Real-time quantitative PCR analysis of human adipose derived stem cells microvesicles
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE59811 | GEO | 2014/08/08
SECONDARY ACCESSION(S): PRJNA256303
REPOSITORIES: GEO
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Project description:Adipose derived stem cells (ASCs) were preconditioned with endothelial differentiation medium (EDM) for four days and then incubated in endothelial basal medium (EBM) supplemented with 1% microvesicle (MV)-free FBS for two days. The conditioned medium was harvested for MV isolation. The small RNA extracted from the MVs was used for microRNA array. qPCR gene expression profiling; Two equal-amount small RNA samples were from two independent experiments.
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Project description:Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MV) derived from human liver stem cells (HLSC) were able to stimulate in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MV in the hepatocytes by an alpha4 integrin-dependent mechanism. However, when treated with RNase, MV despites their internalization were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA dependent effect. Microarray analysis and quantitative RT-PCR demonstrated that MV were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MV were found to accelerate the morphological and functional recovery of liver in a model of 70% hepatectomy in rats by inducing an hepatocytes proliferation that was abolished by RNase treatment. Using human AGO2 gene, which is shuttled by MV, as a reporter gene, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MV. This suggest a translation of the MV shuttled mRNA within hepatocytes of treated rats. Conclusion: these results suggest that MV derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets.
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Project description:Derivation and expansion of human umbilical cord blood-derived endothelial colony forming cells under serum-free conditions - a transcriptome analysis. Endothelial colony forming cells (ECFCs) were isolated from term umbilical cord blood units. ECFCs were expanded under standard, fetal bovine serum (FBS) containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture. Comparison of the expression patterns of ECFCs that were either cultured in FBS-containing medium or in serum-free medium (five replicates each).
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Project description:U937 monocyte cell line was differentiated using PMA. Differentiated U937 monocytes were exposed to MEM+10% FBS medium (untreated cells) and the same medium containing LPS+IFNgamma. The changes in the gene expression of cytokines and chemokines were evaluated using RT2 Profiler qPCR array
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Project description:Human adipose stem cells (ASCs) have been shown, in pre-clinical studies, to have therapeutic applicability in diverse fields, but a standard expansion method for clinical applications remains yet to be established. Isolated ASCs are typically expanded in medium containing fetal bovine serum (FBS). However, sera and other animal-derived culture reagents stage numerous safety issues in clinical therapy, including possible infections and severe immune reactions. By expanding the ASCs in medium containing human serum (HS), the problem can be eliminated. To define how allogeneic HS performs in ASC expansion compared to FBS, we used microarrays to explore differences in gene expression between human adipose stem cells expanded in allogeneic human serum and fetal bovine serum.
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Project description:Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MV) derived from human liver stem cells (HLSC) were able to stimulate in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MV in the hepatocytes by an alpha4 integrin-dependent mechanism. However, when treated with RNase, MV despites their internalization were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA dependent effect. Microarray analysis and quantitative RT-PCR demonstrated that MV were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MV were found to accelerate the morphological and functional recovery of liver in a model of 70% hepatectomy in rats by inducing an hepatocytes proliferation that was abolished by RNase treatment. Using human AGO2 gene, which is shuttled by MV, as a reporter gene, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MV. This suggest a translation of the MV shuttled mRNA within hepatocytes of treated rats. Conclusion: these results suggest that MV derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets. MV contained mRNA was submitted to microarray analysis not to define the amount of mRNA but only to define which transcripts were present. Total RNA was prepared from two independent preparation of vesicles. 250, 500 and 1000 ngs from each preparation were transformed in biotin-labeled cRNA. A simple statistical linear model was used to identify transcript signals linearly correlated to the increment of total RNA concentration used to prepare cRNA.
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Project description:Saliva is a convenient non-invasive source of liquid biopsy to monitor human health and diagnose diseases. In particular, extracellular vesicles (EVs) in saliva can potentially reveal clinically relevant information for systemic health. Recent studies have shown that RNA in saliva EVs could be exploited as biomarkers for disease diagnosis. However, there is no standardized protocol for profiling RNA in saliva EV nor clear guideline on selecting saliva fractions for biomarker analysis. To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers. Methods: To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers.
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Project description:Microvesicles (MV) are small membrane-bound particles comprised of exosomes and various sized extracellular vesicles. These are released by a number of cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and whether they influenced the differentiation of naïve monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including monocytes, endothelial cells, epithelial cells and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. We used GeneChip microarrays to examine changes in gene expression induced by MV in primary monocyte-derived macrophages (MDM) and in THP1 cells, and compare this to cells treated with GM-CSF and PMA, respectively.
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