CORTECON: A Temporal Transcriptome Analysis of In Vitro Human Cerebral Cortex Development From Human Embryonic Stem Cells
ABSTRACT: Many neurological and psychiatric disorders affect the cerebral cortex, and a clearer understanding of the molecular processes underlying human corticogenesis will provide greater insight into such pathologies. To date, knowledge of gene expression changes accompanying corticogenesis is largely based on murine data. Here we present a searchable, comprehensive, temporal gene expression dataset encompassing cerebral cortical development from human embryonic stem cells (hESCs). Using a modified differentiation protocol and RNA-Seq technology with computational analysis, we identified sets of genes and long non-coding RNAs that significantly change during corticogenesis, and those enriched for disease-associations. Numerous alternatively-spliced genes with varying temporal patterns of expression are revealed, including TGIF1, involved in holoprosencephaly and MARK1, involved in autism. We have created a database (http://cortecon.neuralsci.org) that provides online, query-based access to changes in RNA expression and alternatively spliced transcripts during human cortical development. Nine timepoints (days 0,7,12,19,26,33,49,63,77) covering human corticogenesis from embyronic stem cells.
Project description:Forced expression of transcription factors for lineage reprogramming brings hope to cell-based therapy. However, its application is hampered by risks of potential genetic aberrations and tumorigenicity. Using defined small molecules in presence of gastric stromal cells as feeders, we reprogramed human gastric epithelia into induced multipotent endodermal progenitors (hiMEPs) with efficiency of up-to-6%. The hiMEPs expressed genes relative to endodermal lineages but not associating with pluripotency, and could be expanded clonogenically remaining as undifferentiated colonies. Upon induction, hiMEPs were able to give rise to multiple functional endodermal cell types, apart from ectodermal or mesodermal lineages. TGFβ inhibition and particular Wnt signaling activation were crucial in reprogramming process. Collective advantages of availability from donors without age restriction, capabilities in expansion and differentiation, and no concern of tumorigenesis, let hiMEPs have the considerable application potentials on cell therapies of diseases such as liver failure and diabetes, as well as personalized drug-screenings. Gastric epithelial cells (GECs) were isolated from human stomach. Human induced multipotent endodermal progenitors (hiMEPs) were reprogrammed from GECs by small molecules. The hiMEP-Heps were differentiated from hiMEPs under hepatic differentiation protocol. Fetal-Heps were isolated from aborted fetal liver. Definitve endoderm (DE), primitive gut tube (PGT), and posterior foregut (PFG) were endodermal stem cells derived form human enbryonic stem cells (hESCs).We used RNA sequencing and DNA methylation analysis to detail the global gene expression profile of GECs, hiMEPs, hiMEP-Heps, Fetal-Heps, DE, PGT and PFG to delineate the difference of these cells.
Project description:Alternative splicing regulates over 90% of multiexon mammlian genes, but its role in specifying neural progenitor cell (NPC) fates has not been explored. Our analyses of purified mouse NPCs and neurons from developing cerebral cortices revealed hundreds of conserved and differentially spliced exons that add or remove key protein domains, especially in genes regulating the cytoskeleton. Overall design: TBR2-EGFP BAC transgenic mice, generated by GENSAT and outcrossed to FVB strain for 2 generations, drives EGFP expression in the vast majority of intermediate progenitors and neurons at E14.5. We FACS sorted EGFP positive (differentiating and differentiated neurons) and negative (neural progenitors) cells from the dorsal cerebral cortices of E14.5 TBR2-EGFP. Two replicates for each population were subjected to RNA sequencing. Alternatively spliced exons between the two populations (NPCs and neurons) were identified using MISO.
Project description:Stem cell-derived tissues have wide potential for modelling developmental and pathological processes as well as cell-based therapy. However, it has proven difficult to generate several key cell types in vitro, including skeletal muscle. In vertebrates, skeletal muscles derive during embryogenesis from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We differentiated mouse ESCs in serum-free medium supplemented with Rspo3 ( or as an alternative with Chir 9902) and the Bmp inhibitor LDN193189. In vivo, the PSM cells are first expressing Msgn1 (posterior PSM marker) and then mature to express Pax3 (anterior PSM marker). After 4 days of differentiation of mESCs, Msgn1-positive cells were FACS-sorted and their transcriptome analyzed. After 6 days of differentiation, Pax3-positive cells were sorted and their transcriptome analyzed. Mouse ESCs differentiated for 0, 4 and 6 days in serum-free medium containing a Wnt activator, a BMP inhibitor and DMSO, to study paraxial mesoderm in vitro
Project description:Conventional embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) derived from primates resemble mouse epiblast stem cells, raising an intriguing question regarding whether the naïve pluripotent state resembling mouse embryonic stem cells (mESCs) exists in primates and how to capture it in vitro. Here we identified several specific signaling modulators that are sufficient to generate rhesus monkey fibroblast-derived iPSCs with the features of naïve pluripotency in terms of growth properties, gene expression profiles, self-renewal signaling, X-reactivation and the potential to generate cross-species chimeric embryos. Interestingly, together with recent reports of naïve human pluripotent stem cells, our findings suggest several conserved signaling pathways shared with rodents and specific to primates, providing significant insights for acquiring naïve pluripotency from other mammal species. In addition, the derivation of rhesus monkey naïve iPSCs also provides a valuable cell source for use in preclinical research and disease modeling. mRNA expression analysis of 4 rhesus monkey naive iPSC lines and 2 primed iPSC lines were examed.
Project description:The cerebral cortex plays an important role in cognitive function and specialized perception in mammals and its development requires highly specific spatio-temporal control of gene expression. The study identified stage- and region-specific markers throughout cerebral corticogenesis at various important stages of cerebral cortex development; embryonic day (E) 15.5, E17.5, postnatal day (P) 1.5 and 4-6 months old. The study involved the analysis of 12 SAGE libraries, which were generated from the mouse cerebral cortex of E15.5 (n=3), E17.5 (n=2), P1.5 (n=1) and 4-6 month old (n=6). N denotes biological replicates.
Project description:Human lactoferrin (LF) is a multifunctional protein involved in immunomodulation, cell growth, and differentiation. In addition to the secreted form (sLF), an alternatively spliced form (ΔLF) that lacks the signal sequence and downregulated in cancer was found. This study was carried out to identify and compare signaling networks provoked by the two LF isoforms. To do this, the two forms were overexpressed in HEK293 cells using the flp-in/tet-on system and genome-wide expression analysis of 18,367 genes was conducted. Secreted form (sLF) or alternatively spliced form (ΔLF) were overexpressed in HEK293 cells using the flp-in/tet-on system and genome-wide expression analysis of 18,367 genes was conducted.
Project description:The cerebral cortex plays an important role in cognitive function and specialized perception in mammals and its development requires highly specific spatio-temporal control of gene expression. The study identified stage- and region-specific markers throughout cerebral corticogenesis at various important stages of cerebral cortex development; embryonic day (E) 15.5, E17.5, postnatal day (P) 1.5 and 4-6 months old. Overall design: The study involved the analysis of 12 SAGE libraries, which were generated from the mouse cerebral cortex of E15.5 (n=3), E17.5 (n=2), P1.5 (n=1) and 4-6 month old (n=6). N denotes biological replicates.
Project description:We compared the whole mRNA transcript expression from control and homozygous mutant Dhx32 mice by Affymetrix Mouse Exon ST 1.0 Array ST to identify alternatively spliced mRNA transcripts We include exon level expression data from the liver of three control and three Dhx32 homozygous mutant mice Overall design: 6 total samples (three control and three Dhx32 homozygous mutant) were analyzed. The p values for alternatively spliced transcripts were calculated by using the algorithms of Partek Genomics Suite version 6.4, on core probes excluding probe sets with signals less than 3.
Project description:The hedgehog (Hh) signaling pathway is important for various developmental processes during embryogenesis and for homeostasis of adult tissue in vertebrates. Aberrant signaling results in severe birth defects and malignancies, respectively. The small molecules SANT-2 and GANT-61 were identified as potential Hh-pathway inhibitors in a reporter gene assay (Shh-light II cells). We aimed to analyze the effects of these compounds on the gene expression level in zebrafish embryos in comparison with the well known Hh-pathway inhibitor cyclopamine. Zebrafish embryos were treated with Ethanol (0,1%) (control), 10 µM Cyclopamine (C), 10 µM SANT-2 (S), and 10 µM GANT-61 (G), for 24 h (0.5 - 24 hpf), respectively; all in 5 independent replicates with 100 zebrafish embryos per treatment. Total RNA was extracted and processed as recommended by Agilent. One color arrays were used and data is given as normalized log2 of the gprocessed signals.