Dataset Information

Effects of Trichostatin A on the Chondrogenesis from Human Mesenchymal Stem Cells.

ABSTRACT: Histone deacetylase inhibitors (HDACi) are a class of compounds that suppress the function of histone deacetylases (HDACs). This study was performed to examine the effects of Trichostatin A (TSA), a typical HDACi, on chondrogenesis of human bone marrow mesenchymal stem cells (hBMMSCs) and related molecular pathways. After evaluating the concentration for cytotoxicity and HDAC activity, hBMMSCs underwent chondrogenic differentiation in pellet culture with or without TSA for 21 days. The weight of TSA-treated pellets was 25% lower than that of untreated pellets. DNA level was not significantly different, but glycosaminoglycan content per DNA level was lower in TSA-treated pellets than that of untreated pellets. Gene expression of the chondrogenic markers (SOX9, Aggrecan, and Col2A1) decreased by by 12.9-fold, 8.9-fold, and 7.6-fold respectively in TSA-treated pellets compared with that in TSA-untreated pellets. TSA-treated pellets had lower cell density and lower proteoglycan staining content compared with those of TSA-untreated pellets. A microarray analysis from TSA-treated pellets showed that 1,467 chondrogenic-related genes were downregulated and 1,524 were upregulated by more than 2-fold compared with TSA-untreated pellets. Col10A1, TGF-?3, and SOX9 decreased significantly by 10-fold, 2.1-fold, and 3.2-fold respectively in TSA-treated pellets compared with those in untreated pellets, whereas expression of BMP4 and FGFR3 increased significantly by 2.1-fold and 5.4-fold respectively. It is concluded that TSA inhibits chondrogenesis and does not seem to be useful for cartilage tissue engineering of hBMMSCs.

SUBMITTER: Lee J

PROVIDER: S-EPMC6171611 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effects of Trichostatin A on the Chondrogenesis from Human Mesenchymal Stem Cells.

Lee Jimin J Im Gun-Il GI

Tissue engineering and regenerative medicine 20170307 4

Histone deacetylase inhibitors (HDACi) are a class of compounds that suppress the function of histone deacetylases (HDACs). This study was performed to examine the effects of Trichostatin A (TSA), a typical HDACi, on chondrogenesis of human bone marrow mesenchymal stem cells (hBMMSCs) and related molecular pathways. After evaluating the concentration for cytotoxicity and HDAC activity, hBMMSCs underwent chondrogenic differentiation in pellet culture with or without TSA for 21 days. The weight of ...[more]

PMID: 30603496

Similar Datasets

Project description:Chondrogenesis of human mesenchymal stem cells (hMSCs) is an important biological process in many applications including cartilage tissue engineering. We investigated the glucose uptake characteristics of aggregates of hMSCs undergoing chondrogenesis over a 3-week period both experimentally and by using a mathematical model. Initial concentrations of glucose in the medium were varied from 1 to 4.5 g/L to mimic limiting conditions and glucose uptake profiles were obtained. A reaction-diffusion mathematical model was implemented and solved to estimate kinetic parameters. Experimental glucose uptake rates increased with culture time for aggregates treated with higher initial glucose concentrations (3 and 4.5 g/L), whereas they decreased or remained constant for those treated with lower initial glucose concentrations (1 and 2 g/L). Lactate production rate increased by as much as 40% for aggregates treated with higher initial glucose concentrations (2, 3 and 4.5 g/L), whereas it remained constant for those treated with 1 g/L initial glucose concentration. The estimated DNA-normalized maximum glucose uptake rate decreased by a factor of 9 from day 0-2 (12.5 mmol/s/g DNA) to day 6-8 (1.5 mmol/s/g DNA), after which it started to increase. On day 18-20, its value (17.5 mmol/s/g DNA) was about 11 times greater than its lowest value. Further, the extracellular matrix levels of aggregates at day 14 and day 21 correlated with their overall glucose uptake and lactate production. The results suggest that during chondrogenesis, for optimal results, cells require increasing amounts of glucose. Our results also suggest that diffusion limitations play an important role in glucose uptake even in the smaller size aggregate model of chondrogenesis. Further, the results indicate that glucose uptake or lactate production can be a tool for predicting the end quality of tissue during the process of chondrogenesis. The estimated kinetic parameters can be used to model glucose requirements in cartilage tissue engineering applications.

Project description:Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well-defined biological 'blank template' that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three-dimensional system. We customized the backbone of a Streptococcal collagen-like 2 (Scl2) protein with heparin-binding, integrin-binding, and hyaluronic acid-binding peptide sequences previously shown to modulate chondrogenesis and then cross-linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)- and aggrecanase (ADAMTS4)-cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross-linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross-linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel collagen-mimetic protein, cross-linked via multiple enzymatically degradable peptides, provides a highly adaptable and well defined platform to recapitulate a high degree of biological complexity, which could be applicable to numerous tissue engineering and regenerative medicine applications.

Project description:Purpose: Iron oxide based magnetic nanoparticles (MNP) are versatile tools in biology and medicine. Adipose derived mesenchymal stem cells (ADSC) and Wharton Jelly mesenchymal stem cells (WJMSC) are currently tested in different strategies for regenerative regenerative medicine (RM) purposes. Their superiority compared to other mesenchymal stem cell consists in larger availability, and superior proliferative and differentiation potential. Magnetic field (MF) exposure of MNP-loaded ADSC has been proposed as a method to deliver mechanical stimulation for increasing conversion to musculoskeletal lineages. In this study, we investigated comparatively chondrogenic conversion of ADSC-MNP and WJMSC with or without MF exposure in order to identify the most appropriate cell source and differentiation protocol for future cartilage engineering strategies. Methods: Human primary ADSC and WJMSC from various donors were loaded with proprietary uncoated MNP. The in vitro effect on proliferation and cellular senescence (beta galactosidase assay) in long term culture was assessed. In vitro chondrogenic differentiation in pellet culture system, with or without MF exposure, was assessed using pellet histology (Safranin O staining) as well as quantitative evaluation of glycosaminoglycan (GAG) deposition per cell. Results: ADSC-MNP complexes displayed superior proliferative capability and decreased senescence after long term (28 days) culture in vitro compared to non-loaded ADSC and to WJMSC-MNP. Significant increase in chondrogenesis conversion in terms of GAG/cell ratio could be observed in ADSC-MNP. MF exposure increased glycosaminoglycan deposition in MNP-loaded ADSC, but not in WJMSC. Conclusion: ADSC-MNP display decreased cellular senescence and superior chondrogenic capability in vitro compared to non-loaded cells as well as to WJMSC-MNP. MF exposure further increases ADSC-MNP chondrogenesis in ADSC, but not in WJMSC. Loading ADSC with MNP can derive a successful procedure for obtaining improved chondrogenesis in ADSC. Further in vivo studies are needed to confirm the utility of ADSC-MNP complexes for cartilage engineering.

Project description:Assessing the quality of tissue engineered (TE) cartilage has historically been performed by endpoint measurements including marker gene expression. Until the adoption of promoter-driven reporter constructs capable of quantitative and real time non-destructive expression analysis, temporal gene expression assessments along a timeline could not be performed on TE constructs. We further exploit this technique to utilize microRNA (miRNA or miR) through the use of firefly luciferase reporter (Luc) containing a 3' UTR perfect complementary target sequence to the mature miR-145-5p. We report the development and testing of a firefly luciferase (Luc) reporter responsive to miR-145-5p for longitudinal tracking of miR-145-5p expression throughout MSC chondrogenic differentiation. Plasmid reporter vectors containing a miR-145-5p responsive reporter (Luc reporter with a perfect complementary target sequence to the mature miR-145-5p sequence in the 3'UTR), a Luc reporter driven by a truncated Sox9 (one of the targets of miR-145-5p) promoter, or the Luc backbone (control) vector without a specific miRNA target were transfected into MSCs by electroporation. Transfected MSCs were mixed with untransfected MSC to generate chondrogenic pellets. Pellets were imaged by bioluminescent imaging (BLI) and harvested along a preset time line. The imaging signals from miR-145-5p responsive reporter and Sox9 promoter-driven reporter showed correlated time-courses (measured by BLI and normalized to Luc-control reporter; Spearman r=0.93, p=0.0002) during MSC chondrogenic differentiation. Expression analysis by qRT-PCR suggests an inverse relationship between miR-145-5p and Sox9 gene expression during MSC chondrogenic differentiation. Non-destructive cell-pellet imaging is capable of supplementing histological analyses to characterize TE cartilage. The miR-145-5p responsive reporter is relatively simple to construct and generates a consistent imaging signal responsive to miR-145-5p during MSC chondrogenesis in parallel to certain molecular and cellular events.

Project description:BackgroundStem cells are the main choice for seed cells in tissue engineering, but using most traditional stem cells requires invasive and complicated procedures. Human urine-derived stem cells (hUSCs) are an alternative stem cell source with the advantages of being isolated noninvasively and repetitively from the same individual. The aim of this study was to compare chondrogenesis-related biological behaviors between hUSCs and human bone marrow mesenchymal stem cells (hBMSCs) from the same individual.MethodshUSCs and hBMSCs were isolated from six patients who underwent iliac bone grafting. Cell morphology, proliferation, colony-forming, migration, and multidifferentiation analyses were performed in vitro. Then, acellular cartilage extracellular matrix (ACM) scaffolds were fabricated for in vivo implantation. The comparisons of cell viability, morphology, proliferation, and chondrogenesis between hUSCs and hBMSCs cultured on scaffolds were performed before implantation. The scaffolds loaded with hUSCs or hBMSCs were implanted into a rabbit knee model to repair cartilage defects. Magnetic resonance imaging (MRI) and micro-computed tomography (μCT) Analyses, inflammation and toxicity assays, gross observation, and histological evaluation were performed to evaluate the cartilage repair effects.ResultsIn in vitro experiments, hUSCs had better capacity for proliferation, colony-forming, and migration compared to hBMSCs in the same passage, while hBMSCs had greater osteogenic, adipogenic, and chondrogenic abilities compared to hUSCs in the same passage. Both hUSCs and hBMSCs at passage 3 had the strongest potential for proliferation, colony-forming, and multilineage differentiation compared to cells in other passages. The ACM scaffolds loaded with hUSCs or hBMSCs both significantly promoted the repair of cartilage defects in the rabbit knee model at 12 weeks' postimplantation, and the new tissue was mainly hyaline cartilage. However, there was no significant difference in cartilage repair effects between hUSCs and hBMSCs.ConclusionsIn in vitro experiments, hUSCs presented better capacity for proliferation, while hBMSCs had greater chondrogenic ability. However, hUSCs and hBMSCs had similar cartilage repair effects in vivo. Results indicated that hUSCs can be a stem cell alternative for cartilage regeneration and provide a powerful platform for cartilage tissue engineering and clinical transformation.

Dataset Information

Effects of Trichostatin A on the Chondrogenesis from Human Mesenchymal Stem Cells.

Publications

Effects of Trichostatin A on the Chondrogenesis from Human Mesenchymal Stem Cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets