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SRP188088
Homo sapiens
19 Downloadable Samples
Illumina HiSeq 2000
Description
Background & Aims Hepatocytes differentiated from human embryonic stem cells (hESCs) have the potential to overcome the shortage of primary hepatocytes for clinical use and drug development. Many strategies for this process have been reported, but the functionality of the resulting cells is incomplete. We hypothesize that the functionality of hPSC-derived hepatocytes might be improved by making the differentiation method more similar to normal in vivo hepatic development. Methods We tested combinations of growth factors and small molecules targeting candidate signaling pathways culled from the literature to identify optimal conditions for differentiation of hESCs to hepatocytes, using qRT-PCR for stage-specific markers to identify the best conditions. Immunocytochemistry was then used to validate the selected conditions. Finally, induction of expression of metabolic enzymes in terminally differentiated cells was used to assess the functionality of the hESC-derived hepatocytes. Results Optimal differentiation of hESCs was attained using a 5-stage protocol. After initial induction of definitive endoderm (stage 1), we showed that inhibition of the WNT/ß-catenin pathway during the 2nd and 3rd stages of differentiation was required to specify first posterior foregut, and then hepatic gut cells. In contrast, during the 4th stage of differentiation, we found that activation of the WNT/ß-catenin pathway allowed generation of proliferative bipotent hepatoblasts, which then were efficiently differentiated into hepatocytes in the 5th stage by dual inhibition of TGF-ß and NOTCH signaling. Conclusion Here, we show that stage-specific regulation of the WNT/ß-catenin pathway results in improved differentiation of hESCs to functional hepatocytes. Overall design: mRNA profiles of undifferentiated, definitive endoderm, stage 2-5 cell ines were generated by deep sequencing, in duplicate, as well as five liver samples.
Publication Title
Stage-specific regulation of the WNT/β-catenin pathway enhances differentiation of hESCs into hepatocytes.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 4 Downloadable Samples
- Illumina HiSeq 2500
Description
Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-b and BMP signaling, which we found NMD acts through to influence definitive endoderm vs. mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages. Overall design: Examination of differential gene expression in hESCs upon loss of UPF1.
Publication Title
Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 43 Downloadable Samples
- IlluminaHiSeq2500, IlluminaHiSeq2000
Description
Mitochondria are vital due to their principal role in energy production via oxidative phosphorylation (OXPHOS)1. Mitochondria carry their own genome (mtDNA) encoding critical genes involved in OXPHOS, therefore, mtDNA mutations cause fatal or severely debilitating disorders with limited treatment options. 2. Clinical manifestations of mtDNA disease vary based on mutation type and heteroplasmy levels i.e. presence of mutant and normal mtDNA within each cell. 3,4. We evaluated therapeutic concepts of generating genetically corrected pluripotent stem cells for patients with mtDNA mutations. We initially generated multiple iPS cell lines from a patient with mitochondrial encephalomyopathy and stroke-like episodes (MELAS) caused by a heteroplasmic 3243A>G mutation and a patient with Leigh disease carrying a homoplasmic 8993T>G mutation (Leigh-iPS). Due to spontaneous mtDNA segregation in proliferating fibroblasts, isogenic MELAS iPS cell lines were recovered containing exclusively wild type (wt) mtDNA with normal metabolic function. As expected, all iPS cells from the patient with Leigh disease were affected. Using somatic cell nuclear transfer (SCNT; Leigh-NT1), we then simultaneously replaced mutated mtDNA and generated pluripotent stem cells from the Leigh patient fibroblasts. In addition to reversing to a normal 8993G>T, oocyte derived donor mtDNA (human haplotype D4a) in Leigh-NT1 differed from the original haplotype (F1a) at a additional 47 nucleotide sites. Leigh-NT1 cells displayed normal metabolic function compared to impaired oxygen consumption and ATP production in Leigh-iPS cells or parental fibroblasts (Leigh-fib). We conclude that natural segregation of heteroplasmic mtDNA allows the generation of iPS cells with exclusively wild type mtDNA. Moreover, SCNT offers mitochondrial gene replacement strategy for patients with homoplasmic mtDNA disease. Overall design: Duplicate cDNA libraries of fibroblasts from a Leigh patient and a MELAS patient, two sendai produced iPSC lines from the Leigh patient and three sendai produced iPSC lines from the MELAS patient, three fibroblasts lines produced by differentiating three iPS Leigh patient iPSC lines to fibroblasts, two somatic cell nuclear transfer produced NT-ESC lines from the Leigh patient, two fibroblast lines produced by differentiating two Leigh patient NT-ESC lines, four fibroblasts lines produced by differentiating four MELAS patient iPSC lines with the mutation to fibroblasts, four fibroblast lines produced by differentiating two IVF-ESC lines without mutated mtDNA genomes, four fibroblast lines produced by differentiating two somatic cell nuclear transfer NT-ESC lines without mutated mtDNA genomes, and four fibroblasts lines produced by differentiating two MELAS patient iPSC lines without the mutation to fibroblasts. The sequence reads were mapped to hg19 reference genome and hits that passed quality filters were analyzed for differential expression.
Publication Title
Metabolic rescue in pluripotent cells from patients with mtDNA disease.
Sample Metadata Fields
No sample metadata fields
View Samples- Ovis aries
- 52 Downloadable Samples
- Ion Torrent Proton, Illumina HiSeq 2500
Description
RNA seq analysis of laser capture microdissected luminal and glandular epithelium from ewes on day of pregnancy 10, 12, 14, 16 and 20. As well as RNA seq of whole conceptuses, and trophectoderm tissue from day 12, 14, 16 and 20 of pregnancy. Determination of gene expression changes in the uterine epithelium and conceptus during early pregnancy helps to improve our understanding of early pregnancy events and provides a basis of new strategies to improve fertility and reproductive efficiency in ruminants. Overall design: RNA seq analysis of 4 samples of each tissue type (luminal epithelium (LE), glandular epithelium (GE) and conceptus) for 4 animals. Pre-sequencing amplification of LE, GE and day 12 conceptus samples.
Publication Title
Analysis of the Uterine Epithelial and Conceptus Transcriptome and Luminal Fluid Proteome During the Peri-Implantation Period of Pregnancy in Sheep.
Sample Metadata Fields
Specimen part, Subject
View Samples- Bos taurus
- 201 Downloadable Samples
- NextSeq 500
Description
This study relates to pregnancy outcome after assisted reproduction of fertility-classified cattle. The aim is to investigate how the uterine environment impacts and programs conceptus survival and development. The study found that ripple effects of dysregulated conceptus-endometrial interactions elicit post-elongation pregnancy loss in subfertile animals during the implantation period. Overall design: Heifer cows classified as high fertile (HF), subfertile (SF), or infertile (IF) were investigated. The RNA-seq analysis was performed for endometrium samples at day 17 of pregnancy. For comparison, non-pregnant cows were included in the analysis. RNA from conceptus of HF and SF pregnant animals (day 17) were also included in the RNA-seq analysis. A total of 25 endometrium samples (5 non-pregnant of each fertilty group, 5 pregnant HF, and 5 pregnant SF) and 27 conceptus samples (10 SF and 17 HF) were used in the RNA-seq analysis.
Publication Title
Uterine influences on conceptus development in fertility-classified animals.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 31 Downloadable Samples
- Illumina HiSeq 2500
Description
We report a pilot investigation for poly-A RNAs differentially expressed during Mycobacterium tuberculosis infection. Participation in this investigation from March 2010 to July 2013 was voluntary, only subjects that were >18 years old and that informed written consent were considered eligible. The recruitment of tuberculosis (TB) patients was done at public hospitals in Rio de Janeiro, Brazil. The diagnostic criteria for active pulmonary tuberculosis was at least one AFB (acid-fast bacilli) -positive sputum sample for M. tuberculosis and/or positive sputum culture and/or compatible clinical evolution for pulmonary TB and less than 15 days of anti-TB treatment and was in accordance with those of the Brazilian Ministry of Health. Blood was collected from recent close contacts (rCt) and active tuberculosis (TB) index cases (n=6). Latent TB infection (LTBI) was accessed by both tuberculin skin test (TST, cut-off = 5mm) and in house interferon-gamma release assays (IGRA, cut-off = 100 pg/ml), therefore, 12 rCt were classified as uninfected controls and 16 with LTBI. Subsequently, the sequencing was performed following the standard protocols on Illumina HiSeq® 2500 Sequencing System (Illumina, San Diego, CA) running 100 bp paired-end reads (PE100) and generating approximately 30 million reads passing filter for each sample to produce the mRNA reads. Mining these RNAseq data, highly prominent modulation of DOCK9, EPHA4, and NPC2 mRNA expression was observed in the TB samples, indicating that they might have a role in TB pathogenesis. These differential modulations upon M. Tuberculosis infection were further validated by additional evidences in larger cohorts from different geographical areas. Overall design: We collected blood samples from the recent close contacts (rCt) at the recruitment and monitored them for 1-year. All TB participants were treatment-naïve. An infection mRNA signature was derived from whole blood RNA sequencing data by comparing TB and uninfected rCt. We selected the 3 most prominent genes, by area under the ROC curve analysis, for additional validations. Some of the LTBI participants also showed the mRNA infection profile.
Publication Title
Transcriptomic Biomarkers for Tuberculosis: Evaluation of <i>DOCK9. EPHA4</i>, and <i>NPC2</i> mRNA Expression in Peripheral Blood.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 19 Downloadable Samples
Affymetrix Mouse Gene 2.0 ST Array (mogene20st)
Description
In this study, we used conditional knockout and gene expression approaches to understand global molecular and transciptional changes due to ablation of each integrin subunit.
Publication Title
Functional Redundancy between β1 and β3 Integrin in Activating the IR/Akt/mTORC1 Signaling Axis to Promote ErbB2-Driven Breast Cancer.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 11 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
We studied adipose tissue from wild type mice, kinin B1 receptor knockout mice (B1KO), and B1KO mice with rescued expression of kinin B1 receptor selectively in fat.
Publication Title
Kinin B1 and B2 receptor deficiency protects against obesity induced by a high-fat diet and improves glucose tolerance in mice.
Sample Metadata Fields
Sex, Age, Specimen part
View Samples- Mus musculus
- 8 Downloadable Samples
- Illumina HiSeq 2000
Description
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived skin transcriptome profiling (RNA-seq) to determine pathways and networks dependent on retinoic acid during skin development. Methods: Skin mRNA profiles of embryonic day E16.5 wild-type (WT) and Cyp26b1 knockout (Cyp26b1-/-), and of control and of dermal and epidermal skin fractions of Engrailed1cre;Cyp26b1f/- (En1cre;Cyp26b1f/-) conditional knockout mice were generated by deep sequencing, in duplicate, using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level by ANOVA (ANOVA) and TopHat. qRT–PCR validation was performed using TaqMan and SYBR Green assay. Results: RNA-Seq data were generated with Illumina’s HiSeq 2000 system. Raw sequencing data were processed with CASAVA 1.8.2 to generate fastq files. Reads of 50 bases were mapped to the mouse transcriptome and genome mm9 using TopHat 1.3.2. Gene expression values (RPKM) were calculated with Partek Genomics Suite 6.6, which was also used for the ANOVA analysis to determine significantly differentially expressed genes. Conclusions: Our study represents the first detailed analysis of Cyp26b1-/- skin and En1cre;Cyp26b1f/- dermis/epidermic transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Skin mRNA profiles of embryonic-day 16.5 wild type (WT) and Cyp26b1-/- mice and of dermis and epidermis of embryonic day 18.5 control and En1cre;Cyp26b1f/- were generated by deep sequencing, in duplicate, using Illumina HiSeq2000.
Publication Title
Cutaneous retinoic acid levels determine hair follicle development and downgrowth.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Mus musculus
- 4 Downloadable Samples
- Affymetrix Murine Genome U74A Version 2 Array (mgu74av2)
Description
The loss of loricrin, a major component of the cornified envelope, results in a delay of epidermal barrier formation. Therefore, the living layers of the epidermis are aberrantly exposed to late-stage amniotic fluid, which may serve as the signal to upregulate genes that functionally compensate for the loss of loricrin. Consistent with this hypothesis, metabolomic studies revealed marked changes in amniotic fluid between E14.5 and E16.5 dpc. In addition, we discovered that the Nrf2/Keap1 pathway detects these compositional changes and directly upregulates the expression of genes involved in the compensatory response, thus ensuring postnatal survival. In support of this finding, we demonstrate that genetically blocking the Nrf2 pathway abolishes the compensatory response, and preemptively activating Nrf2 pharmacologically rescues the delay in barrier formation in utero. Our findings reveal that the functions of Nrf2 and the composition of amniotic fluid have co-evolved to ensure the formation of a functional barrier.
Publication Title
Amniotic fluid activates the nrf2/keap1 pathway to repair an epidermal barrier defect in utero.
Sample Metadata Fields
Specimen part
View Samples