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Gene expression changes in passage 1 mesenchymal stem cells from bone marrow stroma during 15 day culturing

ABSTRACT: To study the gene expression changes in mesenchymal stem cells from bone marrow stroma (MSCs) during in vitro expansion (from low density), passage 1 MSC were grown in culture for 15 days with medium change every 2-3 days. Samples for microarrays were taken at day 5 (early log-phase), 10 (late log-phase) and 15 (stationary phase). The data was queried for expression changes in Wnt signaling molecules and cell surface markers. Several components of the canonical Wnt signaling pathway were expressed, including Dkk-1; Wnt-5a; alpha-catenin; beta-catenin; frizzled 1, 4, 6, and 7; disheveled; glycogen synthetase kinase 3 beta; and glycogen synthetase kinase 3 alpha. In addition, the expression of over 10 cell surface transcripts decreased and an almost equal number increased during expansion. The two of the transcripts with the largest decreases coded for proteins previously shown to be linked to cell motility and tumor progression: PODXL, and alpha6-integrin (CD49f). As the cultures expanded, the largest increase was for mRNA for the cell adhesion protein VCAM-1. To study the gene expression changes in more detail, real-time RT-PCR, RT-PCR, ELISAs, FACS, and western blotting were performed for additional MSC donors. The results demonstrated dramatic changes in the transcriptome of MSCs during in vitro expansion.

ORGANISM(S): Homo sapiens

PROVIDER: GSE12800 | GEO | 2008/09/19

SECONDARY ACCESSION(S): PRJNA110995

REPOSITORIES: GEO

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Transcription profiling of human passage 1 mesenchymal stem cells from bone marrow stroma during 15 day culturing

Project description:To study the gene expression changes in mesenchymal stem cells from bone marrow stroma (MSCs) during in vitro expansion (from low density), passage 1 MSC were grown in culture for 15 days with medium change every 2-3 days. Samples for microarrays were taken at day 5 (early log-phase), 10 (late log-phase) and 15 (stationary phase). The data was queried for expression changes in Wnt signaling molecules and cell surface markers. Several components of the canonical Wnt signaling pathway were expressed, including Dkk-1; Wnt-5a; alpha-catenin; beta-catenin; frizzled 1, 4, 6, and 7; disheveled; glycogen synthetase kinase 3 beta; and glycogen synthetase kinase 3 alpha. In addition, the expression of over 10 cell surface transcripts decreased and an almost equal number increased during expansion. The two of the transcripts with the largest decreases coded for proteins previously shown to be linked to cell motility and tumor progression: PODXL, and alpha6-integrin (CD49f). As the cultures expanded, the largest increase was for mRNA for the cell adhesion protein VCAM-1. To study the gene expression changes in more detail, real-time RT-PCR, RT-PCR, ELISAs, FACS, and western blotting were performed for additional MSC donors. The results demonstrated dramatic changes in the transcriptome of MSCs during in vitro expansion. Experiment Overall Design: Cells from one MSC donor were used for expansion in vitro and samples were taken at day 5, 10, and 15. Total of 3 samples were run on microarrays (day 5, 10 and 15).

2008-10-25 | E-GEOD-12800 | biostudies-arrayexpress

Epidermal growth factor activates β-catenin via integrin-linked kinase to control proliferation of mesenchymal stromal cells.

Project description:Mesenchymal stromal cells (MSCs) are located in bone marrow where they help to maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal linages. The signalling mechanisms that enable precise control of MSC function remain unclear. Here, we have identified a non-canonical epidermal growth factor (EGF) signalling pathway in MSCs, which acts via integrin-linked kinase (ILK) to activate β-catenin, a key component of Wnt signalling. EGF induces nuclear translocation of β-catenin in MSCs but does not drive T cell factor (TCF)-mediated transactivation of Wnt target genes, and we demonstrate by Design of Experiments statistical analysis that the EGF/β-catenin and Wnt/β-catenin pathways do not cross-talk following co-stimulation with multiple concentrations of both ligands. By examining EGF-regulated genes in MSCs by RNA-Sequencing, we identified gene sets that were exclusively regulated by the EGF/b-catenin pathway, which were distinct from canonical EGF-regulated genes. In contrast, the expression of subsets of canonical EGF signalling gene targets were significantly influenced by b-catenin activation. These newly-identified EGF signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by canonical EGF signalling.

2018-10-24 | GSE121697 | GEO

Expression data from human fetal cardiac MSCs

Project description:The instrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterised. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts with only minor fluctuations over time in culture (from day 15 to day 48). We used microarray to compare the gene-expression profile of cultured human fetal cardiac MSCs over time (from day 15 to day 48). MSCs from human fetal hearts were cultured on GelTrex in a defined medium stimulating the canonical Wnt/beta-catenin pathway. Samples from three different time points (day 15, 27 and 48) were compared on microarray.

2016-02-19 | E-GEOD-78047 | biostudies-arrayexpress

Notch2 controls non-autonomous Wnt-signalling in chronic lymphocytic leukaemia

Project description:The Wnt-signalling pathway is one of the core de-regulated pathways in chronic lymphocytic leukemia (CLL), activated in a subset of patients by somatic coding mutations. Here we describe an alternative mechanism of Wnt-activation in malignant B cells, mediated by Notch2 activity in mesenchymal stromal cells (MSC) in the tumor microenvironment. We identified that tumor cells specifically induce and activate Notch2 in MSCs. Notch2 orchestrates the expression of target genes essential for the activation of canonical Wnt-signaling in CLL cells. Mechanistically, stromal Notch2 mediates the stabilization of â-catenin by inhibiting the activation of Gsk3-â in malignant B cells. Pharmacological inhibition of the Wnt-pathway mitigates microenvironment-mediated survival of malignant B cells in vitro. Similarly, inhibition of Notch-signaling impaired survival of CLL cells and disease engraftment in a PDX mouse model. Notch2 activation in the tumour microenvironment is a pre-requisite for the GSK3-â dependent activation of the canonical Wnt-signaling in tumor cells.

2018-09-19 | PXD010401 | Pride

Transcription profiling of human MSCs during expansion

Project description:Human mesenchymal stem cells or multipotent stromal cells (MSCs) are of interest for clinical therapy, in part because of their capacity for proliferation and differentiation. However, results from clinical trials and in vitro models have been variable, possibly due to MSC heterogeneity and a lack of standardization between MSC in vitro expansion protocols. Here we defined changes in MSCs during expansion in vitro. In low density cultures, MSCs expand through distinct lag, exponential growth and stationary phases. We assayed cultures of passage 2 human MSCs from three donors at low density (50 cells/cm2) at about 5% confluence on Day 2 and after the cultures had expanded to about 70% confluence on Day 7. On Day 2 genes involved in cell division were up-regulated. On Day 7 genes for cell development were up-regulated. The variations between three donors were less than the variation within the expansion of MSCs from a single donor. The microarray data for selected genes were confirmed by real-time PCR, ELISA and FACScan. About 50% of cells at Day 2 were in S-phase compared to 10% at Day 7. The results demonstrated major differences in early and late stage cultures of MSCs that should be considered in using the cells in experiments and clinical applications. Experiment Overall Design: We assayed cultures of passage 2 human MSCs from three donors at low density (50 cells/cm2) at about 5% confluence on Day 2 and after the cultures had expanded to about 70% confluence on Day 7.

2008-06-16 | E-GEOD-9520 | biostudies-arrayexpress

Transcriptome changes in hMSCs during expansion

2007-11-07 | GSE9520 | GEO

RNAseq data of differentiated vs undifferentiated tonsil-derived mesenchymal stem cells into parathyroid-like cells seeded at 100% confluency

Project description:High-cell density/confluency leads to contact inhibition of proliferation, as seen in the majority of epithelial cells, and is associated with the initiation of differentiation. To determine potential effect of In this regard, we analyzed transcriptomes using RNAseq, then performed a large-scale expression profiling of T-MSCs and performed a comparison between un-differentiated T-MSCs and T-MSC-PTHCs. Genes with a mean fold change of > 1.5 (T-MSCs vs. T-MSC-PTHCs) with p < 0.05 were analyzed using gene ontology enrichment analysis.RNAseq analysis showed that total of 228 genes were found to be significantly changed. Of them, the genes involved in the proliferation contributed 28% (total count of 63 genes) was the largest of the altered gene population followed by the genes involved in neurogenesis (30 counts; 13%) and extracellular matrix (16 counts; 7%). Among the genes in differentiation, WNT2 showed the highest fold-increase (2.8-fold) in the T-MSC-PTHCs compared to un-differentiated T-MSCs. When KEGG pathway analyses were performed, we found WNT signaling and stem cell pluripotency-regulating pathways as the pathways, where the WNT2 was involved in. This indicates that the differentiation of the T-MSCs could have induced through both of these pathways.

2022-01-10 | GSE193108 | GEO

RNA-sequencing and ChIP-sequencing in murine mesenchymal stem cells treated with Ezh2 inhibitor GSK126 or β-catenin siRNA

Project description:During mesenchymal stem cell (MSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin is thought to play a critical role in Wnt effects. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chip-seq showed that β-catenin associated with the promoter of EZH2, a key component of the PRC2 complex that governs genome methylation. Knocking down β-catenin lowered EZH2 levels and H3K27me3 at osteogenic loci. Further, when EZH2 was inhibited, β-catenin ’s anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin effects on MSC to preserve MSC multipotentiality.

2019-10-17 | GSE138980 | GEO

LNGFR- and Thy-1-based prospective isolation of human mesenchymal stem cells reveals functionally distinct subpopulations.

Project description:Human multipotent mesenchymal stem cells (MSCs), isolated based on their adherence to plastic, show poor growth and differentiation and frequently contain contaminating cells, which limits to clarify their own characteristics. In this study, the identification of two cell surface markers, LNGFR and Thy-1, allowed the prospective isolation of highly purified clonogenic human MSCs. Furthermore, single cell sorting assays followed by in vitro expansion revealed three distinct MSC subpopulations: rapidly-, moderately- and slowly proliferating MSC clones (RPCs, MPCs and SPCs, respectively). While RPCs exhibited robust multilineage differentiation and self-renewal potency, MPCs and SPCs contained a majority of senescent cells and exhibited frequent genome errors. Single cell sorting assays followed by in vitro expansion revealed three distinct MSC subpopulations: rapidly-, moderately- and slowly proliferating MSC clones (RPCs, MPCs and SPCs, respectively). Sample: RPC, MPC and SPC (n=3).

2011-12-22 | E-GEOD-34484 | biostudies-arrayexpress

Wnt Pathway Contributes to the Protection of Acute Lymphoblastic Leukemia Cells by Bone Marrow Stromal Cells and May Be a Therapeutic Target

Project description:Acute lymphoblastic leukemia (ALL) is the most common cancer in children and one of the most common cancers in adults. Although most children with ALL can be cured, approximately 60% of adults with ALL develop drug resistance or experience relapse and cannot be cured with traditional chemotherapy. One possible reason that treatment fails to eradicate ALL cells is that residual leukemia cells are protected by various components of the bone marrow microenvironment, such as marrow stromal cells (MSCs). To evaluate the effects of MSCs on ALL cell survival and response to chemotherapy, we co-cultured ALL cells with human or murine MSCs. We found that both human and murine MSCs protected human ALL cell lines and primary ALL cells from spontaneous and Ara-C-induced apoptosis. MSCs also modulated the cell cycle and increased ALL cell proliferation. Specifically, we found that Wnt signaling activation contributed to MSC-mediated drug resistance. In contrast, blocking the Wnt pathway led to decreased ALL cell viability. Chemotherapy plus the β-catenin inhibitor XAV939 resulted in a decreased tumor burden and improved overall survival in an ALL mouse model compared with chemotherapy alone. Our data demonstrate that targeting the Wnt pathway may represent an innovative approach to the treatment of ALL.

2014-01-01 | GSE36372 | GEO

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