The goal of this study is to perform RNAseq in different sub-types of the zebrafish embryonic dorsal aorta (DA) at 28-30 hpf using TgBAC(runx1P2:Citrine);Tg(kdrl:mCherry) double-transgenic zebrafish embryos. A min. of 3000 cells per population were collected via FACS. RNA was isolated with the RNeasy Plus Micro Kit (QIAGEN, Cat No. 74034). High quality RNA (RIN > 8.0) was sent for RNAseq to the Wellcome Trust Centre for Human Genetics (WTCHG). 2.2 ng of total RNA was used to generate SMARTer libraries for low-input RNA. Sequencing was performed on an Illumina HiSeq4000 machine with a 75 bp paired end protocol. Sequenced reads were checked for base qualities, trimmed where 20% of the bases were below quality score 20, and filtered to exclude adapters using Trimmomatic (Version 0.32). Sequences were aligned to the Zebrafish Genome Zv10 with STAR with default parameters. Aligned read features were counted using Subread tool: featureCounts method (version 1.4.5-p1). To determine number of mapped reads we used the trimmed data. The alignment has been performed using STAR with default parameters. The number of mapped reads (QC-passed reads count) has been obtain using Samtools mapping statistics (flagstat tool). Overall design: Analysis of 5 different cell types; DN (double negative), SP-kdrl (single positive), DP-R1lo (double positive runx1 low expression), DP-R1med (runx1 medium expressionand) and DP-R1hi (runx1 high expression) in non-injected (Wt) TgBAC(runx1P2:Citrine);Tg(kdrl:mCherry) double-transgenic zebrafish embryos. Analysis was also done of the DN and DP-R1hi populations in runx1-morpholino (MO) injected embryos.
Blood stem cell-forming haemogenic endothelium in zebrafish derives from arterial endothelium.
Specimen part, Subject
View SamplesIn support of the investigation into the response of tissue resident phagocytes to sterile tissue damage in the peritoneal wall, different phagocyte populations were isolated from this tissue and RNA expression was measured using RNAseq. Overall design: Peritoneal wall phagocytes from three Cx3cr1gfp/+Ccr2rfp/+ animals were sorted into Cx3cr1-Ccr2rfp-, Cx3cr1-Ccr2rfp+, and Cx3cr1+Ccr2rfp- groups. Cells from three mice were sorted, and carried through the RNAseq protocol as three replicates per group.
Resident Macrophages Cloak Tissue Microlesions to Prevent Neutrophil-Driven Inflammatory Damage.
Specimen part, Subject
View SamplesAn important cellular defense mechanism against oxidative stress is the induction of genes involved in ROS resistance and DNA damage repair. Under normal conditions, Sod1 is localized mainly in the cytosol. However, we found that Sod1 translocates into the nucleus after oxidative stress.
Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance.
No sample metadata fields
View SamplesWe have employed whole genome microarray expression profiling to identify genes differentially expressed in cord blood enriched CD34+ cells(>95%) after a short-term exposure to the chemokine stromal cell-derived factor-1 (SDF-1).
The tetraspanin CD9 regulates migration, adhesion, and homing of human cord blood CD34+ hematopoietic stem and progenitor cells.
Specimen part
View SamplesPolycystic ovarian syndrome (PCOS) is an endocrine disorder of the reproductive and metabolic axis in women during the reproductive age. In this study, we used a rat model exhibiting reproductive and metabolic abnormalities similar to human PCOS to unravel the molecular mechanisms underlining this complex syndrome.
Polycystic ovarian syndrome is accompanied by repression of gene signatures associated with biosynthesis and metabolism of steroids, cholesterol and lipids.
Specimen part
View SamplesThe study compares gene expression profile at 20 days post amputation of the zebrafish ventricular heart between dusp6 mutant and WT siblings. Overall design: Ventricular resection was performed and 20 dpa, hearts were extracted.
Dusp6 attenuates Ras/MAPK signaling to limit zebrafish heart regeneration.
No sample metadata fields
View SamplesThe purpose of this experiment is to search for the transcriptional target of FRA1. Overall design: We subjected FRA1 knockdown and vector control Bel-7402 cells to RNA-sequencing, and analyzed the genes that were shown to be differentially expressed between the two groups.
Cancer-Associated Fibroblasts Regulate Tumor-Initiating Cell Plasticity in Hepatocellular Carcinoma through c-Met/FRA1/HEY1 Signaling.
No sample metadata fields
View SamplesLow-grade ovarian serous carcinomas are believed to arise via an adenoma-serous borderline tumor-serous carcinoma sequence. In this study, we found that advanced-stage, low-grade ovarian serous carcinomas both with and without adjacent serous borderline tumor shared similar regions of loss of heterozygosity. We then analyzed 91 ovarian tumor samples for mutations in TP53, BRAF, and KRAS. TP53 mutations were not detected in any serous borderline tumors (n = 30) or low-grade serous carcinomas (n = 43) but were found in 73% of high-grade serous carcinomas (n = 18). BRAF (n = 9) or KRAS (n = 5) mutation was detected in 47% of serous borderline tumors, but among the low-grade serous carcinomas (39 stage III, 2 stage II, and 2 stage I), only one (2%) had a BRAF mutation and eight (19%) had a KRAS mutation. The low frequency of BRAF mutations in advanced-stage, low-grade serous carcinomas, which contrasts with previous findings, suggests that aggressive, low-grade serous carcinomas are more likely derived from serous borderline tumors without BRAF mutation. In addition, advanced-stage, low-grade carcinoma patients with BRAF or KRAS mutation have a better apparent clinical outcome. However, further investigation is needed. Low-grade ovarian serous carcinomas are believed to arise via an adenoma-serous borderline tumor-serous carcinoma sequence. In this study, we found that advanced-stage, low-grade ovarian serous carcinomas both with and without adjacent serous borderline tumor shared similar regions of loss of heterozygosity. We then analyzed 91 ovarian tumor samples for mutations in TP53, BRAF, and KRAS. TP53 mutations were not detected in any serous borderline tumors (n = 30) or low-grade serous carcinomas (n = 43) but were found in 73% of high-grade serous carcinomas (n = 18). BRAF (n = 9) or KRAS (n = 5) mutation was detected in 47% of serous borderline tumors, but among the low-grade serous carcinomas (39 stage III, 2 stage II, and 2 stage I), only one (2%) had a BRAF mutation and eight (19%) had a KRAS mutation. The low frequency of BRAF mutations in advanced-stage, low-grade serous carcinomas, which contrasts with previous findings, suggests that aggressive, low-grade serous carcinomas are more likely derived from serous borderline tumors without BRAF mutation. In addition, advanced-stage, low-grade carcinoma patients with BRAF or KRAS mutation have a better apparent clinical outcome. However, further investigation is needed.
BRAF mutation is rare in advanced-stage low-grade ovarian serous carcinomas.
Specimen part
View SamplesErythropoiesis in mammals replenishes the circulating red blood cell (RBC) pool from hematopoietic stem/progenitor cells (HSPCs). Two distinct erythropoietic programs have been described. In the first trimester, hematopoietic precursors in the fetal yolk sac follow a primitive pattern of erythropoiesis. However, in the second trimester, hematopoietic stem cells (HSCs) from the fetal liver and later from the bone marrow differentiate by a definitive program of erythropoiesis to yield enucleated erythrocytes. RBCs can also be derived from human induced pluripotent stem cells (hiPSCs) and can express many of the red cell proteins required for normal erythrocyte function, presaging in vitro RBC production for clinical use. However, expansion and enucleation from hiPSCs is less efficient than with erythroblasts (EBs) derived from adult or cord blood progenitors. We hypothesized that substantial differential gene expression during erythroid development from hiPSCs compared to that from adult blood or cord blood precursors could account for these hitherto unexplained differences in proliferation and enucleation. We have therefore grown EBs from human adult and cord blood progenitors and from hiPSCs. Gene expression during erythroid culture from each erythroblast source was analyzed using algorithms designed to cluster co-expressed genes in an unsupervised manner and the function of differentially expressed genes explored by gene ontology. Using these methods we identify specific patterns of gene regulation for adult- and cord- derived EBs, regardless of the medium used, that are substantially distinct from those observed during the differentiation of EBs from hiPSC progenitors which largely follows a pattern of primitive erythropoiesis.
Distinct gene expression program dynamics during erythropoiesis from human induced pluripotent stem cells compared with adult and cord blood progenitors.
Specimen part
View SamplesThe supply of red blood cells (RBCs) is not sufficient in many developing countries or in developed countries for patients who need chronic transfusion from best-matched donors. Ex vivo expansion and maturation of human erythroid precursor cells (erythroblasts) could represent a potential solution. Proliferating erythroblasts can be expanded from human umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10^6-10^7 fold (in ~50 days) before undergoing senescence. Here, we report that ectopic expression of three to four genetic factors that have been used for iPS cell derivation enables CB-derived erythroblasts to undergo extended ex vivo expansion (10^51 fold in ~9 months) in a defined suspension culture condition without change of cell identity or function. These vastly expanding erythroblasts maintain homogeneously immature erythroblast phenotypes, a normal diploid karyotype and dependence on specific combination of cytokines and hormone for survival and proliferation throughout the continuous expansion period. When switched to a culture condition for terminal maturation, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes. Our result may ultimately lead to the development of unlimited sources of cultured RBCs for optimally-matched or personalized transfusion medicine.
Extensive ex vivo expansion of functional human erythroid precursors established from umbilical cord blood cells by defined factors.
Specimen part
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