Dataset Information

Validation of Osteogenic Properties of Cytochalasin D by High-Resolution RNA-Sequencing in Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissues.

ABSTRACT: Differentiation of mesenchymal stromal/stem cells (MSCs) involves a series of molecular signals and gene transcription events required for attaining cell lineage commitment. Modulation of the actin cytoskeleton using cytochalasin D (CytoD) drives osteogenesis at early timepoints in bone marrow-derived MSCs and also initiates a robust osteogenic differentiation program in adipose tissue-derived MSCs. To understand the molecular basis for these pronounced effects on osteogenic differentiation, we investigated global changes in gene expression in CytoD-treated murine and human MSCs by high-resolution RNA-sequencing (RNA-seq) analysis. A three-way bioinformatic comparison between human adipose tissue-derived MSCs (hAMSCs), human bone marrow-derived MSCs (hBMSCs), and mouse bone marrow-derived MSCs (mBMSCs) revealed significant upregulation of genes linked to extracellular matrix organization, cell adhesion and bone metabolism. As anticipated, the activation of these differentiation-related genes is accompanied by a downregulation of nuclear and cell cycle-related genes presumably reflecting cytostatic effects of CytoD. We also identified eight novel CytoD activated genes-VGLL4, ARHGAP24, KLHL24, RCBTB2, BDH2, SCARF2, ACAD10, HEPH-which are commonly upregulated across the two species and tissue sources of our MSC samples. We selected the Hippo pathway-related VGLL4 gene, which encodes the transcriptional co-factor Vestigial-like 4, for further study because this pathway is linked to osteogenesis. VGLL4 small interfering RNA depletion reduces mineralization of hAMSCs during CytoD-induced osteogenic differentiation. Together, our RNA-seq analyses suggest that while the stimulatory effects of CytoD on osteogenesis are pleiotropic and depend on the biological state of the cell type, a small group of genes including VGLL4 may contribute to MSC commitment toward the bone lineage.

SUBMITTER: Samsonraj RM

PROVIDER: S-EPMC6096350 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Validation of Osteogenic Properties of Cytochalasin D by High-Resolution RNA-Sequencing in Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissues.

Samsonraj Rebekah M RM Paradise Christopher R CR Dudakovic Amel A Sen Buer B Nair Asha A AA Dietz Allan B AB Deyle David R DR Cool Simon M SM Rubin Janet J van Wijnen Andre J AJ

Stem cells and development 20180723 16

Differentiation of mesenchymal stromal/stem cells (MSCs) involves a series of molecular signals and gene transcription events required for attaining cell lineage commitment. Modulation of the actin cytoskeleton using cytochalasin D (CytoD) drives osteogenesis at early timepoints in bone marrow-derived MSCs and also initiates a robust osteogenic differentiation program in adipose tissue-derived MSCs. To understand the molecular basis for these pronounced effects on osteogenic differentiation, we ...[more]

PMID: 29882479

Similar Datasets

Project description:Bone-marrow mesenchymal stem cells (BMSC) are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC). The abundance and ease of harvest make the adipose-derived stem cells (ASC) an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ?0.05 for overall interactions effect and P<0.001 between comparisons were used to determine differentially expressed genes (DEG). Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition, functional analysis data might indicate differences in therapeutic application.

Project description:Ex vivo regional gene therapy strategies using animal mesenchymal stem cells genetically modified to overexpress osteoinductive growth factors have been successfully used in a variety of animal models to induce both heterotopic and orthotopic bone formation. However, in order to adapt regional gene therapy for clinical applications, it is essential to assess the osteogenic capacity of transduced human cells and choose the cell type that demonstrates the best clinical potential. Bone-marrow stem cells (BMSC) and adipose-derived stem cells (ASC) were selected in this study for in vitro evaluation, before and after transduction with a lentiviral two-step transcriptional amplification system (TSTA) overexpressing bone morphogenetic protein 2 (BMP-2; LV-TSTA-BMP-2) or green fluorescent protein (GFP; LV-TSTA-GFP). Cell growth, transduction efficiency, BMP-2 production, and osteogenic capacity were assessed. The study demonstrated that BMSC were characterized by a slower cell growth compared to ASC. Fluorescence-activated cell sorting analysis of GFP-transduced cells confirmed successful transduction with the vector and revealed an overall higher but not statistically significant transduction efficiency in ASC versus BMSC (90.2 ± 4.06% vs. 80.4 ± 8.51%, respectively; p = 0.146). Enzyme-linked immunosorbent assay confirmed abundant BMP-2 production by both cell types transduced with LV-TSTA-BMP-2, with BMP-2 production being significantly higher in ASC versus BMSC (239.5 ± 116.55 ng vs. 70.86 ± 24.7 ng; p = 0.001). Quantitative analysis of extracellular deposition of calcium (Alizarin red) and alkaline phosphatase activity showed that BMP-2-transduced cells had a higher osteogenic differentiation capacity compared to non-transduced cells. When comparing the two cell types, ASC/LV-TSTA-BMP-2 demonstrated a significantly higher mineralization potential compared to BMSC/LV-TSTA-BMP-2 7 days post transduction (p = 0.014). In conclusion, this study demonstrates that transduction with LV-TSTA-BMP-2 can significantly enhance the osteogenic potential of both human BMSC and ASC. BMP-2-treated ASC exhibited higher BMP-2 production and greater osteogenic differentiation capacity compared to BMP-2-treated BMSC. These results, along with the fact that liposuction is an easy procedure with lower donor-site morbidity compared to BM aspiration, indicate that adipose tissue might be a preferable source of MSCs to develop a regional gene therapy approach to treat difficult bone-repair scenarios.

Project description:Silica nonwoven fabrics (SNFs) with enough mechanical strength are candidates as implantable scaffolds. Culture of cells therein is expected to affect the proliferation and differentiation of the cells through cell-cell and cell-SNF interactions. In this study, we examined three-dimensional (3D) SNFs as a scaffold of mesenchymal stem cells (MSCs) for bone tissue engineering applications. The interconnected highly porous microstructure of 3D SNFs is expected to allow omnidirectional cell-cell interactions, and the morphological similarity of a silica nanofiber to that of a fibrous extracellular matrix can contribute to the promotion of cell functions. 3D SNFs were prepared by the sol-gel process, and their mechanical properties were characterized by the compression test and rheological analysis. In the compression test, SNFs showed a compressive elastic modulus of over 1 MPa and a compressive strength of about 200 kPa. These values are higher than those of porous polystyrene disks used for in vitro 3D cell culture. In rheological analysis, the elastic modulus and fracture stress were 3.27 ± 0.54 kPa and 25.9 ± 8.3 Pa, respectively. Then, human bone marrow-derived MSCs were cultured on SNFs, and the effects on proliferation and osteogenic differentiation were evaluated. The MSCs seeded on SNF proliferated, and the thickness of the cell layer became over 80 μm after 14 days of culture. The osteogenic differentiation of MSCs on SNFs was induced by the culture in the commercial osteogenic differentiation medium. The alkaline phosphatase activity of MSCs on SNFs increased rapidly and remained high up to 14 days and was much higher than that on two-dimensional tissue culture-treated polystyrene. The high expression of RUNX2 and intense staining by alizarin red s after differentiation supported that SNFs enhanced the osteogenic differentiation of MSCs. Furthermore, permeation analysis of SNFs using fluorescein isothiocyanate-dextran suggested a sufficient permeability of SNFs for oxygen, minerals, nutrients, and secretions, which is important for maintaining the cell viability and vitality. These results suggested that SNFs are promising scaffolds for the regeneration of bone defects using MSCs, originated from highly porous and elastic SNF characters.

Dataset Information

Validation of Osteogenic Properties of Cytochalasin D by High-Resolution RNA-Sequencing in Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissues.

Publications

Validation of Osteogenic Properties of Cytochalasin D by High-Resolution RNA-Sequencing in Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissues.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure