Comparative transcriptomic analysis of self-organized, in vitro generated optic tissues
ABSTRACT: Embryonic stem (ES) cells have a remarkable capacity to self-organize complex, multi-layered optic cups in vitro via a culture technique called SFEBq. During both SFEBq and in vivo optic cup development, Rax (Rx) expressing neural retina epithelial (NRE) tissues utilize Fgf and Wnt/β-catenin signalling pathways to differentiate into neural retina (NR) and retinal-pigmented epithelial (RPE) tissues, respectively. How these signaling pathways affect gene expression during optic tissue formation has remained largely unknown, especially at the transcriptome scale. We generated Day 10 Rx+ optic tissue using SFEBq, exposed these tissues to either Fgf or Wnt/β-catenin stimulation, and assayed their gene expression at Days 12 and 15 using RNA-Seq. We measured gene expression in these 5 sample groups in biological triplicate using RNA-seq (Illumina HiSeq) .
Project description:To define molecular mechanisms underlying rod and cone differentiation, we generated H9 human embryonic stem cell line carrying a GFP reporter that is controlled by the promoter of cone-rod homeobox (CRX) gene, the first known marker of post-mitotic photoreceptor precursors. CRXp-GFP reporter in H9 line replicates endogenous CRX expression when induced to form self-organizing 3-D retina-like tissue. We define temporal transcriptome dynamics of developing photoreceptors during the establishment of cone and rod cell fate. Our studies provide an essential framework for delineating molecules and cellular pathways that guide human photoreceptor development and should assist in chemical screening and cell-based therapies of retinal degeneration. Undifferentiated CRXp-GFP HP hES cells and 3D-neural retina were collected at days 37, 47, 67 and 90 and dissociated into single cells. Cells were sorted at 4°C and by FACSAria (Becton Dickinson). GFP+ and GFP- cells were separately collected. Total RNA was extracted by RNA purification kit (Norgen Biotek) and analyzed by 2100 Bioanalyzer (Agilent Technologies Genomics). High quality of total RNA (RIN: 7.7-9.2) was subjected to libraries construction using 40-60 ng of total RNA as input. Libraries were constructed using a stranded modification of the Illumina TruSeq mRNA (Brooks, et al. Meth Mol Biol 2012). Each library was single-end sequenced in an independent lane of a GAIIx at a length of 76 bases. Fastq files were generated from reads passing chastity filter.
Project description:Mice that are mutant for both Fgfr1 and Fgfr2 specifically in the developing retina develop coloboma. To analyze the transcripts that are affected by defective FGF signaling, we micro-dissected the optic fissure region from the control and FGFR condtional mutant mice and did microarray analysis. E11.5 control and Six3Cre+; Fgfr1fx/fx;Fgfr2 fx/fx optic fissures are dissected using laser-assisted micro-dissection microscope, RNAs are extracted and labelled and hybridyzed to chips
Project description:Using stem cell–based therapies to treat retinal abnormalities is becoming a likely possibility; therefore, identifying the key factors and the relevant mechanisms controlling optic vesicle morphogenesis and neuroretina (NR) differentiation is important. Recent advances in self-organizing, 3-dimensional (3D) tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided a valuable in vitro model for characterizing regulatory cascades and signaling pathways controlling mammalian retinal development. Using Rx-GFP expressing ESCs and Six3–/– iPSCs we identified R-spondin 2 (Rspo2)-mediated repression of Wnt signaling as a novel required step during optic vesicle morphogenesis and NR differentiation. Furthermore, we also show that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to arrest NR differentiation. ChIP assays identified Six3-responsive elements in the Rspo2-promoter region, indicating that Six3-mediated repression of Rspo2 is required to restrict Wnt signaling in the developing anterior neuroectoderm and allow eye development to proceed. Overall design: We used microarrays to measure changes in gene expression profiles in mouse embryonic stem cells after retinal differentiation for 4 or 7 days. Mouse ES cells were derived from the mouse embryonic stem cell line EB5 (129/Ola) in which GFP gene is knocked-in under Rx promoter (Eiraku and Sasai, Nat Protocols 7:69-79, 2012; RIKEN cell line AES0145). Expression profiles were compared at day 4 and day 7 of retinal differentiation between WT Rx-GFP ESC.
Project description:Bmp4 was conditionally deleted from the mouse optic vesicle with Rx-Cre. The heads of wild type and knockout E10.5 mouse embryos were laser micodissected to isolate the lens ectoderm and prospective retina from three embryos of each genotype. Total RNA was purified and reverse transcribed and amplified using a NuGEN Ovation Pico WTA system V2 kit. cDNA was biotinylated and hybridized to Illumina Mouse6 V2 bead arrays. Three wild type and three knockout embryos were used. RNA obtained from the lens ectoderm or prospective retina (distal optic vesicle) was pooled, amplified and used for microarray analysis. Statistical analysis was performed using the values from the 25-50 beads on each array.
Project description:We report RNA-sequencing data of 283 blood platelet samples, including 228 tumor-educated platelet (TEP) samples collected from patients with six different malignant tumors (non-small cell lung cancer, colorectal cancer, pancreatic cancer, glioblastoma, breast cancer and hepatobiliary carcinomas). In addition, we report RNA-sequencing data of blood platelets isolated from 55 healthy individuals. This dataset highlights the ability of TEP RNA-based 'liquid biopsies' in patients with several types with cancer, including the ability for pan-cancer, multiclass cancer and companion diagnostics. Blood platelets were isolated from whole blood in purple-cap BD Vacutainers containing EDTA anti-coagulant by standard centrifugation. Total RNA was extracted from the platelet pellet, subjected to cDNA synthesis and SMARTer amplification, fragmented by Covaris shearing, and prepared for sequencing using the Truseq Nano DNA Sample Preparation Kit. Subsequently, pooled sample libraries were sequenced on the Illumina Hiseq 2500 platform. All steps were quality-controlled using Bioanalyzer 2100 with RNA 6000 Picochip, DNA 7500 and DNA High Sensitivity chips measurements. For further downstream analyses, reads were quality-controlled using Trimmomatic, mapped to the human reference genome using STAR, and intron-spanning reads were summarized using HTseq. The processed data includes 285 samples (columns) and 57736 ensemble gene ids (rows). The supplementary data file (TEP_data_matrix.txt) contains the intron-spanning read counts, after data summarization by HTseq.
Project description:Human embryonic stem cell (HES3) aggregates were differentiated into cardiomyocytes using biphasic WNT pathway modulation (CHIR99021 and IWP2) in 20mL scale using agitated Erlenmeyer Flask. On day 6 of differentiation, cells were treated with 1mM 1-EBIO or solvent control (DMSO) for 10 days.
Project description:Purpose: Genetic and clinical association studies have identified disrupted-in-schizophrenia 1 (DISC1) as a candidate risk gene for major mental illness. DISC1 is interrupted by a balanced chr(1;11) translocation in a Scottish family, in which the translocation predisposes to psychiatric disorders. We investigate the consequences of DISC1 interruption in human neural cells using TALENs or CRISPR-Cas9 to target the DISC1 locus. We sought to compare the gene expression profiles of human neural progenitor cells (NPCs) and neurons with interruption of the DISC1 gene in exon 2 (affecting all known coding transcripts) or exon 8 (near the site of the Scottish translocation, affecting longer transcripts). Methods: Wild-type and DISC1-targeted iPSCs (wild-type = "WT", exon 8 single allelic frameshift mutant = "ex8_wm", exon 8 biallelic frameshift mutant = "ex8_mm", exon 2 biallelic frameshift mutant = "ex2mm") were differentiated to NPCs and neurons using an embryoid aggregate method. NPC or neuronal cultures were used for RNA harvest and subsequent paired-end stranded sequencing of >50M reads/sample and 3-6 biological replicates per group. Results: We find that a subset of genes related to neuronal differentiation and development are dysregulated with DISC1 disruption at the NPC timepoint, whereas expression of genes related to neuronal function and signaling are altered at the neuronal timepoint. This study implicates DISC1 as a regulator of neuronal development. mRNA profiles of wild-type and DISC1-targeted human iPSC-derived neural progenitor cells (day 17) and neurons (day 50) by paired-end sequencing, with 3-6 biological replicates, using Illumina HiSeq
Project description:H3K4me3 ChIP-Seq profiling of a directed differentiation time course converting human embryonic stem cells (hES) into immature pancreatic beta cell precursors. CyT49, a Viacyte proprietary male hESC line with normal karyotype was used.
Project description:Failure of remyelination in multiple sclerosis (MS) is associated with inhibition of oligodendrocyte precursor (OPC) differentiation, but the cellular and molecular mechanisms involved remain poorly understood. We now report inflammatory demyelination in MS is associated with localized expression of fibroblast growth factor 9 (FGF9) by oligodendrocytes and to a lesser extent astrocytes, and demonstrate FGF9 inhibits myelination and remyelination in vitro. This inhibitory activity is reversible and due to an off target FGF9-dependent effect on astrocytes that disrupts in the growth factor milieu required to support myelination. We identify multiple downstream events induced by FGF9 associated with this effect including increased expression of leukaemia inhibitory growth factor (LIF) and FGF2, both of which are shown to inhibit myelination if present in excess. These studies identify FGF9-dependent signal transduction in astrocytes as a novel target for therapeutic strategies designed to enhance remyelination by endogenous OPC in MS. Gene expression profiles of rat myelinating cultures grown in the presence or absence of FGF9 (100 ng/ml) for 24h and 10 days were generated using Affymetrix GeneChip® Rat Gene 1.0 ST Arrays. Each time point (T1: 24 hrs, and T2: 10 days) has Control (CTR) and Treatment (FGF) groups, with two replicates in each group. In total, 8 arrays were generated from the four groups (CTR-T1, CTR-T2, FGF-T1 and FGF-T2).
Project description:We aim to understand the role that Cdx2 plays in specifying the rostro-caudal identity of differentiating motor neurons. We find that expressing Cdx2 in combination with FGF signaling is sufficient to produce motor neurons with a more caudal identity. ChIP-seq analysis of Cdx2 finds that it binds extensively throughout the Hox regions in progenitor motor neurons. Analysis of polycomb-associated chromatin over Hox regions in the subsequently generated motor neurons finds that Cdx2 binding corresponds to chromatin domains encompassing de-repressed caudal Hox genes. These results suggest a direct role for Cdx2 in specifying caudal motor neuron identity. ChIP-seq studies: We characterize the binding of Cdx2 in progenitor motor neurons using a V5 tagged doxycycline inducible Cdx2 ESC line (iCdx2). Progenitor motor neurons were generated after 4 days of in vitro differentiation of mouse embryonic stem cells using retinoic acid (RA) and hedgehog (Hh) signaling exposure at day 2. On day 3, the cells are exposed to Dox with and without accompanying FGF signaling. The genome-wide binding of the induced Cdx2 transcription factor is profiled using ChIP-seq with an anti-V5 antibody. An appropriate whole-cell extract control experiment for these ChIP-seq experiments is also included. We also examine the effect of induced Cdx2 expression on polycomb-associated chromatin structure in the resulting cellular populations by profiling the H3K27me3 chromatin mark using ChIP-seq. H3K27me3 experiments were performed after 5 days of in vitro differentiation using cells exposed to either: 1) RA & Hh to derive progenitor motor neurons, followed by Dox & FGF; 2) Dox & FGF alone; or 3) RA and Hh alone. There are 6 Illumina sequencing datasets included in this submission: two biological replicates of iCdx2 ChIP-seq in the presence of FGF; one sample of iCdx2 ChIP-seq in the absence of FGF; one H3K27me3 ChIP-seq in the presence of RA, Hh, Dox, and FGF; one H3K27me3 ChIP-seq in the presence of Dox and FGF; and one H3K27me3 ChIP-seq in the presence of RA and Hh.