels of intracellular reactive oxygen species (ROS), localization of mitochondria, activity of antioxidant enzymes, alkaline phosphatase (ALP) and mineralization, the secretion of Col I and expression of osteogenic markers. The percentages of apoptosis were determined by flow cytometric analysis; apoptosis-related protein levels, including nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1) with or without Nrf2 inhibitor were analyzed via western blot. Hematoxylin and eosin (H&E) and ALP staining revealed in vivo FLJC effect on mice LPS-induced bone loss. ResultsFive chemical components in FLJC were identified, and fingerprint analysis showed good reproducibility. FLJC pretreatment significantly reduced H2O2-induced ROS levels in osteoblasts and increased antioxidant enzyme activities to reduce oxidative damage. With regard to osteoblast differentiation, FLJC pretreatment increased ALP expression, as well as levels of mineralization and osteoblast markers. Additionally, FLJC protected against H2O2-induced apoptosis by inhibiting changes in expression of major Bcl-2 family effector proteins of the mitochondrial apoptosis pathway. Furthermore, FLJC protected cells from H2O2-induced oxidative damage by up-regulating Nrf2 and HO-1 protein levels. Finally, we confirmed that FLJC administration could reverse the bone loss in LPS-induced mice. ConclusionThese results indicate that FLJC may significantly attenuate oxidative damage of osteoblasts induced by H2O2 via the Nrf2/HO-1 signaling pathway, providing new insights to guide development of treatments for osteoporosis induced by oxidative injury.

Effect of dedifferentiation on tendon differentiation of rat bone marrow mesenchymal stem cells

Objective To investigate the differentiation potential of rat bone marrow mesenchymal stem cells in dedifferentiation process. Methods Bone marrow mesenchymal stem cells (BMSCs) of Sprague Dawley rats were isolated and cultured using a whole bone marrow adherent method. BMSCs were induced to dedifferentiate with culture medium containing growth differentiation factor (GDF-5), and then its tendon differentiation was further induced with culture medium containing GDF-5. After 3 weeks, the expression of type Ⅰ (Col Ⅰ), Collagen type Ⅲ (ColⅢ), tenascin-C (TNC), basic Helix-Loop-Helix (scleraxis, SCX) and Tenomodulinwas detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Results are given as mean±standard deviation. Statistical analysis was performed using Student’s t-test. Multiple comparisons were done using one way ANOVA test. Statistical signicance was set at P<0.05. Results The gene expression of Scx, Col Ⅰ, Col Ⅲ and TNC in experimental group (0.635±0.557, 0.590±0.020, 0.447±0.063, 0.613±0.061 respectively) was significantly higher than that in induced differentiation group (0.416±0.054, 0.406±0.173, 0.294±0.013, 0.373±0.042, t=17.857, P<0.01; t=12.948, P<0.01; t=7.747, P<0.01; t=-5.212, P<0.05) and control group (0.197±0.019, 0.201±0.014, 0.216±0.037, 0.190±0.014, t=31.077, P<0.01; t=14.419, P<0.01; t=15.054, P<0.01; t=8.013, P<0.05). Western blotting indicated that the expression of Col Ⅰ, TNC and Tenomodulin in experimental group (1.705±0.159, 1.587±0.018, 1.616±0.019 respectively)was higher than that in positive control group (1.160±0.018, 1.164±0.016, 1.092±0.022, t=4.825, P<0.05; t=17.395, P<0.05; t=3.961, P<0.05 respectively) and negative group (0.643±0.047, 0.441±0.02l, 0.673±0.015 respectively, t=13.299, P<0.05; t=37.618, P<0.05; t=9.349, P<0.05 respectively). Conclusion GDF-5 can induce the differentiation of bone marrow mesenchymal stem cells into tendon cells, and the dedifferentiation process can improve the ability of bone marrow mesenchymal stem cells to differentiate into tendon cells. Key words: Bone mesenchymal stem cells; Dedifferentiation; Growth differentiation factor-5; Differentiation; Tendon cells

MiR‑204 inhibits the osteogenic differentiation of mesenchymal stem cells by targeting bone morphogenetic protein 2.

Mesenchymal stem cells (MSCs) are used to investigate regeneration and differentiation. MicroRNA-204 (miR-204) in involved in the Runt-related transcription factor 2/alkaline phosphatase/bone morphogenic protein 2 (Runx2/ALP/BMP2) signaling pathway that regulates bone marrow mesenchymal stem cell (BMSC) differentiation; however, the mechanisms underlying the effects of miR-204 are yet to be determined. The aim of the present study was to investigate the effects of miR-204 on BMSC differentiation. BMSCs were derived from rat bone marrow. The expression levels of Runx2, ALP and BMP2 were measured via reverse transcription-quantitative polymerase chain reaction and western blot analyses following transfection of BMSCs with miR-204 agomir or BMP2 expression vector. The ability of the miR-204 gene to directly bind BMP2 mRNA was assessed using dual-luciferase assays. Ossification was measured via alizarin red stain assays. It was observed that the expression levels of Runx2 and ALP increased over time, whereas those of miR-204 decreased; additionally, miR-204 agomir upregulation inhibited the expression of Runx2, ALP and BMP2 in BMSCs. It was revealed that miR-204 directly interacted with BMP2 mRNA, and that transfection with miR-204 agomir suppressed ossification in BMSCs by targeting the BMP2/Runx2/ALP signaling pathway.

Conformation changes of albumin and lysozyme on electrospun TiO2 nanofibers and its effects on MSC behaviors

Protein adsorption plays a key role in bone repair and regeneration by affecting cell behavior. In this study, TiO2 nanofibers (TiO2 NFs) with different structures, including anatase TiO2 nanofibers (A-NFs), anatase TiO2 nanofibers with beads (B-NFs), anatase-rutile TiO2 nanofibers (AR-NFs) and rutile TiO2 nanofibers (R-NFs), were prepared by electrospinning method. Bovine serum albumin (BSA) and lysozyme (LYZ) were used to explore the adsorption behaviors of TiO2 NFs and then the effects of materials with protein on bone marrow mesenchymal stem cells (MSCs) were studied. Pure titanium metal (PT) was used as control. The results displayed that the adsorption amounts of BSA on samples were B-NFs > AR-NFs > A-NFs ≈ R-NFs > PT, and that for LYZ were B-NFs > AR-NFs > R-NFs > A-NFs > PT. The conformation of proteins changed remarkably when they were adsorbed on meterials. Soaking the TiO2 NFs with and without protein into SBF revealed that the BSA and LYZ on B-NFs, A-NFs and AR-NFs could accelerate the HA deposition on its surface, but it had no promoting effect on HA deposition on B-NFs. MTT and PCR tests showed that the BSA and LYZ adsorbed on materials could promote the proliferation and osteogenic differentiation of MSCs to different degrees due to their different adsorption amount and conformation changes on different TiO2 NFs. The current work demonstrated that the surface properties and crystal structure of TiO2 NFs could influence the adsorption behavior and conformational change of BSA and LYZ, and then further regulate MSCs biological behavior.

MicroRNA-130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate.

ObjectivesWith age, bone marrow mesenchymal stem cells (BMSC) have reduced ability of differentiating into osteoblasts but have increased ability of differentiating into adipocytes which leads to age‐related bone loss. MicroRNAs (miRNAs) play major roles in regulating BMSC differentiation. This paper explored the role of miRNAs in regulating BMSC differentiation swift fate in age‐related osteoporosis.Material and methodsMice and human BMSC were isolated from bone marrow, whose miR‐130a level was measured. The abilities of BMSC differentiate into osteoblast or fat cell under the transfected with agomiR‐130a or antagomiR‐130a were analysed by the level of ALP, osteocalcin, Runx2, osterix or peroxisome proliferator‐activated receptorγ (PPARγ), Fabp4. Related mechanism was verified via qT‐PCR, Western blotting (WB) and siRNA transfection. Animal phenotype intravenous injection with agomiR‐130a or agomiR‐NC was explored by Micro‐CT, immunochemistry and calcein double‐labelling.ResultsMiR‐130a was dramatically decreased in BMSC of advanced subjects. Overexpression of miR‐130a increased osteogenic differentiation of BMSC and attenuated adipogenic differentiation in BMSC, conversely, Inhibition of miR‐130a reduced osteogenic differentiation and facilitated lipid droplet formation. Consistently, overexpression of miR‐130a in elderly mice dropped off the bone loss. Furthermore, the protein levels of Smad regulatory factors 2 (Smurf2) and PPARγ were regulated by miR‐130a with an negative effect through directly combining the 3'UTR of Smurf2 and PPARγ.ConclusionsThe results indicated that miR‐130a promotes osteoblastic differentiation of BMSC by negatively regulating Smurf2 expression and suppresses adipogenic differentiation of BMSC by targeting the PPARγ, and supply a new target for clinical therapy of age‐related bone loss.

Effect of Chordin-Like 1 on Differentiation of Bone Marrow Mesenchymal Stem Cells Through Wnt5/TGF-β Signaling Pathway

Chordin-like 1 (CHRDL1) functions in multiple tissues and organs. However, whether CHRDL1 affects bone marrow mesenchymal stem cells (BMSCs) differentiation remain unclear. Rat BMSCs were isolated and divided into control group, CHRDL1 group and CHRDL1 siRNA group followed by analysis of CHRDL1 level by real time PCR and ELISA, cell proliferation by MTT assay, Caspase 3 activity, ALP activity, expression of o Runx2, OC and PPARγ2 by Real time PCR, TGF-β secretion by ELIS, and Wnt5 protein expression by Western blot. CHRDL1 expression was significantly increased in CHRDL1 group, along with significantly promoted cell proliferation, decreased Caspase 3 activity, increased ALP activity and expression of Runx2 and OC, decreased PPARγ2 expression, increased TGF-β secretion and Wnt5 expression compared to control group (P &lt; 0.05). However, CHRDL1 siRNA transfection significantly decreased CHRDL1 expression, inhibited cell proliferation, increased Caspase 3 activity, decreased ALP activity and Runx2 and OC expression, increased PPARγ2 expression, decreased TGF-β secretion and Wnt5 expression. (P &lt; 0.05). Down-regulation of CHRDL1 expression in BMSCs promotes Wnt5/TGF-β signaling transduction, which in turn increases BMSCs proliferation and osteogenic differentiation. Up-regulation of CHRDL1 expression in BMSCs inhibited the activation of Wnt5/TGF-β signaling pathway, promoted BMSCs apoptosis, and inhibited BMSCs proliferation and osteogenic differentiation.

Mechanism and research progress of long non-coding RNA in osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells

With the continuous development of high throughput transcriptome analysis and further research of epigenetics, more and more studies have shown that long non-coding RNA (lncRNA) have involved in the osteogenesis and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), which have also become a hot spot in the field of orthopedic research. This review summarizes the research progress in the role and mechanism of lncRNA in regulating osteogenesis and adipogenic differentiation of BMSCs. Key words: Long non-coding RNA; Bone marrow mesenchymal stem cells; Osteogenic differentiation; Adipogenic differentiation

Teriparatide Promotes Bone Marrow Mesenchymal Stem Cell Proliferation and Differentiation by Down-Regulating miR-339 to Target DLX5

Osteoporosis (OP) is a metabolic bone disease which is characterized as loss of bone mineral content, decreased bone density, bone microstructural destruction, and reduced bone biomechanical evaluation index. Proliferation and differentiation of bone marrow mesenchymal stem cells (MSCs) is important for the treatment of OP. It is proposed that down-regulation of miR-339 can target DLX to promote the proliferation and differentiation of MSCs. However, whether teriparatide promotes osteogenic differentiation through this pathway remains unclear. MSCs were isolated from 4–6 week-old Wister rats. The effect of teriparatide on cell proliferation was evaluated by tetrazolium salt colorimetric method (MTT) and BrdU staining. Cell differentiation was measured by alkaline phosphatase staining. miR-339 expression was detected by real-time quantitative PCR (qRT-PCR). DLX5 expression was detected by Western-blot. It was found that teriparatide can promote the proliferation and differentiation of MSCs. MiR-339 level was significantly decreased and DLX5 expression was significantly upregulated after teriparatide treatment. After knocking down miR-339, teriparatide-induced upregulation of DLX5 by was attenuated. Teriparatide promotes the proliferation and differentiation of MSCs by down-regulating miR-339 to improve osteoporosis. It provides theoretical and experimental basis for the treatment of osteoporosis by teriparatide.

Long noncoding RNA UCA1 promotes chondrogenic differentiation of human bone marrow mesenchymal stem cells via miRNA-145-5p/SMAD5 and miRNA-124-3p/SMAD4 axis

Long noncoding RNA (lncRNAs) UCA1 has been known to be critical for the chondrogenic differentiation of marrow mesenchymal stem cells (MSCs). In this study, we explore the effects and mechanisms of UCA1 on the promotion of chondrogenesis of MSCs. During the processes of chondrogenic differentiation of MSCs, UCA1, miRNA-145-5p or miRNA-124-3p was overexpressed into MSCs. UCA1 substantially improved chondrogenesis of MSCs. Furthermore, UCA1 obviously down-regulated the expression of miRNA-145-5p and miRNA-124-3p, which attenuated the chondrogenic differentiation of MSCs. In addition, UCA1 significantly stimulated TGF-β pathway member SMAD5 and SMAD4, which is targeted by miRNA-145-5p and miRNA-124-3p. Collectively, these outcomes suggest that UCA1 enhances chondrogenic differentiation of MSCs via the miRNA-145-5p/SMAD5 and miRNA-124-3p/SMAD4 axis.

Indirect 3D printing technology for the fabrication of customised \u03b2-TCP/chitosan scaffold with the shape of rabbit radial head\u2014an in vitro study

BackgroundWith the development of indirect three-dimensional (3D) printing technology, it is possible to customise individual scaffolds to be used in bone transplantation and regeneration. In addition, materials previously limited to the 3D printing (3DP) process due to their own characteristics can also be used well in indirect 3DP. In this study, customised β-TCP/chitosan scaffolds with the shape of rabbit radial head were produced by indirect 3D printing technology.MethodsSwelling ability, porosity, mechanical characterisation, and degradation rate analysis were performed, and in vitro studies were also implemented to evaluate the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) on the scaffolds. CCK8 cell proliferation assay kit and alkaline phosphatase (ALP) staining solution were used to study cell proliferation and early ALP content at the scaffold surface. Moreover, the osteogenic differentiation of MSCs on scaffolds was also evaluated through the scanning electron microscopy analysis.Resultsβ-TCP/chitosan scaffold has good performance and degradation rate, and in vitro cell experiments also confirm that the scaffold has adequate cytocompatibility and bioactivity.ConclusionThis study provides a promising new strategy for the design of customised scaffolds for the repair of complex damaged tissues.

MicroRNA‐26b‐5p Regulates Bone Marrow Mesenchymal Stem Cell Differentiation to Pericytes via Targeting Platelet‐Derived Growth Factor Receptor‐β and Interacting with Long non‐Coding RNA Maternally Expressed Gene 3 in Liver Fibrosis and Angiogenesis

Background/AimsPericytes are important regulators of vascular morphogenesis and function in multiple biological processes, including liver fibrosis, which is closely associated with angiogenesis. However, the origin and functional role of pericytes during liver fibrosis have not been deciphered yet.MethodsMice received irradiation and transplantation of EGFP‐labeled bone marrow (BM) mesenchymal stem cells (BMSCs), which obtained from EGFP transgenic mice and had been sorted and purified by immunomagnetic cell sorting with anti‐CD146 microbeads. Four weeks later, the BM was reconstructed and the chimera mice with EGFP‐labeled BMSCs were fed with methionine‐choline‐deficient and high fat (MCDHF) diet to build liver injury model. Expression of pericyte markers or fibrosis markers was characterized by qRT‐PCR, Western blot, immunofluerescent staining and microarray analysis. Biotin‐avidin pull‐down and lucifer reporter assay were performed to validate the binding of microRNA (miR)‐26b‐5p and its target platelet‐derived growth factor receptor beta (PDGFR‐β). GO, KEGG and Reactome enrichment analysis was conducted to clarify the function of differentially expressed genes.ResultsIncreased expression of pericyte markers (e.g. PDGFR‐β, neural glial antigen 2, regulator of G protein signaling 5) was detected in MCDHF mice with a positive correlation with fibrosis markers (e.g. TGF‐β1, α‐smooth muscle actin, procollagen α1(I) and (III)). Numerous PDGFR‐β+ pericytes was found in the fibrotic liver not only around mature vessels but also around nascent vessels, and a significant proportion (72.5%) of pericytes in mouse fibrotic liver derived from BMSCs. BMSCs differentiated into pericytes under TGF‐β1 treatment in vitro. Long non‐coding RNA maternally expressed gene 3 (LncMEG3), which had been focused on its effects on angiogenesis recently, positively regulated PDGFR‐β expression during BMSC differentiation to pericytes. Bioinformatics analysis from three databases (miRanda, TargetScan, and RNA22v2) suggested that miR‐26b‐5p was predicted to target PDGFR‐β. MiR‐26b‐5p negatively regulated BMSC differentiation to pericytes via directly targeting PDGFR‐β in cultured BMSCs. GO, KEGG and Reactome enrichment analysis revealed that miR‐26b‐5p overexpression affected a list of genes associated with cell differentiation, angiogenesis and extracellular matrix. LncMEG3 acted as a molecular sponge of miR‐26b‐5p thus contributed to the de‐repression of miRNA target PDGFR‐β. MiR‐26b‐5p agomir blocked BMSC differentiation to pericytes and attenuates liver fibrosis and angiogenesis in vivo.ConclusionmiR‐26b‐5p regulates BMSC differentiation to pericytes via targeting PDGFR‐β and interacting with lncMEG3, which may represent an effective therapeutic strategy for liver fibrosis.Support or Funding InformationThis work was supported by grants from the National Natural and Science Foundation of China (81430013, 81500465) and the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality(IDHT20150502)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of mir-124-3p in inhibiting osteogenic differentiation of bone marrow mesenchymal stem cell and reducing bone quality in senile osteoporosis

Objective To investigate different expression levels between young and old bone marrow mesenchymal stem cells in microRNAs (miRNAs) that are significantly conserved between humans and mice. Additional studies have been conducted to discover changes in miRNA expression in old mice relative to that in young adults and discussed the roles of miRNAs in primary osteoporosis. Methods MiRNAs that are highly conserved between human and mice, and are expressed at significantly different levels in the bone marrow mesenchymal stem cells of young and old people were identified by searching the Gene Expression Omnibus (GEO) database. Human bone mesenchymal stem cells (hBMSCs) were transfected with miRNA mimics, and their relative alkaline phosphatase (ALP) activity levels were then determined. Micro-CT scanning was employed to quantitatively characterize cortical and cancellous bones of young and old mice, and to confirm that these mice accurately modeled natural aging osteoporosis. Simultaneously, we investigated differences in expression levels of miRNAs that influence ALP activity in hBMSCs in the two groups of mice. Correlations between miRNA expression levels, and parameters of bone mass and bone strength were studied. Results 28 miRNAs were found to be more than 2 fold up-regulated (down-regulated) with statistical significance (P<0.05) in the GEO database. We also found that ALP activity was lower in hBMSCs transfected with 4 miRNAs (mir-124-3p, mir-126-3p, mir-128-3p, mir-424-5p, P<0.05 or P<0.01). The micro-CT scans indicated that the mice are accurately modeled natural aging osteoporosis. Expression of mir-124-3p increased significantly in older mice. This upregulation correlated positively with trabecular separation, and negatively with trabecular pattern factor in trabecular bone. However, in cortical bone, its expression correlated positively with trabecular separation, and negatively with bone volume fraction, trabecular number, and bone mineral density (P<0.05). Conclusion Hsa-mir-124-3p, which is expressed differently in young and old bone marrow stromal cells, inhibited the osteogenic differentiation of hBMSCs. Upregulation of this miRNA in the bone tissue of aged mice may be related to the development of osteoporosis. Key words: Human bone marrow mesenchymal stem cells; mir-124-3p; Osteogenic differentiation

Exogenous Nitric Oxide Induced Early Mineralization in Rat Bone Marrow Mesenchymal Stem Cells via Activation of Alkaline Phosphatase

Background:Since the low concentration and short-time treatment with sodium nitroprusside (SNP), a nitric oxide (NO)–donor, cause no harm to rat bone marrow mesenchymal stem cells (MSCs), we studied the impact of SNP on MSCs differentiation. Methods:MSCs were treated with 100 and 1000 µM of SNP for 1 hour in every 48 hours and after 5, 10, 15, and 21 days in osteogenic media. The viability and the level of mineralization were determined using MTT assay and alizarin red staining, respectively. Morphology of the cells was studied using fluorescent dye. Concentration of calcium and the activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were evaluated by commercial kits. Results:SNP with the concentration of 1000 µM significantly reduced viability from day 5 to day 20, but 100 µM did not affect the viability until the day 15. The low concentration of SNP increased matrix deposition from day 10 and reached almost its maximum (4.40 ± 2.4) at the day 15. Also, increasing the activity of ALP (419 ± 2.2), due to low concentration of SNP, started at day 10 and continued till the day 20, while LDH (2026 ± 11) and AST (25.6 ± 0.4) elevations were observed from day 5 onwards. In case of ALT, we observed a significant decrease (36%) from day 5 till day 20. Conclusion:Based on our findings, low concentrations of SNP might be useful in the promotion of bone repair.