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Background: We studied the potential of human bone marrow-derived mesenchymal stem cell conditioned media (hBMSC CM) in protecting endothelial cell properties (viability, proliferation, and migrations) from the deleterious effects produced by the inflammatory environment of H2O2. Additionally, we investigated their impact on the endothelial cells' gene expression of some inflammatory-related genes, namely, TGF-ß1, FOS, ATF3, RAF-1, and SMAD3. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured individually under three conditions: alone, with varying concentrations of H2O2, or with varying concentrations of H2O2 and hBMSC CM. HUVEC adhesion, proliferation, and migration were evaluated using the xCELLigence system. The HUVECs' gene expressions were evaluated by real-time polymerase chain reaction (RT-PCR). Results: Generally, we observed enhanced HUVEC viability, proliferation, and migration when cultured in media supplemented with H2O2 and hBMSC CM. Furthermore, the CM modulated the expressions of the studied inflammatory-related genes in HUVECs, promoting a more robust cellular response. Conclusion: This study has illuminated the protective role of hBMSC CM in mitigating the damaging effects of H2O2 on endothelial cell function. Our data demonstrate that hBMSC CM enhances the viability, proliferation, and migration of HUVECs even under oxidative stress conditions. Additionally, the conditioned medium was found to modulate the gene expression of pivotal markers related to inflammation, suggesting a favorable influence on cellular response mechanisms.
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Aterosclerose , Movimento Celular , Proliferação de Células , Células Endoteliais da Veia Umbilical Humana , Peróxido de Hidrogênio , Células-Tronco Mesenquimais , Humanos , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/toxicidade , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Aterosclerose/genética , Aterosclerose/metabolismo , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Bone formation is regulated by numerous factors, such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. We found that an hHNR called NR4A1 was the most highly expressed after human MSC differentiation into osteoblasts by whole-genome microarray. NR4A1 knockout decreased the osteoblastic differentiation of hMSCs in terms of ALPL expression and key marker gene expression. Whole-genome microarray analysis further confirmed the decrease in key pathways when we knocked down NR4A1. Further studies with small molecule activators identified a novel molecule called Elesclomol (STA-4783), which could activate and enhance osteoblast differentiation. Elesclomol activation of hMSCs also induced the gene expression of NR4A1 and rescued the phenotype of NR4A1 KD. In addition, Elesclomol activated the TGF-ß pathway by regulating key marker genes. In conclusion, we first identified the role of NR4A1 in osteoblast differentiation and that Elesclomol is a positive regulator of NR4A1 through activation of the TGF-ß signalling pathway.
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Osteoblastos , Osteogênese , Humanos , Regulação para Baixo , Fenótipo , Osteoblastos/metabolismo , Diferenciação Celular , Fatores de Transcrição/genética , Proteínas de Transporte/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismoRESUMO
Background: Smoking and the severity of periodontal disease have long been associated. In Saudi Arabia, tobacco smoking is rising, contributing to the increased demand for products that counter its detrimental effects. The antioxidant properties of vitamin C (vit C) make it a powerful countermeasure to tobacco toxicity. Observation of these effects on human gingival fibroblasts (hGFs) would suggest use of vitamin C in future dental applications. Aim: To examine the proliferation, adhesion, and expression of extracellular RNA in human gingival fibroblasts extracted from cigarette smokers when compared to never-smokers, in association with vitamin C. Materials and Methods: Human gingival fibroblasts were extracted from Periodontal free sites of healthy adult male participants. Group 1; consisted of Heavy cigarette smokers (n = 1) while group 2 was never-smokers (n = 1). Collected cells were cultured and subcultured in supplemented growth medium. Vitamin C was then induced in the medium at the experimental sixth passage. RNA expression analysis using quantitative reverse transcriptase-polymerase chain reaction was performed to analyze the adhesion, proliferation, and extracellular matrix expression. Results: Expression of the adhesion gene (CD44) in the smoker group was significantly downregulated than never-smoker group (p-value = 0.024). After the induction of vitamin C, the smoker samples showed a significant improvement in their gene expression levels. The extracellular genes involved in this study (COL1A1, LAMA3, and TGFB3) were significantly affected by the smoking status. In addition, the proliferation of MK167 and CCNB1 genes in smokers and never-smokers was increased. Conclusion: Cigarette smoking affects the overall properties of human gingival fibroblasts' adhesion, proliferation, and extra-cellular matrix formation. Furthermore, the addition of vitamin C affects these cellular properties in a positive manner.
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BACKGROUND/PURPOSE: Iloprost has been proposed as a potential biomaterial owing to angiogenic and odontogenic properties. However, the liquid form can limit its use during clinical applications. Mineral trioxide aggregate (MTA) has been used for various dental applications in which cell-material interaction is essential. This study aimed to investigate additive effects of iloprost on the biological properties of MTA on the viability, attachment, migration and differentiation of human mesenchymal stem cells (hMSCs). MATERIALS AND METHODS: Standardized human dentin disks were prepared. MTA was prepared by mixing distilled water or iloprost solution, and the lumen of the disks was filled with MTA or MTA-iloprost. hMSCs on disk alone and hMSCs on culture plates were used as controls. Cell viability and attachment were measured after 1, 7 and 14 days using AlamarBlue assay and scanning electron microscopy (SEM). Cell migration in MTA or MTA-iloprost extracts was determined using a wound-healing model.Osteogenic differentiation was evaluated by real-time reverse transcriptase polymerase chain reaction for alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OSP) gene expressions after 7 and 14 days of osteogenic induction. RESULTS: Cells on MTA-iloprost surface showed similar viability with MTA at 1 and 14 days but enhanced cellular viability and cell spreading compared to MTA at 7 days (pâ¯<â¯0.05). Cell migration was similar by MTA-iloprost and MTA extracts (pâ¯>â¯0.05). MTAiloprost significantly upregulated BSP, OCN and OSP expressions compared to MTA (pâ¯<â¯0.05). CONCLUSION: The addition of iloprost to MTA improved the initial cell viability and osteogenic potential of hMSCs.
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Background: Osteoporosis is associated with a metabolic imbalance between adipogenesis and osteogenesis. We hypothesized that implanting a carrier for differentiated stem cells and signaling molecules inside adipose tissues could be used to enable transdifferentiation between cells, upregulate osteogenesis, and support bone formation, which may regain the balance between osteogenesis and adipogenesis. Methodology: A CL1 human mesenchymal stem cell line was grown in an osteogenic medium to differentiate into osteoblasts, and the differentiated cells were then exposed to an adipogenic medium to stimulate differentiation into adipocytes. Osteogenic and adipogenic differentiation were confirmed by the following assays: alkaline phosphatase staining, Nile red Staining, and quantitative real-time polymerase chain reaction (qPCR). The ratio of adipocytes to osteocytes for both cases was calculated. To evaluate bone induction in vivo, a calcium sulfate/hydroxyapatite cement was prepared in a syringe and then seeded with 106 cells/mL of rat bone marrow stromal cells (rMSCs) and covered with 1 mL of tissue culture media containing 0.1 mg of bone morphogenetic protein 7 (BMP-7). The construct was injected into the abdominal fat tissue of 10 male Sprague-Dawley rats. Results: The conversion of osteocytes to adipocytes was 20-fold greater than the reverse conversion, and the area of bone regeneration was 15.7 ± 3.7%, the area of adipose tissue was 65.8 ± 13.1%, and the area of fibrous tissue was 18.3 ± 7.8%. Conclusion: Adipogenic interconversion and associated bone formation demonstrate the potential of a new therapy for balancing osteogenesis and adipogenesis.
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Tecido Adiposo , Osteogênese , Engenharia Tecidual , Adipogenia , Animais , Osso e Ossos , Diferenciação Celular , Células Cultivadas , Masculino , Osteoblastos , Ratos , Ratos Sprague-DawleyRESUMO
Colorectal cancer (CRC) is the fourth most common cancer type globally. Investigating the signaling pathways that maintain cancer cell phenotype can identify new biomarkers for targeted therapy. Aberrant transforming growth factor-ß (TGFß) signaling has been implicated in CRC progression, however, the exact mechanism by which TGFß exerts its function is still being unraveled. Herein, we investigated TAGLN expression, prognostic value, and its regulation by TGFß in CRC. While TAGLN was generally found to be downregulated in CRC, elevated expression of TAGLN was associated with advanced CRC stage and predicted poor overall survival (hazard ratio (HR) = 1.8, log-rank test P-value = 0.014) and disease-free survival (HR = 1.6, log-rank test P-value = 0.046), hence implicating TAGLN as poor prognostic factor in CRC. Forced expression of TAGLN was associated with enhanced CRC cell proliferation, clonogenic growth, cell migration and in vivo tumor formation in immunocompromised mice, while targeted depletion of TAGLN exhibited opposing biological effects. Global gene expression profiling of TAGLN-overexpressing or TAGLN-deficient CRC cell lines revealed deregulation of multiple cancer-related genes and signaling pathways. Transmission electron microscopy (TEM) revealed ultrastructural changes due to loss of TAGLN, including disruption of actin cytoskeleton organization and aberrant actin filament distribution. Hierarchical clustering, principle component, and ingenuity pathway analyses revealed distinct molecular profile associated with TAGLNhigh CRC patients with remarkable activation of a number of mechanistic networks, including SMARCA4, TGFß1, and P38 MAPK. The P38 MAPK was the top predicted upstream regulator network promoting cell movement through regulation of several intermediate molecules, including TGFß1. Concordantly, functional categories associated with cellular movement and angiogenesis were also enriched in TAGLNhigh CRC, supporting a model for the molecular mechanisms linking TGFß-induced upregulation of TAGLN and CRC tumor progression and suggesting TAGLN as potential prognostic marker associated with advanced CRC pathological stage.
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Movimento Celular , Proliferação de Células , Neoplasias Colorretais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/ultraestrutura , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Células HCT116 , Células HT29 , Humanos , Camundongos Nus , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Invasividade Neoplásica , Estadiamento de Neoplasias , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Carga Tumoral , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The Arabian camel is the largest known mammal that can survive in severe hot climatic conditions. We provide the molecular explanation for the thermotolerance of camel granulosa somatic cells after exposure to 45 °C for 2 (acute heat shock) or 20 h (chronic heat shock). The common features of the cellular responses to acute heat stress were the increase of heat shock proteins and DNA repair enzymes expression. Actin polymerization and Rho signaling were critically activated as a cellular defense against heat shock. Cells exposed to chronic heat shock showed altered cell architecture with a decrease in total detected proteins, metabolic enzymes, and cytoskeletal protein expression. Treatment with transforming growth factor beta (TGFß) pathway inhibitor SB-431542 suppressed the morphological alterations of cells exposed to chronic heat shock. Moreover, during the recovery stage at 38 °C for 24 h, proteomic changes were partially restored with an exponential increase in HSP70 expression, and the cells restored their normal cellular morphology on the 9th day of recovery. Full proteomics data are available via ProteomeXchange with identifier PXD012159. The strategies of cellular defense and tolerance to both thermal conditions reflect the flexible adaptability of camel somatic cells to conserve life under extremely hot conditions.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Targeting regulatory signaling pathways that control human bone marrow stromal (skeletal or mesenchymal) stem cell (hBMSC) differentiation and lineage fate determination is gaining momentum in the regenerative medicine field. Therefore, to identify the central regulatory mechanism of osteoblast differentiation of hBMSCs, the molecular phenotypes of two clonal hBMSC lines exhibiting opposite in vivo phenotypes, namely, bone forming (hBMSC+bone) and non-bone forming (hBMSC-Bone) cells, were studied. Global transcriptome analysis revealed significant downregulation of several TGFß responsive genes, namely, TAGLN, TMP1, ACTA2, TGFß2, SMAD6, SMAD9, BMP2, and BMP4 in hBMSC-Bone cells and upregulation on SERPINB2 and NOG. Transcriptomic data was associated with marked reduction in SMAD2 protein phosphorylation, which thereby implies the inactivation of TGFß and BMP signaling in those cells. Concordantly, activation of TGFß signaling in hBMSC-Bone cells using either recombinant TGFß1 protein or knockdown of SERPINB2 TGFß-responsive gene partially restored their osteoblastic differentiation potential. Similarly, the activation of BMP signaling using exogenous BMP4 or via siRNA-mediated knockdown of NOG partially restored the differentiation phenotype of hBMSC-Bone cells. Concordantly, recombinant NOG impaired ex vivo osteoblastic differentiation of hBMSC+Bone cells, which was associated with SERBINB2 upregulation. Our data suggests the existence of reciprocal relationship between TGFB and BMP signaling that regulates hBMSC lineage commitment and differentiation, whilst provide a plausible strategy for generating osteoblastic committed cells from hBMSCs for clinical applications.
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Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Clonais , Regulação para Baixo/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Serpinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
All-trans retinoic acid (RA) is a metabolite of vitamin A and has pleiotropic actions on many different biological processes, including cell growth and differentiation, and is involved in different aspects of fertility and developmental biology. In the current study, we investigated the effects of RA on camel (Camelus dromedarius) cumulus-oocyte complex in vitro maturation (IVM). IVM medium was supplemented with 0, 10, 20, and 40 µM RA. Application of 20 µM RA significantly reduced the proportion of degenerated oocytes and significantly improved oocyte meiosis and first polar body extrusion compared to the control and other experimental groups. Retinoic acid significantly reduced the mRNA transcript levels of apoptosis-related genes, including BAX and P53, and reduced the BAX/BCL2 ratio. In addition, RA significantly reduced the expression of the Transforming growth factor beta (TGFß) pathway-related transcripts associated with the actin cytoskeleton, ACTA2 and TAGLN; however, RA increased TGFß expression in cumulus cells. The small molecule SB-431542 inhibits the TGFß pathway by inhibiting the activity of activin receptor-like kinases (ALK-4, ALK-5, and ALK-7); however, combined supplementation with RA during IVM compensated for the inhibitory effect of SB-431542 on cumulus expansion, oocyte meiosis I, and first polar body extrusion in activated oocytes. The current study shows the beneficial effects of RA on camel oocyte IVM and provides a model to study the multifunctional mechanisms involved in cumulus expansion and oocyte meiosis, particularly those involved in the TGFß pathway.
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Células do Cúmulo/citologia , Técnicas de Maturação in Vitro de Oócitos , Oócitos/citologia , Tretinoína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Blastocisto/citologia , Camelus , Meios de Cultura , Células do Cúmulo/efeitos dos fármacos , Dioxóis/farmacologia , Técnicas de Cultura Embrionária , Feminino , Fertilidade , Técnicas de Transferência Nuclear , Oócitos/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
Endochondral ossification is the process by which long bones are formed; the process of long bone formation is regulated by numerous factors such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone Nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. Whole genome microarray data from our previous study revealed that most hHNR's are up-regulated during osteoblast differentiation in hMSCS. NR2F1 was among the highest expressed hHNR during osteogenesis, NR2F1 belongs to the steroid/thyroid hormone nuclear receptor superfamily. NR2F1 is designated as an orphan nuclear receptor because its ligands are unknown. NR2F1 plays a wide range of roles, including cell differentiation, cancer progression, and central and peripheral neurogenesis. Identifying signaling networks involved in osteoblast differentiation is important in orchestrating new therapeutic and clinical applications in bone biology. This study aimed to identify alterations in signaling networks mediated by NR2F1 in osteoblast differentiation. siRNA-mediated down-regulation of NR2F1 leads to impairment in the differentiation of hBMSC-TERT to osteoblast; gene-expression results confirmed the down-regulation of osteoblast markers such as RUNX2, ALPL, OSC, and BSP. Global whole gene expression analysis revealed that most down-regulated genes were associated with osteoblast differentiation (DDIT3, BMP2). Pathway analysis revealed prominent signaling pathways that were down-regulated, including the TGFß pathway and MAPK pathway. Functional studies on NR2F1 transfected cells, during osteoblast differentiation in combination with TGFß1 and BMP-2, showed that TGFß1 does not recover osteoblast differentiation, whereas BMP-2 rescues osteoblast differentiation in NR2F1 siRNA transfected cells. Thus, our results showed that BMP-2 could intervene in NR2F1 down-regulated signaling pathways to recover osteoblast differentiation.
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Proteína Morfogenética Óssea 2/genética , Fator I de Transcrição COUP/genética , Diferenciação Celular/genética , Fator de Crescimento Transformador beta1/genética , Desenvolvimento Ósseo/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , TransfecçãoRESUMO
The mammalian ovary is a highly dynamic organ, in which proliferation and differentiation occur constantly during the entire life span, particularly in camels that are characterized by a follicular wave pattern and induced ovulation. Granulosa cells are the main cells of mature follicles. Two distinct cell types, namely, the mural and cumulus granulosa cells are distinguished on the basis of antral fluid increase. The multipotency of follicular fluid and the luteinizing cell were recently demonstrated. However, reports regarding the plasticity of cumulus cells are lacking. We obtained cumulus cells from cumulus-oocyte complexes and showed that camel cumulus cells expressed stem cell mRNA transcripts (POU5A1, KLF4, SOX2, and MYC) and were able to differentiate into other non-ovarian follicular cell types in vitro, such as neurons, osteoblasts, and adipocytes. In contrast, removal of the ooplasm (oocytectemy) showed no effect on cumulus cell proliferation and differentiation. This is the first report to identify an invaluable source of multipotent stem cells, which is routinely discarded during in vitro embryo production. The plasticity and transdifferentiation capability of camel cumulus cells definitely requires attention as it provides a cheap biological experimental model for basic research in stem cells and for understanding ovarian differentiation, both of which are relevant for use in regenerative medicine and tissue engineering in humans and animals.
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Camelus , Células do Cúmulo/fisiologia , Células-Tronco Multipotentes/fisiologia , Folículo Ovariano/citologia , Animais , Diferenciação Celular , Plasticidade Celular/genética , Proliferação de Células , Células Cultivadas , Feminino , Genes myc/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Células-Tronco Multipotentes/química , Fator 3 de Transcrição de Octâmero/genética , RNA Mensageiro/análise , Fatores de Transcrição SOXB1/genéticaRESUMO
The dromedary camel (Camel dromedarius) is physiologically well adapted to life in hot, dry and barren land. In the present study, we report the tolerance of camel oocytes and cumulus cells to acute and chronic heat shock. Camel oocytes and cumulus cells were exposed to acute (45⯰C for 2â¯h) and chronic (45⯰C for 20â¯h) heat shock. Our results demonstrated that acute and chronic heat shock altered malondialdehyde concentration, which is a marker for oxidative stress. Furthermore, the heat shock reduced glutathione levels during in vitro oocyte maturation. The expression of two well-known heat shock proteins HSP70 and HSP90 were increased similarly in oocytes and cumulus cells after acute heat shock. Oocytes were less tolerant to the short acute heat shock, and showed decreased maturation, which leads to reduction in ooplasmic diameter and an increase in chromosomal count abnormalities. Furthermore, the pro-apoptotic genes P53 and BAX had increased expression levels, whereas for the anti-apoptotic gene such as BCL2 expression levels was decreased. On the other hand, the cumulus cells tolerated acute and chronic heat shock, as evident by the increase in HSP70 and HSP90 expression and steady expression levels of P53, BAX, and BCL2 after acute hyperthermia. Cumulus cells regained their vitality and ability to proliferate after chronic hyperthermia and showed wound healing capabilities after 9 days of chronic hyperthermia. Collectively, these results indicate the adaptive tolerance of camel somatic cells to acute and chronic heat shock, which is lethal to cells in many other mammals.
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Camelus/metabolismo , Células do Cúmulo/metabolismo , Febre/metabolismo , Resposta ao Choque Térmico , Oócitos/metabolismo , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Feminino , Proteínas de Choque Térmico HSP72/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Peroxidação de LipídeosRESUMO
TGFß is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs) is currently poorly understood. In the present study, we demonstrate that a single dose of TGFß1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGFß-mediated enhancement of lineage commitment, we compared the gene expression profiles of TGFß1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGFßl treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGFß1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGFß1 and cytochalasin D. Our study demonstrates that TGFß1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.
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We isolated and characterized trophoblast from in vivo-derived camel embryos and compared with embryonic stem-like cells. Camel embryos were flushed on day 8 post-insemination and used to derive trophectoderm and embryonic stem-like cells under feeder-free culture conditions using a basement membrane matrix. Embryos were evaluated for the expression of POU5F1, MYC, KLF4, SOX2, CDX2, and KRT8 mRNA transcripts by relative quantitative polymerase chain reaction. Camel embryos grew and expanded to â¼4.5 mm and maintained their vesicular shape in vitro for 21 days post-insemination. Trophoblast and embryonic stem-like cell lines grew under feeder-free culture conditions and showed distinct morphological criteria and normal chromosomal counts. Embryonic stem-like cells showed positive staining in the alkaline phosphatase reaction. Trophoblast cells showed a significant increase in CDX2, KRT8, KLF4, and SOX2 expression compared with embryonic stem-like cells and whole embryos. Embryonic stem-like cells showed a significant decrease in CDX2 expression and increase in SOX2 and KRT8 expression compared to embryonic expression. POU5F1 and MYC expression showed no difference between embryos and both cell lines. We characterized embryo survival in vitro, particularly the derivation of trophectoderm and embryonic stem-like cells, providing a foundation for further analysis of early embryonic development and placentation in camels.
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Camelus/embriologia , Embrião de Mamíferos/citologia , Animais , Técnicas de Cultura Embrionária , Feminino , GravidezRESUMO
TGF-ß1, a multifunctional regulator of cell growth and differentiation, is the most abundant bone matrix growth factor. During differentiation of human bone stromal cells (hBMSCs), which constitute bone marrow osteoblast (OS) and adipocyte (AD) progenitor cells, continuous TGF-ß1 (10 ng/ml) treatment enhanced OS differentiation as evidenced by increased mineralised matrix production. Conversely, pulsed TGF-ß1 administration during the commitment phase increased mature lipid-filled adipocyte numbers. Global gene expression analysis using DNA microarrays in hBMSCs treated with TGF-ß1 identified 1587 up- and 1716 down-regulated genes in OS-induced, TGF-ß1-treated compared to OS-induced hBMSCs (2.0 fold change (FC), p < 0.05). Gene ontology (GO) analysis revealed enrichment in 'osteoblast differentiation' and 'skeletal system development-associated' genes and up-regulation of several genes involved in 'osteoblastic-differentiation related signalling pathways'. In AD-induced, TGF-ß1-treated compared to AD-induced hBMSCs, we identified 323 up- and 369 down-regulated genes (2.0 FC, p < 0.05) associated with 'fat cell differentiation', 'fatty acid derivative biosynthesis process', 'fatty acid derivative metabolic process', and 'inositol lipid-mediated'. Serpin peptidase inhibitor, clade B (ovalbumin), member 2 (SERPINB2) was down-regulated 3-fold in TGF-ß1-treated hBMSCs. siRNA-mediated SERPINB2 inhibition enhanced OS and AD differentiation. Thus, TGF-ß signalling is important for hBMSC OS and AD differentiation and SERPINB2 is a TGF-ß-responsive gene that plays a negative regulatory role in hBMSC differentiation.
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Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Serpinas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Fenótipo , RNA Interferente Pequeno/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Understanding the regulatory networks underlying lineage differentiation and fate determination of human bone marrow stromal cells (hBMSC) is a prerequisite for their therapeutic use. The goal of the current study was to unravel the novel role of the low-density lipoprotein receptor-related protein 3 (LRP3) in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during the osteogenic induction of the imCL1 clone. Data from functional and gene expression assays demonstrated the role of LRP3 as a molecular switch promoting hBMSC lineage differentiation into osteoblasts and inhibiting differentiation into adipocytes. Interestingly, microRNA (miRNA) expression profiling identified miR-4739 as the most under-represented miRNA (-36.11 fold) in imCL1 compared to imCL2. The TargetScan prediction algorithm, combined with functional and biochemical assays, identified LRP3 mRNA as a novel target of miR-4739, with a single potential binding site for miR-4739 located in the LRP3 3' UTR. Regulation of LRP3 expression by miR-4739 was subsequently confirmed by qRT-PCR, western blotting, and luciferase assays. Over-expression of miR-4739 mimicked the effects of LRP3 knockdown on promoting adipogenic and suppressing osteogenic differentiation of hBMSCs. Hence, we report for the first time a novel biological role for the LRP3/hsa-miR-4739 axis in balancing osteogenic and adipocytic differentiation of hBMSCs. Our data support the potential utilization of miRNA-based therapies in regenerative medicine.
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Adipócitos/metabolismo , Células da Medula Óssea/citologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Regiões 3' não Traduzidas , Adipócitos/citologia , Adipogenia , Antagomirs/metabolismo , Sequência de Bases , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Proteínas Relacionadas a Receptor de LDL/antagonistas & inibidores , Proteínas Relacionadas a Receptor de LDL/genética , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Osteoblastos/citologia , Osteogênese , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Alinhamento de SequênciaRESUMO
Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional heterogeneity of hBMSC cultures. In the present study, we characterized in detail two hTERT-BMSC clonal cell populations termed here CL1 and CL2 that represent an opposing phenotype with respect to morphology, markers expression: alkaline phosphatase (ALP) and CD146, and ex vivo differentiation potential. CL1 differentiated readily to osteoblasts, adipocytes, and chondrocytes as shown by expression of lineage specific genes and proteins. Whole genome transcriptome profiling of CL1 versus CL2 revealed enrichment in CL1 of bone-, mineralization-, and skeletal muscle-related genes, for example, ALP, POSTN, IGFBP5 BMP4, and CXCL12. On the other hand, CL2 transcriptome was enriched in immune modulatory genes, for example, CD14, CD99, NOTCH3, CXCL6, CFB, and CFI. Furthermore, gene expression microarray analysis of osteoblast differentiated CL1 versus CL2 showed significant upregulation in CL1 of bone development and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures.
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BACKGROUND: Debridement and disinfection of the root canal system is a crucial step in endodontic procedures. The effectiveness of irrigation relies on both the mechanical flushing action and the ability of irrigants to dissolve tissue and kill bacteria. The objective of the present study is to evaluate and compare the cytotoxicity of QMix™ root canal irrigating solution on immortalized human bone marrow mesenchymal stem cells (hTERT-MSC-C1) and to compare it with that of sodium hypochlorite (NaOCl). METHODS: Immortalized human bone marrow mesenchymal stem cells (hTERT-MSCs) were exposed to QMix™ and NaOCl. Cell viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alamarBlue assays. The cell morphology was studied after two hours of exposure to QMix™ and NaOCl. Scanning electron microscopy (SEM) analyses were performed after 2- and 4-hour incubation periods. Finally, ethidium bromide/acridine orange (EB/AO) fluorescent stain was applied to the cells in the 8-chamber slides after they were incubated with the testing agents for 2 hours to detect live and dead cells. The observations were tabulated and analyzed statistically. RESULTS: QMix™ exposure resulted in a significantly higher percentage of cell viability than NaOCl in the MTT and alamarBlue assays at three time points compared to the control. The SEM analysis demonstrated minimal morphological changes associated with cells that were exposed to the QMix™ solution, with little shrinkage and fragmentation of the cell wall. The live/dead analysis showed that the number of live cells after exposure to QMix™ was similar to that of the untreated control. No cell structure could be observed with the NaOCl group, indicating cell lysis. CONCLUSION: Both the QMix™ and NaOCl solutions were toxic to human bone marrow MSCs. Each solution might have induced cell death in a different way as evidenced in the cell viability, SEM and fluorescent studies. The slower cell death induced by QMix™ might therefore be less aggressive and more acceptable to living tissues.