Bulk and single-cell AML RNA-seq post ex vivo culture
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ABSTRACT:
PROVIDER: EGAS00001006265 | EGA |
REPOSITORIES: EGA
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Project description:Analyses of circulating tumor cells (CTC) cultured from blood of patients with cancer may allow individualized testing for susceptibility to therapeutic regimens. We established ex vivo cultures of CTCs from six patients with metastatic estrogen receptor-positive breast cancer and performed RNA-Seq on those cultures.
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Project description:Analyses of circulating tumor cells (CTC) cultured from blood of patients with cancer may allow individualized testing for susceptibility to therapeutic regimens. We established ex vivo cultures of CTCs from six patients with metastatic estrogen receptor-positive breast cancer and performed RNA-Seq on those cultures. One sample each from six metastatic estrogen receptor positive breast cancer patients
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Project description:Mouse LT-HSCs were co-cultured with polymer nanospheres for ex vivo expansion
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Project description:To further examine the gene expression of isolated primary mouse proximal tubular epithelial cells (mPTECs) during ex vivo culture, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish the key regulatory transcription factor which was significantly altered. Primary culuture of mPTECs were cultured on culture dishes for 1 and 3 days. As compared to freshly isolated mPTECs, a 1801-gene consensus signature was identified that distinguished between day 1 and and day 3 samples. Within these genes, 78 transcriptal factors were dramatically altered. Expression of three genes (KLF5, KLF4, and CCND1) from this signature was quantified in the same RNA samples by RT-PCR. The proliferation of mouse proximal tubular epithelial cells in ex vivo culture depends on matrix stiffness. Combined analysis of the microarray and experimental data revealed that Krüppel-like factor 5 (Klf5) was the most upregulated transcription factor accompanied by Krüppel-like factor 4 (Klf4) downregulation when cells on stiff matrix. These changes were reversed by soft matrix via ERK inactivation. Knockdown of Klf5 or forced-expression of Klf4 inhibited stiff matrix-induced cell spreading and proliferation, suggesting that Klf5/Klf4 act as positive/negative regulators, respectively. Moreover, stiff matrix-activated ERK increased the protein level and nuclear translocation of mechanosensitive Yes-associated protein 1 (YAP1), which is reported to prevent Klf5 degradation. Finally, in vivo model of unilateral ureteral obstruction (UUO) revealed that matrix stiffness-regulated Klf5/Klf4 is related to the pathogenesis of renal fibrosis. In the dilated tubules of obstructed kidney, ERK/YAP1/Klf5/Cyclin D1 axis were upregulated and Klf4 was downregulated. Inhibition of collagen crosslinking by lysyl oxidase inhibitor alleviated UUO-induced tubular dilatation and proliferation with preserving Klf4 and suppressing the ERK/YAP1/Klf5/Cyclin D1 axis. This study unravels a novel mechanism how matrix stiffness regulates cellular proliferation and highlights the importance of matrix stiffness-modulated Klf5/Klf4 in the regulation of renal physiological functions and fibrosis progression. Gene expression in cultured mPTECs was measured at 0, 1 and 3 days after culturing on culture dishes.
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Project description:Single-shot proteome analysis of 4 AML cell lines; 2 selinexor sensitive cell lines (GDM-1, MV4-11) and 2 resistant cell lines (NOMO-1 and PL-21) and ex vivo AML cells from 30 patients treated with DMSO (B; before treatment) or 1 uM selinexor (A; after treatment) for 6 hours.
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Project description:An ex vivo approach to studying hepatic schistosomiasis used thick naive mouse liver sections and sterile culturing. This tissue is unfixed, unfrozen, and alive for subsequent in vitro studies. We have cultured and monitored these sections for up to 48 hrs noting unchanged gross hepato-cellular morphology, and no significant increases in hepatotoxicity as demonstrated by media liver enzyme concentrations. Liver slices were treated with soluble egg antigen from Schistosoma japonicum eggs and cultured for 48hrs. Transcriptional changes were then analysed by microarray.
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Project description:Wide inter-individual variation in terms of outcome and toxic side effects of treatment exist among patients with AML receiving chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Drug resistance and relapse are considered major causes of treatment failure. Gene expression profiling was undertaken to address possible mechanisms of Ara-C/Dnr resistance. Based on ex vivo Ara-C cytotoxicity at diagnosis, Ara-C sensitive (IC50 <3uM AraC) and Dnr sensitive samples (IC50 < 0.5 uM) (5 samples each) were included for microarray analysis. These were compared with the samples which were drug resistant ex vivo at diagnosis. Our microarray experiment resulted in indentifying differentially expressed genes under ex vivo Ara-C sensitive as well as Dnr sensitive samples compared to ex vivo Drug resistant samples. One-color experiment,Organism: Homo sapiens, Custom Human Whole Genome 8x60k Array designed by Genotypic Technology Private Limited (AMADID: 27114), Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
2016-07-04 | E-GEOD-52919 | biostudies-arrayexpress
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.
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