Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes.
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes. Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.
Project description:Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.
Project description:Pluripotent cells emerge as a naïve founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively represent the initial naïve and final primed phases of pluripotency. Here we investigated the intermediate formative stage. Using minimal exposure to specification cues, we derived stem cells from formative mouse epiblast. Unlike ES cells or EpiSCs, formative stem (FS) cells responded directly to germ cell induction. They colonised somatic tissues and germline in chimaeras. Whole transcriptome analyses showed similarity to pre-gastrulation formative epiblast. Signal responsiveness and chromatin accessibility features reflect lineage capacitation. Furthermore, FS cells showed distinct transcription factor dependencies, relying critically on Otx2. Finally, FS cell culture conditions applied to human naïve cells or embryos supported expansion of similar stem cells, consistent with a conserved staging post on the trajectory of mammalian pluripotency.
Project description:Pluripotent cells emerge as a naïve founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively represent the initial naïve and final primed phases of pluripotency. Here we investigated the intermediate formative stage. Using minimal exposure to specification cues, we derived stem cells from formative mouse epiblast. Unlike ES cells or EpiSCs, formative stem (FS) cells responded directly to germ cell induction. They colonised somatic tissues and germline in chimaeras. Whole transcriptome analyses showed similarity to pre-gastrulation formative epiblast. Signal responsiveness and chromatin accessibility features reflect lineage capacitation. Furthermore, FS cells showed distinct transcription factor dependencies, relying critically on Otx2. Finally, FS cell culture conditions applied to human naïve cells or embryos supported expansion of similar stem cells, consistent with a conserved staging post on the trajectory of mammalian pluripotency.
Project description:Pluripotent cells emerge as a naïve founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively represent the initial naïve and final primed phases of pluripotency. Here we investigated the intermediate formative stage. Using minimal exposure to specification cues, we derived stem cells from formative mouse epiblast. Unlike ES cells or EpiSCs, formative stem (FS) cells responded directly to germ cell induction. They colonised somatic tissues and germline in chimaeras. Whole transcriptome analyses showed similarity to pre-gastrulation formative epiblast. Signal responsiveness and chromatin accessibility features reflect lineage capacitation. Furthermore, FS cells showed distinct transcription factor dependencies, relying critically on Otx2. Finally, FS cell culture conditions applied to human naïve cells or embryos supported expansion of similar stem cells, consistent with a conserved staging post on the trajectory of mammalian pluripotency.
Project description:Pluripotent cells emerge as a naïve founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively represent the initial naïve and final primed phases of pluripotency. Here we investigated the intermediate formative stage. Using minimal exposure to specification cues, we derived stem cells from formative mouse epiblast. Unlike ES cells or EpiSCs, formative stem (FS) cells responded directly to germ cell induction. They colonised somatic tissues and germline in chimaeras. Whole transcriptome analyses showed similarity to pre-gastrulation formative epiblast. Signal responsiveness and chromatin accessibility features reflect lineage capacitation. Furthermore, FS cells showed distinct transcription factor dependencies, relying critically on Otx2. Finally, FS cell culture conditions applied to human naïve cells or embryos supported expansion of similar stem cells, consistent with a conserved staging post on the trajectory of mammalian pluripotency.