A small molecule modulator of prion protein enhances proliferation and engraftment of mesenchymal stem cells and extends their lifespan upon serial passage in culture
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE31205 | GEO | 2012/01/30
SECONDARY ACCESSION(S): PRJNA145989
REPOSITORIES: GEO
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Project description:Human mesenchymal stem cells (hMSC) play an important role in the maintenance of bone and blood. Most protocols rely on their in vitro expansion prior to clinical use. However, several groups including our own have shown that hMSC lose proliferation and differentiation ability with serial passage in culture, limiting their clinical applications. Here we show that targeting prion protein (PrP) by chemical intervention delays this process. When PrP expression was knocked down, cultures showed significant reduction in proliferation and differentiation capacity. In contrast hMSC expanded in the presence of small molecule modulator of PrP expression, 3/689, showed extended lifespan up to 10 population doublings. Upon re-plating cultures contained more than double the number of clonogenic progenitors and showed a 10 fold increase in engraftment levels in bone marrow 5 weeks post-transplant. Human MSC treated with 3/689 showed enhanced protection from DNA damage and enhanced cell cycle progression. Gene expression profiling revealed upregulation of superoxide dismutase 2 in cells treated with 3/689, dependent on PrP expression, and suggests increased scavenging of reactive oxygen species as mechanism of action. These data point to PrP as a good target to delay loss of proliferation and differentiation of hMSC with expansion in culture. Gene expression changes in hMSC expanded in the presence of 10uM small molecule modulator of prion protein 3/689 or DMSO were analysed at passage 2 and passage 8 from 3 separate donors.
2012-01-30 | E-GEOD-31205 | biostudies-arrayexpress
Project description:Adult human mesenchymal stem cells (hMSCs) have shown promise as a valuable new therapeutic tool in a wide range of diseases. hMSCs from bone marrow stroma are currently isolated by their adherence to tissue culture treated polystyrene (TCP) and passaged multiple times on the same plastics until they are able to produce enough cells to be useful for research or clinical therapeutic trials. However, evidence in the literature has shown that culture on TCP can negatively alter hMSC function. Our aim was to expand hMSCs in an in vitro environment more closely resembling that of the hMSCs' native microenvironment to maximize proliferation while retaining therapeutic potential. We used decellularized hMSC-derived extracellular matrix (hMSC-ECM) to test hMSCs' maintenance of stem cell properties during in vitro expansion. We found that hMSC-ECM was able to increase hMSC proliferation while retaining the stem cells‘ immature state and increasing differentiation potential. In addition, the hMSC-ECM could be covalently cross-linked to polymer substrates and was effective in the isolation and expansion of hMSCs in the presence of fetal bovine serum and human serum. Using proteomics and transcriptomics, we were able to determine the mechanism behind the hMSCs’ increased proliferation was due to their ability to downregulate their otherwise required gene expression for ECM proteins by on hMSC-ECM. The effects of hMSC-ECM were largely hMSC specific and were not found with several other types of human cells. Providing a pre-formed in vitro niche for hMSCs can provide the cells with the critical components required for hMSC function during in vitro expansion.
2020-09-01 | GSE119096 | GEO
Project description:Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases, including osteoporosis and bone tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (MSCs). CASC2 knockdown using small interfering RNA (siCASC2) increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining levels, or the expression of early osteogenic transcripts including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at either transcriptional and protein level. Inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP levels. The results indicate that lncRNA CASC2 regulates late osteogenesis and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests lncRNA CASC2 as a potential new therapeutic target in bone mineralization.
2023-06-14 | PXD040463 | Pride
Project description:The aim of this study was to describe the gene expression patterns related to the differentiation and mineralization of bone-forming cells, including activation and/or repression of osteogenic or non-osteogenic pathways, remodeling of cell architecture, cell adhesion, cell communication, and assembly of extracellular matrix. The study implied patient selection, tissue collection, isolation and culture of human marrow stromal cells (hMSC) and osteoblasts (hOB), and characterization of bone-forming cells. RNA samples were collected at defined time points, in order to understand the regulation of gene expression during the processes of cell differentiation/mineralization that occur during bone repair. Transcriptome analysis was performed by using the Affymetrix GeneChip microarray technology platform and GeneChip® Human Genome U133 Plus 2.0 Array. Our results help to design a gene expression profile of bone-forming cells during specific steps of osteogenic differentiation. These findings offer an useful tool to monitor the behaviour of osteogenic precursors cultured in presence of exogenous stimuli, i.e. growth factors, or onto 3D scaffolds for bone engineering. Moreover, they can contribute to identify and clarify the role of new genes for a better understanding of the molecular mechanisms regulating osteogenesis. Experiment Overall Design: hMSC were derived from mononuclear cells (MNC) of bone marrow aspirates of four patients. MNC cultures were maintained in differentiation medium containing ascorbic acid-2 phosphate and dexamethasone, and hMSC were collected at different time points. The experimental protocol was specifically devised to mark five steps of hMSC differentiation (MD). The reference sample consisted in MNCs before the addition of differentiation medium (MD1).
2008-10-26 | E-GEOD-12265 | biostudies-arrayexpress
Project description:Abstract: Stem cell therapies are severely limited by poor cell survival and engraftment rates. A major hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human mesenchymal stem cells (hMSC) transplanted in murine bone defects. Integrin-specific hydrogels regulated hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a triple helical peptide targeting α2β1 integrin, prolonged hMSC survival and engraftment in a segmental bone defect and resulted in improved bone repair compared to other adhesive and control peptides. Moreover, integrin-specific hydrogels had diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin specificity can be engineered into synthetic hydrogel systems resulting in improved tissue healing by directing mesenchymal stem cell survival, engraftment, paracrine immunomodulatory potential, and reparative activities. Radial segmental defects were treated with hydrogels functionalized with adhesive peptide and encapsulated hMSC. The tissue within the defect space was explanted at 1 week post-transplantation and stored in RNAlater solution (Qiagen) until further processing. Samples were placed in Qiazol solution (Qiagen), lysed by probe sonication, and homogenized in QIAshredder columns (Qiagen). Total RNA was isolated using an RNAeasy Plus Micro kit (Qiagen), and RNA content and purity were measured by spectrophotometry (NanoDrop 1000). cDNA synthesis was performed on total RNA (100 ng) using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher). Quantitative PCR was performed using Fluidigm 96×96 nanofluidic arrays targeting a set of 96 transcripts (human or murine) to observe changes in bone, survival, inflammation, vascularization, and matrix markers. Primers used are listed in Supplementary Table 4. The genes were pre-amplified in a single 13-cycle PCR reaction for each sample with EvaGreen Mastermix (Fluidigm BioMark) following the manufacturer’s protocol. Sixty-three gene targets resulted in detectable qPCR results. All subsequent statistical analyses were carried out using JMP-Genomics (SAS Institute) using the basic gene expression workflow 62. Raw Ct values were imported into JMP-Genomics and normalized to mean Ct values across all genes for each sample for principal components analysis (PCA), assessment of the biological principal variance component contributions (PVCA), and hierarchical clustering using Ward’s method to identify sub-types of expression profile. Finally, one-way ANOVA was used to detect statistical differences followed by False Discovery Rate analysis using two-state linear step-up procedure of Benjamini, Krieger and Yekutieli. Results are presented as raw Ct values normalized to mean Ct values across all genes for a sample.
2019-12-09 | GSE141517 | GEO
Project description:The aim of this study was to describe the gene expression patterns related to the differentiation and mineralization of bone-forming cells, including activation and/or repression of osteogenic or non-osteogenic pathways, remodeling of cell architecture, cell adhesion, cell communication, and assembly of extracellular matrix. The study implied patient selection, tissue collection, isolation and culture of human marrow stromal cells (hMSC) and osteoblasts (hOB), and characterization of bone-forming cells. RNA samples were collected at defined time points, in order to understand the regulation of gene expression during the processes of cell differentiation/mineralization that occur during bone repair. Transcriptome analysis was performed by using the Affymetrix GeneChip microarray technology platform and GeneChip® Human Genome U133 Plus 2.0 Array. Our results help to design a gene expression profile of bone-forming cells during specific steps of osteogenic differentiation. These findings offer an useful tool to monitor the behaviour of osteogenic precursors cultured in presence of exogenous stimuli, i.e. growth factors, or onto 3D scaffolds for bone engineering. Moreover, they can contribute to identify and clarify the role of new genes for a better understanding of the molecular mechanisms regulating osteogenesis. Experiment Overall Design: hMSC were obtained from bone marrow aspirates of four patients. hMSC cultures were maintained in mineralization medium containing β-glycerophosphate, and collected at different time points. The experimental protocol was specifically devised to mark three steps of hMSC mineralization (MM). The reference sample consisted in confluent hMSCs before the addition of mineralization medium (MD4).
2008-10-21 | E-GEOD-12266 | biostudies-arrayexpress
Project description:Extracellular nucleotides are potent signaling molecules mediating cell-specific biological functions. We previously demonstrated that adenosine 5'-triphosphate (ATP) inhibits the proliferation while stimulating the migration, in vitro and in vivo, of human bone marrow-derived mesenchymal stem cells (BM-hMSC). Here, we investigated the effects of ATP on BM-hMSC differentiation capacity. Molecular analysis showed that ATP treatment modulated the expression of several genes (e.g. wnt-pathway-related genes) governing osteoblastic and adipogenic differentiation of MSCs. Functional studies demonstrated that ATP, under specific culture conditions, stimulated adipogenic and osteogenic differentiation by significantly increasing the lipid accumulation and the expression levels of the adipogenic master gene PPARγ (peroxisome proliferator activated receptor-gamma) and by promoting the mineralization and the expression of the osteoblast-related gene RUNX2 (Runt-related transcription factor 2), respectively.
2013-04-11 | GSE37237 | GEO
Project description:Extracellular nucleotides are potent signaling molecules mediating cell-specific biological functions. We previously demonstrated that adenosine 5'-triphosphate (ATP) inhibits the proliferation while stimulating the migration, in vitro and in vivo, of human bone marrow-derived mesenchymal stem cells (BM-hMSC). Here, we investigated the effects of ATP on BM-hMSC differentiation capacity. Molecular analysis showed that ATP treatment modulated the expression of several genes (e.g. wnt-pathway-related genes) governing osteoblastic and adipogenic differentiation of MSCs. Functional studies demonstrated that ATP, under specific culture conditions, stimulated adipogenic and osteogenic differentiation by significantly increasing the lipid accumulation and the expression levels of the adipogenic master gene PPARγ (peroxisome proliferator activated receptor-gamma) and by promoting the mineralization and the expression of the osteoblast-related gene RUNX2 (Runt-related transcription factor 2), respectively. BM-hMSCs cells were transiently exposed to ATP 1mM for 24 hours (ATP pre-treatment) before starting differentiation induction. Then, BM-hMSCs were cultured under adipogenic/osteogenic conditions. Gene Expression Profile was performed on differentiate cells after 3 weeks of induction culture.
2013-04-11 | E-GEOD-37237 | biostudies-arrayexpress
Project description:Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow-derived mesenchymal stromal cells (hMSCs) stimulate myeloma cell expansion (e.g., IL-6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination. We present an in vitro model using primary hMSCs co-cultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNAseq, cell viability and apoptosis. Results were correlated with gene expression data (n=837) and survival of myeloma patients (n=536). On dispersed hMSCs, INA-6 saturate hMSC-surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC-INA-6 interactions, which modulated apoptosis time-dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6). Isolated nMA-INA6 indicated hMSC-autonomy through superior viability after IL6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples. Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow-retentive phenotype with potential clinical relevance.
2024-04-11 | GSE261423 | GEO
Project description:We have employed whole genome microarray to identify changes in gene expression in human bone marrow MSCs (hMSC-TERT) sorted from seven days co-cultures with breast cancer cell lines (MCF7) and (BT-20)
2015-06-23 | GSE70103 | GEO