Novel ex vivo culture of purified mouse mesenchymal stromal cells reveals tissue-dependent niche heterogeneity in bone marrow and functional interaction with macrophages
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ABSTRACT:
PROVIDER: PRJNA951395 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24043081.fastq.gz | Fastqsanger.gz | |||
| SRR24043082.fastq.gz | Fastqsanger.gz | |||
| SRR24043083.fastq.gz | Fastqsanger.gz | |||
| SRR24043084.fastq.gz | Fastqsanger.gz | |||
| SRR24043085.fastq.gz | Fastqsanger.gz |
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2025-05-14 | GSE228749 | GEO
Project description:Comprehensive transcriptional characterization of bone marrow stromal cells by RNA sequencing was performed to determine the molecular properties/signatures of endothelium during niche formation.Here, we identify a rare subset of cells in the human fetal BM that co-express endothelial and stromal markers, including low-affinity nerve growth factor receptor (LNGFR/CD271). They display transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT), reflected in their potential to generate stromal cells with in vivo BM niche forming capacity.
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Project description:Ex-vivo expanded mesenchymal stromal cells (MSCs) are increasingly used for paracrine support of hematopoietic stem cell (HSC) regeneration, but inconsistent outcomes have been the huddle for on-going clinical trials. Here, we hypothesized that the heterogeneity in the niche activity of manufactured MSCs can be a parameter for variable outcomes in MSC-based cell therapy. We first screened MSC culture medium and found that serum batches caused larger variations in colony forming unit-fibroblast (CFU-F) content of MSCs than individual donor variations. The culture conditions supporting high (stimulatory) and low (non-stimulatory) CFU-F caused distinct niche activity of MSCs; MSCs under stimulatory condition exhibited higher level expression of cross-talk molecules (Jagged-1 and CXCL-12) and higher support for HSCS during long-term culture than MSCs under non-stimulatory culture. Moreover, the effects of MSCs enhancing hematopoietic engraftment were only visible when HSCs were co-transplanted with MSCs expanded under stimulatory, but not non-stimulatory conditions. However, these differences of MSCs were readily reversed by switching the culture mediums, indicating their distinct functional state, rather than clonal heterogeneity. Accordingly, transcriptomic analysis showed distinct gene set enrichment between the different MSCs and revealed distinct upstream signaling pathways such as inhibition of P53 and activation of ATF4 for MSCs under stimulatory conditions. Taken together, our study shows that the heterogeneity in the niche activity of MSCs can be created during ex-vivo expansion to cause a difference in the hematopoietic engraftment and raise the possibility that MSCs can be pre-screened for more predictable outcomes in clinical trials of MSCs. Total RNA obtained from isolated human mesenchymal stromal cells. To compare stimulatory (SS) serum and non-stimulatory (NSS) serums, MSCs had been maintained in each serum media were sub-cultured for at least two passages before analysis.
2016-07-25 | E-GEOD-84754 | biostudies-arrayexpress
Project description:Direct contact with mesenchymal stromal impacts on migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex-vivo expansion Objective: To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133+ hematopoietic stem cells (HSCs) in terms of expansion potential differentiation, migratory capacity and gene expression profile. Methods: CD133+ purified HSCs were cultured for 7 days on subconfluent MSCs supplemented with growth factor containing medium. After ex-vivo expansion, non-adherent and adherent cells were collected and analyzed separately. Results: The adherent cells were found to have a more immature phenotype compared to the non-adherent fraction. CXCR4 was up regulated in the adherent fraction which was associated with a higher migration capacity towards a SDF-1 gradient. CFU-GM and LTC-IC assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell cycle control, motility, self-renewal and apoptosis were expressed at a higher level in the adherent fraction. Conclusion: Adhesion and direct cell-cell contact with a MSC feeder layer supports ex-vivo expansion, migratory potential and stemness of CD133+ HSCs. Keywords: co-culture hematopoietic stem cells (HSCs) on mesenchymal stromal cells (MSCs)
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Project description:Wnt/b-catenin signaling supports intestinal homeostasis by regulating proliferation in the crypt. Multiple Wnts are expressed in Paneth as well as other intestinal epithelial and stromal cells. Ex vivo, Wnts secreted by Paneth cells can support intestinal stem cells when Wnt signaling is enhanced with supplemental R-Spondin 1 (RSPO1). However, in vivo, the source of Wnts in the stem cell niche is less clear. Genetic ablation of Porcn, an endoplasmic reticulum resident O-acyltransferase that is essential for the secretion and activity of all vertebrate Wnts, confirmed the role of intestinal epithelial Wnts in ex vivo culture. Unexpectedly, mice lacking epithelial Wnt activity (PorcnDel/Villin-Cre mice) had normal intestinal proliferation and differentiation, as well as successful regeneration after radiation injury, indicating epithelial Wnts are dispensable for these processes. Consistent with a key role for stroma in the crypt niche, intestinal stromal cells endogenously expressing Wnts and Rspo3 support the growth of PorcnDel organoids ex vivo without RSPO1 supplementation. Conversely, increasing pharmacologic PORCN inhibition, affecting both stroma and epithelium, reduced Lgr5 intestinal stem cells, inhibited recovery from radiation injury, and at the highest dose fully blocked intestinal proliferation. We conclude that epithelial Wnts are dispensable, and that stromal production of Wnts can fully support normal murine intestinal homeostasis. Microarray was performed on samples enriched for stromal or epithelial cells from small intestine from Porcn(Del)/Villin-Cre and Porcn(WT)/Villin-Cre male C57Bl/6 mice.
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Project description:Ascites or solid tumour from patients with ovarian cancer was collected and grown in culture as ex vivo models. Each sample has a mixture of tumour and stromal cells which were separated into individual cultures. Therefore each patient has tumour and stromal cultures originating from the same tissue collection. Variant calling (exome-seq) analysis was performed on these matched models to establish tumour specific mutations. Stromal cells were used to rule out germline mutations.
2020-03-13 | E-MTAB-7225 | biostudies-arrayexpress
Project description:Haematopoietic stem cells (HSCs) reside in bone marrow (BM) niches where they are maintained to replenish all blood cell lineages throughout life. Among the constituents of the murine HSC niches, various cell types such as CXCL12-abundant reticular (CAR) cells, Nestin-GFP+ perivascular cells, leptin receptor (LepR)+ cells, endothelial cells, osteoblasts, sympathetic nerves, macrophages and megakaryocytes in the BM have been proposed to contribute to HSC niche activity. When niche cells are removed from their natural habitat, the ability to maintain HSCs ex vivo is markedly diminished. Mesenchymal-derived stromal cells (MSCs) identified by Nestin-GFP express high levels of niche factors in vivo, but their expression is downregulated rapidly upon culture, suggesting that alterations in transcriptional rewiring may contribute to the reduced HSC maintenance potential. We found that the enforced expression of 5 genes (Klf7, Ostf1, Xbp1, Irf3 and Irf7) markedly restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited boosted synthesis of HSC niche factors while retaining their mesenchymal lineage differentiation capacity. By contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed significantly higher repopulation capacity. To evaluate to what extent rMSCs are reprogrammed toward freshly isolated MSCs, we compared, by RNA-seq analysis, the transcriptome of freshly sorted CD45(-) Ter119(-) CD31(-) Scf-GFP(-) cells, CD45(-) Ter119(-) CD31(-) Scf-GFP(+) cells, rMSCs and control vector-transduced stroma. HSC niche-associated genes were highly expressed in both native Scf-GFP(+) stromal cells and rMSCs compared to the Scf-GFP cell fraction and control MSCs. With the motif analysis of ATAC-seq data, we found that myocyte enhancer factor 2c (Mef2c) is one of the key factor in the revitalization of MSCs. These results suggest that rMSC may provide a promising platform for curative transplantation therapies.
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Project description:Haematopoietic stem cells (HSCs) reside in bone marrow (BM) niches where they are maintained to replenish all blood cell lineages throughout life. Among the constituents of the murine HSC niches, various cell types such as CXCL12-abundant reticular (CAR) cells, Nestin-GFP+ perivascular cells, leptin receptor (LepR)+ cells, endothelial cells, osteoblasts, sympathetic nerves, macrophages and megakaryocytes in the BM have been proposed to contribute to HSC niche activity. When niche cells are removed from their natural habitat, the ability to maintain HSCs ex vivo is markedly diminished. Mesenchymal-derived stromal cells (MSCs) identified by Nestin-GFP express high levels of niche factors in vivo, but their expression is downregulated rapidly upon culture, suggesting that alterations in transcriptional rewiring may contribute to the reduced HSC maintenance potential. We found that the enforced expression of 5 genes (Klf7, Ostf1, Xbp1, Irf3 and Irf7) markedly restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited boosted synthesis of HSC niche factors while retaining their mesenchymal lineage differentiation capacity. By contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed significantly higher repopulation capacity. To evaluate to what extent rMSCs are reprogrammed toward freshly isolated MSCs, we compared, by RNA-seq analysis, the transcriptome of freshly sorted CD45(-) Ter119(-) CD31(-) Scf-GFP(-) cells, CD45(-) Ter119(-) CD31(-) Scf-GFP(+) cells, rMSCs and control vector-transduced stroma. HSC niche-associated genes were highly expressed in both native Scf-GFP(+) stromal cells and rMSCs compared to the Scf-GFP cell fraction and control MSCs. With the motif analysis of ATAC-seq data, we found that myocyte enhancer factor 2c (Mef2c) is one of the key factor in the revitalization of MSCs. These results suggest that rMSC may provide a promising platform for curative transplantation therapies.
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Project description:Ascites or solid tumour samples from patients with ovarian cancer were collected and grown in culture as ex vivo models of purified tumour cells. RNA-seq was performed on these models to establish gene expression profiles, which allow identification of genes that are differentially expressed between patients with differing tumour intrinsic properties. These samples have been interrogated for the presence of a gene expression signature indicative of sensitivity to an inhibitor of poly(ADP-ribose) glycohydrolase (PARG). These samples are processed in the same manner as previous studies: “E-MTAB-7223 - RNA-seq of human ex vivo ovarian cancer models with matched stromal cells” and “E-MTAB-10801 - RNA-seq of human ex vivo ovarian cancer models with matched stromal cells - part II” with no stromal counterparts included in this current sequencing batch.
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Project description:Direct contact with mesenchymal stromal impacts on migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex-vivo expansion Objective: To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133+ hematopoietic stem cells (HSCs) in terms of expansion potential differentiation, migratory capacity and gene expression profile. Methods: CD133+ purified HSCs were cultured for 7 days on subconfluent MSCs supplemented with growth factor containing medium. After ex-vivo expansion, non-adherent and adherent cells were collected and analyzed separately. Results: The adherent cells were found to have a more immature phenotype compared to the non-adherent fraction. CXCR4 was up regulated in the adherent fraction which was associated with a higher migration capacity towards a SDF-1 gradient. CFU-GM and LTC-IC assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell cycle control, motility, self-renewal and apoptosis were expressed at a higher level in the adherent fraction. Conclusion: Adhesion and direct cell-cell contact with a MSC feeder layer supports ex-vivo expansion, migratory potential and stemness of CD133+ HSCs. Keywords: co-culture hematopoietic stem cells (HSCs) on mesenchymal stromal cells (MSCs) Non-adherent and adherent fractions from three independent experiments were isolated and collected. Cells were stabilized in PreProtct™ buffer (Miltenyi Biotec, Germany) and stored at -80ºC. Samples were shipped to Miltenyi Biotec (Bergisch Gladbach, Germany) a Whole Human Genome expression analysis. Briefly RNA was extracted and overall quality of total RNA samples was checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). RNA samples were amplified and labelled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies). Non-adherent samples were pooled as “Non-adherent pool” and adherent samples were pooled as “adherent pool” and labelled with Cy3 and Cy5, respectively. Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner and the microarray image files were processed with The Agilent Feature Extraction Software (FES).
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