Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of differentiating adipose cells by high-throughput single-cell RNA-Seq

ABSTRACT: Directed differentiation of cells in vitro is a powerful approach for dissection of developmental pathways, disease modeling and regenerative medicine, but analysis of such systems are complicated by heterogeneous and asynchronous cellular responses to differentiation-inducing stimuli. To enable deep characterization of heterogeneous cell populations, we developed an efficient digital gene expression profiling protocol that enables surveying of mRNA in thousands of single cells at a time. We then applied this protocol to profile 11,116 cells collected during directed differentiation of human adipose-derived stem/stromal cells. The resulting data reveals the major axes of cell-to-cell variation within and between time points and suggests a link between incomplete adipogenesis in vitro and adipocyte dysfunction in vivo. High-throughput single cell RNA-seq method applied to human adipose tissue-derived stromal/stem cells during differentiation towards an adipogenic fate

ORGANISM(S): Homo sapiens

SUBMITTER: Magali Soumillon

PROVIDER: E-GEOD-53638 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The side population (SP), recently identified in several normal tissues and in a variety of tumors, may comprise cells endowed with stem cell features. In this study, we investigated the presence of SP in epithelial ovarian cancer (EOC) and found it in 4 out of 6 primary cultures from xenotransplants, as well as in 9 out of 25 clinical samples analyzed. SP cells from one xenograft bearing a large SP fraction were characterized in detail and they were capable of recreate the full repertoire of cancer cell populations observed in the parent tumor. Moreover, SP cells had higher proliferation rates, were much less apoptotic compared to non-SP cells, and generated tumors more rapidly than non-SP cells. We also investigated the effects of interferon-alfa (IFN-alpha), a cytokine which has been widely used to treat solid tumors, on EOC cells and observed that IFN-alpha exerts marked anti-proliferative and pro-apoptotic effects on primary cultures containing SP cells. IFN-alfa-treatment invariably caused a dramatic reduction in SP size in tumor cell lines of different origin and in normal bone-marrow SP cells, associated with a distinctive transcriptional profile. Gene therapy with human IFN-alpha resulted in regression of established tumors bearing a large SP fraction, which was not observed when tumors bearing low SP levels were treated. These findings could have relevant clinical implications since they imply that tumors bearing large SP numbers - albeit rare - could be sensitive to IFN-alpha treatment. Experiment Overall Design: 4 Affymetrix microarrays: Experiment Overall Design: 2 technical replicates: Ovarian cancer, untreated Side Population, rep1 Experiment Overall Design: Ovarian cancer, untreated Side Population, rep2 Experiment Overall Design: + Experiment Overall Design: 2 technical replicates: Ovarian cancer, IFN-alpha treated Side Population, rep1 Experiment Overall Design: Ovarian cancer, IFN-alpha treated Side Population, rep2

Project description:From the results of microarray, 7616 miRNAs with significantly different expression ratios were selected when CNE-2-S-18/SP cells were treated with DC120. Among these, 80 miRNAs were up-regulated and 78 were down-regulated. We then chose 20 miRNAs through the qRT M-bM-^@M-^S PCR, and our results showed that 9 miRNAs were up-regulated after treatment with DC120, which was consistent with the results of the microarray. Among these, miR-30a were significantly up-regulated. These results indicated that miR 30a expression was up-regulated by DC120. We then investigated the role of miR 30a in DC120-treated cells, we explored its potential targets using a bioinformatics approach of complementary base pairing. The relative luciferase activity of the reporter that contained the wild type 3' UTR was significantly suppressed when miR 30a was contransfected. In contrast, the luciferase activity of the mutant reporter was unaffected by the contransfection of miR 30a, indicating that miR 30a suppressed Sox2 gene expression using the miR 30a binding sequence at the 3' UTR of the Sox2 gene. Additionally, the ectopic expression of miR 30a caused a decrease in Sox2 protein expression and mRNA levels in CNE-2-S-18 cells. These data demonstrate that miR 30a may directly target the Sox2 gene via seed matches on both 3' UTR. Taken together, our results demonstrate that DC120 regulates Sox2 via up-regulation of miR 30a. In this study, two samples were used to acquire the miRNA expression profiling. We analyzed the differences in miRNA profiles between control (DMSO solution) and DC120 treatment in CNE-2-S-18/SP cells from miRNA microarrays. The CNE-2-S-18/SP cells were separately cultured with a DMSO solution or 10M-NM-<mol/L DC120 for 15h, and total RNA from all of the cells was extracted. Then we chose 20 up-regulated miRNAs through the qRT M-bM-^@M-^S PCR, and our results showed that 9 miRNAs were up-regulated after treatment with DC120. Among these, miR-30a were significantly up-regulated. Then we investigated the role of miR 30a in DC120-treated cells by bioinformatic methods and Dual luciferase reporter system, the function of miR-30a was analyzed through ectopic expression of miR 30a. Taken together, our results demonstrate that DC120 regulates Sox2 via up-regulation of miR 30a.

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Characterization of differentiating adipose cells by high-throughput single-cell RNA-Seq

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