Dataset Information

Enhancement of Glycosaminoglycan-Rich Matrix Production in Human Marrow-Derived Mesenchymal Stem Cell Chondrogenic Culture by Lithium Chloride and SB216763 Treatment.

ABSTRACT: OBJECTIVE:Cartilage mass produced from mesenchymal stem cell (MSC) differentiation would be a suitable candidate for use in regenerative medicine. Since the proper function of cartilage tissue is largely dependent on matrix glycosaminoglycan (GAG) contents, the objective of this study was to investigate the enhancing effect of two GSK3 inhibitors on the GAG content of cartilage produced by human marrow MSCs in vitro chondrogenesis. MATERIALS AND METHODS:MSCs that were used in this experimental study were derived from human marrow aspirates and confirmed using standard assays. Optimal concentrations of Lithium chloride and SB216763 were determined based on the yield of viable cell numbers in MSC cultures treated with varying concentrations of either Lithium chloride or SB216763. Passaged-3 MSCs were then centrifuged into small aggregates and provided with a chondrogenic medium supplemented with either lithium or SB216763 reagent at the optimal concentration determined in the previous experiment. Three weeks after, GAG contents of the culture were quantified and compared to each other and the control. RESULTS:According to our data, the cultures treated with 5 mM Lithium and 1 µM SB216763 tended to have comparatively more viable cells; therefore these concentrations were used in the differentiation experiments. The addition of either SB216763 or lithium to chondrogenic cultures appeared to significantly enhance cartilage matrix production. In SB216763 and Lithium-treated cultures average GAG concentrations were 6.17 ± 0.7 and 6.12 ± 1.1 µg/ml compared to 2.00 ± 0.3 µg/ml in the control (p<0.05). CONCLUSION:Using SB216763 and Lithium as supplements in human marrow MSC chondrogenic culture can lead to the production of cartilage mass high in GAG content.

SUBMITTER: Baghaban Eslaminejad M

PROVIDER: S-EPMC3584459 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Enhancement of Glycosaminoglycan-Rich Matrix Production in Human Marrow-Derived Mesenchymal Stem Cell Chondrogenic Culture by Lithium Chloride and SB216763 Treatment.

Baghaban Eslaminejad Mohamadreza M Karimi Negar N Shahhoseini Maryam M

Cell journal 20110824 2

<h4>Objective</h4>Cartilage mass produced from mesenchymal stem cell (MSC) differentiation would be a suitable candidate for use in regenerative medicine. Since the proper function of cartilage tissue is largely dependent on matrix glycosaminoglycan (GAG) contents, the objective of this study was to investigate the enhancing effect of two GSK3 inhibitors on the GAG content of cartilage produced by human marrow MSCs in vitro chondrogenesis.<h4>Materials and methods</h4>MSCs that were used in this ...[more]

PMID: 23508136

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Project description:BackgroundIndian hedgehog (IHH) and Sonic hedgehog (SHH) are important regulators of chondrogenesis. However, activation of IHH and SHH also promotes chondrocyte hypertrophy and ossification during chondrogenesis. The aims of this study were to investigate the effect of microgravity on IHH- and SHH-induced chondrogenic differentiation and to elucidate the role of microgravity in this process.MethodsAdenovirus plasmids encoding the rabbit IHH gene and SHH genes were constructed in vitro and transfected into rabbit bone marrow-derived mesenchymal stem cells (BMSCs). A rotary cell culture system (RCCS), in which a dynamic three-dimensional culture system combines the mechanical environment with a three-dimensional culture surface, was used for cell culture and differentiation. During the induction of differentiation, expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Toluidine blue and collagen II immunohistochemical staining and annexin V-Cy3 staining were used to indicate investigate cartilage matrix synthesis and hypertrophic hypertrophy, respectively, on day 21 after induction of differentiation.ResultsIn this study, IHH and SHH were shown to be equipotent inducers of chondrogenesis in rabbit BMSCs, as evidenced by strong staining for proteoglycans and collagen II, and increased expression of mRNAs and proteins associated with chondrogenesis in an RCCS environment. More importantly, chondrogenic hypertrophy and aging were effectively inhibited in the RCCS environment. In addition, levels of cartilage-related markers in the IHH and SHH transfection groups were initially increased and later decreased in the traditional two-dimensional environment, while cartilage hypertrophy-related factors revealed higher mRNA expression levels during induction.ConclusionsIn summary, microgravity significantly promoted chondrogenic differentiation of BMSCs induced by IHH and SHH and attenuated chondrogenic hypertrophy and aging during chondrogenesis. Furthermore, exogenous IHH and SHH had the same effect on chondrogenic differentiation of BMSCs in the RCCS environment. This study provides further evidence of chondrogenic induction of BMSCs in vitro via IHH and SHH gene delivery.

Project description:Epithelial?to?mesenchymal transition (EMT) confers stem cell?like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E?cadherin decreases, resulting in loss of cell?cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approaches. Western blot assay, quantitative real?time PCR and immunofluorescence were performed to investigate the expression of E?cadherin, vimentin, ??catenin, cytokeratin?20 and ?18, Twist1, Snail, CD44, cyclooxygenase?2 (COX2), Sox2, Oct4 and Nanog. Moreover, cell differentiation was induced by incubation with LiCl?containing medium for 10 days. We observed that these primary colorectal cancer (CRC) cells lost expression of the E?cadherin epithelial marker, which was instead expressed in cancer and normal colon mucosa of the same patient, while overexpressed vimentin (mesenchymal marker), Twist1, Snail (EMT markers) and COX2. Cytokeratin?18 was expressed both in tissues and cell cultures. Expression of stem cell markers, such as CD44, Oct4 and Nanog, were also observed. Following differentiation with the glycogen synthase kinase 3? (GSK3?) inhibitor LiCl, the cells began to express E?cadherin and, at once, Twist1 and Snail expression was strongly downregulated, suggesting a MET?reverting process. In conclusion, we established primary colon mesenchymal cancer cell cultures expressing mesenchymal and epithelial biomarkers together with high level of EMT transcription factors. We propose that they could represent a good model for studying EMT and its reverting mechanism, the mesenchymal?to?epithelial transition (MET). Our observation indicates that LiCl, a GSK3? inhibitor, induces MET in vitro, suggesting that LiCl and GSK3? could represent, respectively, interesting drug, and target for CRC therapy.

Dataset Information

Enhancement of Glycosaminoglycan-Rich Matrix Production in Human Marrow-Derived Mesenchymal Stem Cell Chondrogenic Culture by Lithium Chloride and SB216763 Treatment.

Publications

Enhancement of Glycosaminoglycan-Rich Matrix Production in Human Marrow-Derived Mesenchymal Stem Cell Chondrogenic Culture by Lithium Chloride and SB216763 Treatment.

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