Distinctive Biological Properties Between Mesenchymal Stem Cells Spheroids and Clumps of Mesenchymal Stem Cells/Extracellular Matrix Complexes in 3D Culture Systems.
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE247989 | GEO | 2023/11/28
REPOSITORIES: GEO
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Project description:Background: Mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC), named EMSC here, have been found efficacious in animal models of autoimmune, inflammatory, and degenerative diseases. However, all the EMSC derivation methods reported so far are in two-dimensional (2D) culture systems, which are of low efficiency and high cost, difficult for large-scale production for research and therapeutic applications. Methods: We established a 3D system that allowed differentiation of hESC spheroids into MSC spheroids (EMSCSp) following treatment with BMP4 and A8301 for 5 days and subsequent culture in a MSC medium for about 15 days. All the procedures were conducted in one vessel without intermediate passaging. Results: EMSCsp cells were efficiently derived from hESC spheroids within 20 days in the 3D culture system, which could be scaled up from a small culture vessel to a 100-ml plastic bag. EMSCSp could further differentiate into spheroids of chondrocytes or adipocytes. EMSCSp could also reattach and form a 2D-monolayer culture (EMSCSp-ML). Compared to EMSC differentiated in monolayer, EMSCSp-ML had faster proliferation and higher yield, and developed less apoptosis and slower senescence. EMSCSp-ML also retained immune-modulatory effects in vitro and therapeutic effects on two mouse models of colitis. Conclusions: The 3D method provides a simple and economic system for large-scale production of EMSC as an unlimited source of the therapeutically promising cells.
2018-07-03 | GSE93218 | GEO
Project description:Compared to conventional monolayer cell culture, three-dimensional (or spheroid) cultures are more reflective of the in vivo environment and represent a better means of representing normal tissue. Therefore, understanding the biology of spheroid-derived cells is important for a more complete appreciation of in vivo tissue function. Although it has been shown that culture conditions such as nutrients, oxygen, cell passage etc. alter gene expression patterns of the cells, the gene expression profile of spheroid as compared to conventional monolayer culture in primary cells has not been studied. To investigate the effects of spheroid culture system on gene expression of primary cells, single cell RNA sequencing (scRNAseq) was performed on mouse dermal fibroblasts (tail/ear fibroblasts, TEFs) cultured as monolayers on cell culture dishes or grown as spheroids. After quality control, a total of 11,515 cells (8,022 spheroid cells and 3,493 cells of monolayer fibroblasts) were analyzed using Uniform Manifold Approximation and Projection (UMAP) which identified 2 largely separated libraries. These libraries contained cells with 2,491 and 8722 detected genes (nFeature_RNA) for monolayer and spheroid cells respectively. UMAP clustering of the integrated datasets identified 8 significant cell clusters (resolution=0.3) in which clusters 0, 1, 2, 4 and 7 had a higher number of cells in the spheroid library. Differential gene expression analyses revealed that each cluster was characterized by a specific transcriptional profile. Among cell clusters, cluster 4 exhibited significantly increased expression of collagen family genes, including Col1a2, Col3a1, Col1a1, Col5a2, Col4a, Col4a2, Col5a3, Col5a1, Col6a1, Col12a1 and Col15a1. Furthermore, expression of mesenchymal stem cell genes [Sca-1 (Ly6a), CD29 (Itgb1), CD44, CD90.1 (Thy1)] as well as a group of genes important for stem cell self-renewal including Notch2, Sox4, Sox9 Klf2, and Foxp1 were significantly increased in cluster 4. Immunofluorescence (IF) staining was performed to validate the protein expression of selected genes. Our work provides a significant advance in characterizing the global gene expression profile of spheroid culture of mouse TEFs compared to monolayer culture. We reached a high resolution at single-cell level, which enabled us to identify specific clusters associated with mesenchymal stem-like cells.
2023-10-30 | GSE241814 | GEO
Project description:Spheroids are 3D multi-cell aggregates formed in non-addherent culture conditions. In ovarian cancer (OC), they serve as a vehicle for cancer cell dissemination in the peritoneal cavity. We investigated genes and networks upregulated in three dimensional (3D) versus two-dimensional (2D) culture conditions by Affymetrix gene expression profiling and identified ALDH1A1, a cancer stem cell marker as being upregulated in OC spheroids. Network analysis confirmed ALDH1A1 upregulation in spheroids in direct connection with elements of the beta-catenin pathway. A parallel increase in the expression levels of beta-catenin and ALDH1A1 was demonstrated in spheroids vs. monolayers an in successive spheroid generations by using OC cell liness and primary OC cells. The percentage of Aldefluor positive cells was significantly higher in spheroids vs. monolayers in IGROV1, A2780, SKOV3, and primary OC cells. B-catenin knock-down decreased ALDH1A1 expression and chromatin immunoprecipitation demonstrated that beta-catenin directly binds to the ALDH1A1 promoter. Both siRNA mediated beta-catenin knock-down and a novel ALDH1A1 small molecule enzymatic inhibitor described here for the first time, decreased the number of OC spheroids (p<0.001) and cell viability. These data strongly support the role of beta-catenin regulated ALDH1A1 in the maintenance of OC spheroids and of a stem cell phenotype and propose new ALDH1A1 inhibitors targeting this cell population. Different gene profiles were observed in ovarian cancer spheroids versus ovarian cancer monolayers. Nine samples were analyzed in triplicate. Each group is a reference.
2014-01-03 | E-GEOD-53759 | biostudies-arrayexpress
Project description:Spheroids are 3D multi-cell aggregates formed in non-addherent culture conditions. In ovarian cancer (OC), they serve as a vehicle for cancer cell dissemination in the peritoneal cavity. We investigated genes and networks upregulated in three dimensional (3D) versus two-dimensional (2D) culture conditions by Affymetrix gene expression profiling and identified ALDH1A1, a cancer stem cell marker as being upregulated in OC spheroids. Network analysis confirmed ALDH1A1 upregulation in spheroids in direct connection with elements of the β-catenin pathway. A parallel increase in the expression levels of β-catenin and ALDH1A1 was demonstrated in spheroids vs. monolayers an in successive spheroid generations by using OC cell liness and primary OC cells. The percentage of Aldefluor positive cells was significantly higher in spheroids vs. monolayers in IGROV1, A2780, SKOV3, and primary OC cells. B-catenin knock-down decreased ALDH1A1 expression and chromatin immunoprecipitation demonstrated that β-catenin directly binds to the ALDH1A1 promoter. Both siRNA mediated β-catenin knock-down and a novel ALDH1A1 small molecule enzymatic inhibitor described here for the first time, decreased the number of OC spheroids (p<0.001) and cell viability. These data strongly support the role of β-catenin regulated ALDH1A1 in the maintenance of OC spheroids and of a stem cell phenotype and propose new ALDH1A1 inhibitors targeting this cell population.
2014-01-03 | GSE53759 | GEO
Project description:We encapsulated common marmoset pluripotent stem cells (cmPSCs) in agarose microgels and optimised culture conditions to generate epiblast and amnion spheroids. Using this system, we investigate the effects of signalling perturbations on epiblast and amnion spheroids as a model for early lineage specification
2022-07-06 | E-MTAB-10639 | biostudies-arrayexpress
Project description:For definitive endoderm differentiation, iPS cells (day0) were treated with 100 ng ml-1 activin A and 3μM CHIR99021 for 1 day and 100 ng ml-1 activin A for the following two days. Definitive endoderm was subsequently treated with 3uM CHIR99021 and 500ng ml-1 FGF4 for mid/hindgut differentiation. Mid/hindgut floating spheroids (fl; t-Spheroids) were collected from culture medium from day6 to day 8. On day6, mid/hindgut cells were dissociated into single cells and seeded onto EZSPHERE plate to generate suspension spheroids(EZ; s-Spheroids). RNA of fl spheroids and EZ spheroids were extracted using QIAGEN RNeasy kit.
2022-11-15 | GSE182230 | GEO
Project description:Therapeutic benefits of mesenchymal stem/stromal cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. The cellular environment of MSCs is of critical importance when directing paracrine activity. Typical 2D cultivation of stem cells on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion and cargo proteome composition and functional assessment. The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.
2021-11-17 | PXD029766 | Pride
Project description:To understand how interactions of myeloma cells with osteoclasts and mesenchymal stem cells in the bone marrow affect the clinical course of myeloma, we used microarrays to study changes in gene expression in freshly isolated myeloma plasma cells following co-cultures with osteoclasts (8 experiments) or with mesenchymal stem cells (13 experiments). Interaction with osteoclasts induced changes in the expression of 675 genes, and interaction with mesenchymal stem cells induced changes in the expression of 296 genes. Expression of only 58 genes commonly and similarly changed in both co-culture systems. Among these, we identified genes associated with overall, progression-free, and post-relapse survival, and developed survival prediction models. Gene expression data from 347 patients treated with total therapy 2 protocol, 433 with total therapy 3, and 98 patients who received various treatments (91 of them high-dose therapy with autologous stem cell support) were used for the analysis. Good predictive models were developed only for post-relapse survival, using genes involved in interaction with osteoclasts or with mesenchymal stem cells. The best predictive model used expression of first relapse of 33 probesets whose expression changed in myeloma cells following interaction with osteoclasts, with hazard ratios of 24, 20, and 12 for patients who relapsed following total therapy 2, total therapy 3 and the various other treatments, respectively. Among the probesets used for prediction, only 10, representing 8 genes, were commonly changed after both co-culture systems. These could present favorable target for therapy. Global gene expression profiling of primary multiple myeloma plasma cells (MMPCs) and mesenchymal stem cells (MSCs) before and after co-culture was done using Affymetrix microarrays. Thirteen MMPC and MSC co-culture experiments using MMPCs from 8 patients and MSCs from 5 healthy donors were performed.
2015-08-13 | E-GEOD-31159 | biostudies-arrayexpress
Project description:Human mesenchymal stem cells (MSC) derived from perirenal adipose tissue (PV) of living kidney donors were cultured under various conditions, namely (1) control (medium+foetal bovine serum(FBS)) or (2) control (medium+heat-inactivated FBS); (3) with mixed-lympohocyte reactions (MLR) in transwell culture systems for 4 days; (4) with mixed-lympohocyte reactions (MLR) in transwell culture systems for 7 days; or (5)with pro-inflammatory cytokines(IFNgamma, TNFalpha and interleukin 6). We used microarrays to detail the effect of the various culture conditions on MSC.
2010-10-01 | GSE18662 | GEO
Project description:Intestinal epithelial spheroids were isolated from mouse jejunum as described in our previous publication (Miyoshi and Stappenbeck. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nature Protocols. 2013. PMID: 24232249) and then grown in 50% L-WRN conditioned medium (for stem cells) or differentiation medium containing dmPGE2 (for wound-associated epithelial cells) or differentiation medium containing EP4 inhibitor (for enterocytes). Transcription profiling was performed to gain insight into the characteristics distinguishing these cell types.
2016-10-25 | E-MTAB-3952 | biostudies-arrayexpress