Characterization of canine mesenchymal stromal cell lines (MSC)
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ABSTRACT: The bone marrow microenvironment is a complex mixture of cells that function in concert to regulate hematopoiesis. Cellular components include fixed nonhematopoietic stromal elements (MSC) as well as monocytes and resident macrophages, which are derived from the hematopoietic stem cells. Clinical studies have shown healing effects, direct or indirect, from the injection of MSC int Keywords: Cell type comparison Canine bone marrow stromal cells were immortalized by transduction with a replication-defective recombinant retrovirus containing the human papilloma virus E6/E7 genes (pLXSN-16 E6E7), as described for human stromal cell lines in Roecklein and Torok-Storb (Blood 85:997-1005, 1995). Transduced clones were selected with 50 ug/ml G418 and resistant clones were isolated, grown to confluency and characterized for morphology, cytokine production and cell surface molecule expression. These cells are intended for clinical research in the canine transplantation model, as a complement for existing human stromal cell lines, for mitigation of graft-versus-host disease and radiation effects.
Project description:The bone marrow microenvironment is a complex mixture of cells that function in concert to regulate hematopoiesis. Cellular components include fixed nonhematopoietic stromal elements as well as monocytes and resident macrophages, which are derived from the hematopoietic stem cells. Although these monocyte-lineage cells are reported to modify stromal cell function, the reverse also occurs. Given the secretory capability of the monocyte/macrophage and their various potential functions, it is not surprising that stromal cells contained within a particular niche can modify monocyte gene expression and functional maturation. Experiment Overall Design: Monocytes were isolated from peripheral blood mononuclear cells from 2 different normal donors and cultured for 48h in conditioned medium (CM). The CM was collected from each of two functionally distinct human bone marrow stromal cell lines (HS5 and HS27a) representing different compartments of the bone marrow microenvironment (Roecklein BA, Torok-Storb B. Blood. 1995;85:997-1005). Four samples were analyzed, with two biological replicates for each CM.
Project description:Expression analysis of migrating and non-migrating mesenchymal stromal cells (MSC) in fetal bone marrow Keywords: fetal bone marrow, mesenchymal stromal cells, migration, gene expression, genomics Three biological replates for both migrating and non-migrating mesenchymal stromal cells (MSC) in fetal bone marrow
Project description:Human bone marrow mesenchymal stromal cells (BM-MSC) could be committed toward a functional lymphoid-like stroma by a combination of TNFalpha (TNF) and Lymphotoxin alpha1/beta2 (LT) (Amé-Thomas et al Blood 2007). Bone marrow and lymph node stromal cells support FL malignant cell recruitment and growth in particular after comittment to a lymphoid-like differentiation in vitro. In addition, more than 70% of FL patients exhibit a bone marrow involvment at diagnosis. We delineate using Affymetrix U133+2.0 microarrays the gene expression profile of BM-MSC obtained from FL patients (FL-MSC) and age-matched healthy donors (HD-MSC) in order to identify a specific FL-MSC signature. In addition, we used Affymetrix microarrays to define the gene expression signature of lymphoid-like stromal cells obtained from HD-MSC by treatment with TNF/LT in vitro. This TNF/LT signature was then used to interpret the gene expression profile of FL-MSC. GEP was performed on 10 BM-MSC from FL patients and 6 from healthy donors, treated or not with TNF(10 ng/mL)/LT(100ng/mL)
Project description:Reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is an epigenetic phenomenon. We have reprogrammed mesenchymal stromal cells (MSC) from human bone marrow by retrovirus-mediated overexpression of OCT-3/4, SOX2, c-MYC, and KLF4. This series summarizes gene expression profiles of eight iP-MSC clones derived from three different donors. These datasets were subsequently used for PluriTest analysis (Muller FJ, Schuldt B et al., Nat. Methods 2011; 8: 315-317) demonstrating that all iP-MSC clones were clearly associated with pluripotent cells. Eight iP-MSC clones derived from three different donors.
Project description:Tumor infiltrating neutrophils (TAN) have been shown to exert both pro- and anti-tumoral activities and their recruitment and polarization are triggered by tumor-derived signals. Resident mesenchymal stromal cells (MSC) could contribute to tumor-supportive cell niche and have been shown to display tumor-specific transcriptomic, phenotypic, and functional features compared to normal tissue. In our study, we investigate whether these two cell subsets establish a bidirectional crosstalk in the context of B-cell lymphoma. We used microarrays to explore how neutrophils could trigger the polarization of tumor-supportive stromal cells. Gene expression analysis were performed on stromal cells (MSC) derived from bone marrow (BM) or tonsil (Resto) of healthy donors. These BM-MSC (n=3) or Resto (n=3) were primed or not with neutrophils for 1 day to induce stromal modification.
Project description:Reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is an epigenetic phenomenon. We have reprogrammed mesenchymal stromal cells (MSC) from human bone marrow by retrovirus-mediated overexpression of OCT-3/4, SOX2, c-MYC, and KLF4. This series summarizes gene expression profiles of eight iP-MSC clones derived from three different donors. These datasets were subsequently used for PluriTest analysis (Muller FJ, Schuldt B et al., Nat. Methods 2011; 8: 315-317) demonstrating that all iP-MSC clones were clearly associated with pluripotent cells.
Project description:Bone-marrow mesenchymal stem cells (MSCs) are plastic adherent cells that can differentiate into various tissue lineages, including osteoblasts, adipocytes and chondrocytes. However, this progenitor property is not shared by all cells within the MSC population. In addition, MSCs vary in their proliferation capacities and expression of markers. Because of heterogeneity of CD146 expression in the MSC population, we compared CD146-/Low and CD146High cells under clonal and non-clonal (sorted MSCs) conditions to determine whether this expression is associated with specific functions. CD146-/Low and CD146High MSCs did not differ in colony-forming unit-fibroblast number, osteogenic and adipogenic differentiation or in vitro hematopoietic supportive activity. However, CD146-/Low clones proliferated slightly but significantly faster than did CD146High clones. In addition, a strong expression of CD146 molecule was associated with a commitment towards a vascular smooth muscle cell lineage with upregulation of calponin-1 expression. Thus, within a bone-marrow MSC population, certain subpopulations characterized by high expression of CD146, are committed toward a vascular smooth muscle cell lineage. Clonal MSC were selected on the basis of CD146 level at their surface (CD146Low and CD146 High) and non-clonal MSC were compared from 4 different donors.
Project description:Mesenchymal stromal cells (MSC) are ideal candidates for cell therapies, due to their immune-regulatory and regenerative properties. We have previously reported that lung-derived MSC are tissue-resident cells with lung-specific properties compared to bone marrow-derived MSC. Assessing relevant molecular differences between lung-MSC and bone marrow-MSC is important, given that such differences may impact their behavior and potential therapeutic use. Here, we present an in-depth mass spectrometry (MS) based strategy to investigate the proteomes of lung-MSC and bone marrow-MSC. The MS-strategy relies on label free quantitative data-independent acquisition (DIA) analysis and targeted data analysis using a MSC specific spectral library. We identified several significantly differentially expressed proteins between lung-MSC and bone marrow-MSC within the cell layer (352 proteins) and in the conditioned medium (49 proteins). Bioinformatics analysis revealed differences in regulation of cell proliferation, which was functionally confirmed by decreasing proliferation rate through Cytochrome P450 stimulation. Our study reveals important tissue-specific differences within proteome and matrisome profiles between lung- and bone marrow-derived MSC that may influence their behavior and affect the clinical outcome when used for cell-therapy.
Project description:Comparison of gene expression profiles of canine adipose and bone marrow derived mesenchymal stem cells by microarray Mesenchymal stem cells (MSC) from rodents and humans have been shown to suppress T cells by distinct primary pathways, with NO-dependent pathways dominating in rodents and IDO-dependent pathways dominating in humans. However, the immune suppressive pathways utilized by canine MSC have not been as thoroughly studied, nor have BM-MSC and Ad-MSC been directly compared for their immune modulatory potency or pathway utilization. Therefore, canine BM-MSC and Ad-MSC were generated in vitro and their potency in suppressing T cell proliferation and cytokine production was compared, as well as differential gene expression. Mechanisms of T cells suppression were also investigated for both MSC types. We found that BM-MSC and Ad-MSC were roughly equivalent in terms of their ability to suppress T cell activation. However, the two MSC types used both shared and distinct biochemical pathways to suppress T cell activation. Adipose-derived MSC utilized, TGF-β signaling pathways and adenosine signaling to suppress T cell activation, whereas BM-MSC used cyclooxygenase TGF-β, and adenosine signaling pathways to suppress T cell activation. These results indicate that canine MSC are distinct from human and rodent MSC terms of their immune suppressive pathways, relying primarily on cyclooxygenase and TGF-β pathways for T cell suppression, rather than on NO or IDO-mediated pathways.