RNA sequencing of conventional and reset human pluripotent stem cells
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR590396_1.fastq.gz | Fastqsanger.gz | |||
| ERR590396_2.fastq.gz | Fastqsanger.gz | |||
| ERR590397_1.fastq.gz | Fastqsanger.gz | |||
| ERR590397_2.fastq.gz | Fastqsanger.gz | |||
| ERR590398_1.fastq.gz | Fastqsanger.gz |
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Project description:Human pluripotent cells were reset to ground state pluripotency by transient overexpression of NANOG and KLF2 and subsequent inhibition of ERK and protein kinase C. Transcriptional profiling of reset cells and conventional pluripotent stem cell cultures was carried out by RNA-seq, in tandem with mouse embryonic stem cells propagated under similar conditions to assess the combinatorial effects of MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021 and PKC inhibitor Go6983.
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Project description:Human pluripotent cells were reset to ground state pluripotency by transient overexpression of NANOG and KLF2 and subsequent inhibition of ERK and protein kinase C. Transcriptional profiling of H9 parental and clonal lines, and induced pluripotent stem cells derived from adult fibroblast and adipose cell types was carried out in reset and conventional culture conditions.
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Project description:A simple method is presented to reset human pluripotent cells to a naive state via transient histone deacetylase inhibition and maintenance in chemically-defined naive stem cell culture media. Cells can be reset without transgenes and expanded continuously either on feeders or alternative substrates in feeder-free conditions. Multiple cell lines of varying origin were reset and characterised in parallel with conventionally cultured counterparts.
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Project description:RNA-seq of chemically reset human naive pluripotent stem cells
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Project description:In human embryos, naive pluripotent cells of the inner cell mass (ICM) generate epiblast, primitive endoderm and trophectoderm (TE) lineage, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and efficiently generate trophoblast stem cells (TSCs), while conventional PSCs form TSCs at low efficiency. Transient histone deacetylase and MEK inhibitions with LIF stimulation is used to chemically reset conventional to naive PSCs. Here we report that chemical resetting induces expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allows for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a plastic intermediate state, characterised by co-expression of naive and TSC markers, after which cells steer towards one of the two fates in response to the signalling environment. The efficiency and rapidity of our system will be useful to study cell fate transitions, and to generate models of placental disorders.
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Project description:Functional-assay limitations are an emerging issue in characterizing human pluripotent stem cells (hPSCs). With rodent PSCs, chimera formation, using pre-implantation embryos, is the gold-standard assay of pluripotency. In hPSCs, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-animal chimera. To circumvent this issue, we established a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay). The assay uses mouse pre-implantation embryos and human PSCs to make interspecies chimeras cultured in vitro to the early egg cylinder stage. When hiPSCs, both conventional and naive type, which called M-bM-^@M-^\reset cellM-bM-^@M-^], were injected into mouse embryos and cultured. The cells were never integrated into the epiblast of egg cylinder stage-embryo. These results suggest that hPSCs, including naM-CM-/ve type, are unable to form chimera with mouse embryo. Reset cells were converted from conventional human iPSC line PB004, and then compared their gene expression profile with or without transgene overexpression induced by doxycyclin treatment.
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Project description:Generation of T cells from pluripotent stem cells (PSC) has the potential to transform adoptive immunotherapy for cancer into universal donor, off-the-shelf cellular therapies. However, differentiation of human PSCs into fully mature T cells has been challenging with existing methods. We report that a 3D organoid method permitted efficient differentiation of human embryonic stem cell and induced pluripotent stem cell-derived mesoderm progenitors to mature, functional conventional T cells with a diverse T cell receptor (TCR) repertoire. This continuous culture system supported both hematopoietic induction and terminal differentiation to naïve, conventional CD3+CD8αβ+ and CD3+CD4+ T cells. Introduction of a Class I-restricted TCR in PSCs produced antigen-specific CD8αβ+ T cells lacking endogenous TCR expression. Functional assays and RNA sequencing aligned PSC-derived T cells with primary naïve conventional CD8+ T cells. The organoid system presented here provides an effective and scalable platform to generate functional mature T cells from human PSCs.
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Project description:Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C signalling sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signalling, are phenotypically stable and karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed in reset cells with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground state transcription factors, TFCP2L1 or KLF4 has marginal impact on conventional human pluripotent stem cells, but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground state pluripotency in human cells. DNA methylation analysis in Conventional and Reset human embryonic stem cells by whole genome bisulfite sequencing, in triplicate, using the Illumina platform
2014-09-10 | E-GEOD-60945 | biostudies-arrayexpress
Project description:Organoid-induced differentiation of conventional T cells from human pluripotent stem cells
| PRJNA476793 | ENA
Project description:Functional-assay limitations are an emerging issue in characterizing human pluripotent stem cells (hPSCs). With rodent PSCs, chimera formation, using pre-implantation embryos, is the gold-standard assay of pluripotency. In hPSCs, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-animal chimera. To circumvent this issue, we established a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay). The assay uses mouse pre-implantation embryos and human PSCs to make interspecies chimeras cultured in vitro to the early egg cylinder stage. When hiPSCs, both conventional and naive type, which called “reset cell”, were injected into mouse embryos and cultured. The cells were never integrated into the epiblast of egg cylinder stage-embryo. These results suggest that hPSCs, including naïve type, are unable to form chimera with mouse embryo.
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