Dynamics of Telomere Rejuvenation During Chemical Induction to Pluripotent Stem Cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE113921 | GEO | 2018/05/01
REPOSITORIES: GEO
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Project description:The ERK5 MAP kinase signalling pathway was recently discovered as a driver of naïve pluripotency in mouse Embryonic Stem Cells (mESCs). However, the molecular functions of ERK5 in mESCs have not been investigated. Here, we employ combinatorial mESC proteomics to identify ERK5 target genes and substrates. Global proteomic profiling reveals ZSCAN4 and other 2-cell stage genes as transcriptional targets of the ERK5 pathway. ZSCAN4 expression is specifically induced by ERK5-dependent transcription of the core pluripotency factor KLF2. Additionally, ERK5 directly phosphorylates tandem KLF2 Thr-Pro/Ser-Pro motifs identified by phosphoproteomics to recruit the FBXW7-CUL1 E3 ligase, promoting KLF2 ubiquitylation and degradation. ERK5 phosphorylation of KLF2 thereby provides negative-feedback control to restrain transcriptional induction of ZSCAN4. Our data uncover an auto-regulatory module by which ERK5 co-opts KLF2 to pattern ZSCAN4 expression. This study provides the first molecular insight into ERK5 functions in mESCs, and suggests a novel role for ERK5 signalling in stem cell rejuvenation
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Project description:Pluripotent stem cells can be generated by pure small molecule compounds. However, only fibroblasts, a heterogeneous cell population, were reported for use in chemical reprogramming, and the efficiency is relatively low, raising the possibility that chemically induced pluripotent stem cells (CiPSCs) are derived from a specific cell subpopulation residing in fibroblast culture. Thus, it is of interest to know whether chemical reprogramming can be induced in other cell types, even using the same chemical cocktail. Here, using lineage tracing, we verify the generation of CiPSCs from fibroblasts. We further demonstrate that neural stem cells (NSCs) and small intestinal epithelial cells (IECs), cell types from the ectoderm and endoderm, respectively, can be chemically reprogrammed into pluripotent stem cells. CiPSCs derived from NSCs and IECs resemble mouse embryonic stem cells (ESCs) in terms of proliferation rate, global gene expression profiling, epigenetic status, self-renewal and differentiation capacity, and germline transmission competency. Interestingly, in the chemical reprogramming process from different cell types, a pluripotency gene Sall4 was commonly expressed in the initial stage, and the same core small molecules were required, suggesting conservatism underlying chemical reprogramming from different cell types. Moreover, the use of these small molecules should be fine-tuned to meet the requirement of reprogramming from different cell types. Together, these findings demonstrate that our reported chemical reprogramming approach can be reproduced in different cell types and suggest that chemical reprogramming is a promising strategy with the potential to be extended to more initial cell types. We analyzed the gene expression profiles of NSC-derived CiPSCs and IEC-derived CiPSCs ,using RNA-Sequencing. NSCs, IECs, and embryonic stem cells (R1) are controls.
2016-07-03 | E-GEOD-75522 | biostudies-arrayexpress
Project description:Naïve and primed pluripotent states represent two different states of pluripotency. Mouse naïve pluripotent stem cells (PSCs) exhibit germline competence as determined by chimera production test and can generate all-PSC mice by tetraploid embryo complementation (TEC) test, the most stringent functional test of developmental potency. By contrast, primed PSCs fail in germline chimeras and TEC test. Unfortunately, these tests cannot be applied to characterization of developmental pluripotency of human PSCs due to ethic issue. Extensive studies demonstrated that naïve and primed pluripotent state exhibits clear epigenome differences. Here we uncover surprising differences in telomere maintenance, retrotransposon activity and genomic stability between these two pluripotent states. Although both states express high telomerase activity, naïve PSCs show robust telomere elongation capacity, associated with higher telomere recombination, consistent with sporadic expression of two-cell (2C) genes including Zscan4. Distinctively, primed PSCs only can maintain their telomere length but without elongation, in association with repression of 2C genes, and increased telomere fragility and telomeric DNA damage with passages. RNA-seq revealed that DNA repair and especially recombination repair pathways are down-regulated in primed state compared to naïve state cells, corroborating the robust DNA repair capacity and genome stability in naïve PSCs. These data suggest that vigorous telomere elongation of naïve state may act to minimize DNA damage to the genome. Furthermore, we identified LINE1 family integrant L1Md_T as naïve-specific retrotransposon and ERVK family integrant IAPEz-int to define primed PSCs, distinguishing the two pluripotent states. Heterochromatin modifications and Dnmt3b differentially regulate transcription of the 2C genes and retrotransposons. Notably, aberrant expression of retrotransposons links to increased genomic stability of primed PSCs. Hence, our data reveals that telomere regulation and retrotransposon activity markedly distinguishes naïve from primed pluripotent state. These new criteria may facilitate induction of high quality and additional characterization of developmental pluripotency and scrutinizing the genomic stability of human naïve PSCs for regenerative medicine
2020-08-22 | GSE140665 | GEO
Project description:Naïve and primed pluripotent states represent two different states of pluripotency. Mouse naïve pluripotent stem cells (PSCs) exhibit germline competence as determined by chimera production test and can generate all-PSC mice by tetraploid embryo complementation (TEC) test, the most stringent functional test of developmental potency. By contrast, primed PSCs fail in germline chimeras and TEC test. Unfortunately, these tests cannot be applied to characterization of developmental pluripotency of human PSCs due to ethic issue. Extensive studies demonstrated that naïve and primed pluripotent state exhibits clear epigenome differences. Here we uncover surprising differences in telomere maintenance, retrotransposon activity and genomic stability between these two pluripotent states. Although both states express high telomerase activity, naïve PSCs show robust telomere elongation capacity, associated with higher telomere recombination, consistent with sporadic expression of two-cell (2C) genes including Zscan4. Distinctively, primed PSCs only can maintain their telomere length but without elongation, in association with repression of 2C genes, and increased telomere fragility and telomeric DNA damage with passages. RNA-seq revealed that DNA repair and especially recombination repair pathways are down-regulated in primed state compared to naïve state cells, corroborating the robust DNA repair capacity and genome stability in naïve PSCs. These data suggest that vigorous telomere elongation of naïve state may act to minimize DNA damage to the genome. Furthermore, we identified LINE1 family integrant L1Md_T as naïve-specific retrotransposon and ERVK family integrant IAPEz-int to define primed PSCs, distinguishing the two pluripotent states. Heterochromatin modifications and Dnmt3b differentially regulate transcription of the 2C genes and retrotransposons. Notably, aberrant expression of retrotransposons links to increased genomic stability of primed PSCs. Hence, our data reveals that telomere regulation and retrotransposon activity markedly distinguishes naïve from primed pluripotent state. These new criteria may facilitate induction of high quality and additional characterization of developmental pluripotency and scrutinizing the genomic stability of human naïve PSCs for regenerative medicine
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Project description:We analyzed the genome-wide DNA methylation in Zscan4 overexpressing ES cells. Zscan4 overexpression induced slight DNA demethylation in telomere and major satellite regions. Subsequent genome-wide analysis of non-repeated regions revealed the significant reduction of DNA methylation at Zscan4-dependent hyperacetylation sites. This result indicates that Zscan4 is not only a marker of the Zscan4+ state of ES cells, but also indispensable for the dramatic epigenetic modifications occurring in the Zscan4+ state. DNA methylation in Zscan4 overexresssing ES cells
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Project description:Telomere shortening is universally acquired with aging in humans, but the contribution of telomere shortening to immune aging is not fully understood. Here, we studied T cells derived from short telomere syndrome patients and compared their T cell profile and function to controls and elderly individuals who had normal age-adjusted telomere length. The short telomere syndrome patients were all under age 40 and carried mutant telomerase or telomere genes and had telomere lengths below the 1st age-adjusted percentile. The T cell profile of young short telomere syndrome patients resembled those that were five decades older including a depletion of naive T cell population and accumulation of terminally differentiated effector cells. To test whether short telomeres affect the quality of these cells, we performed a gene expression microarray. The data are summarized here. We found that although numerically expanded in both short telomere and elderly cases, the gene expression microarrays were distinct with short telomere cells predominately upregulating gene expression of DNA damage pathways and elderly T cells upregulating cell extrinsic apoptotic pathways. The goal of the experiment is to highlight important genes and pathways that may drive immune aging in short telomere syndromes and which may also contribute to normal immune aging.
2018-09-14 | GSE77525 | GEO
Project description:Pluripotent stem cells can be induced from somatic cells, providing an unlimited cell resource for regenerative medicine. However, genetic manipulation and difficult-to-manufacture strategies used in reprogramming limit their clinical applications. Here, we show pluripotency can be induced from mouse somatic cells by specific small-molecule compounds. The completely chemically-induced pluripotent stem cells (CiPSCs) can be stably maintained in embryonic stem cell (ESC) culture medium and resemble ESCs in terms of their gene expression profiles, epigenetic status, and potential for differentiation and germline transmission. These findings suggest that exogenous master genes are dispensable for cell fate reprogramming and pave the way for the clinical application of somatic reprogramming techniques. Chemicals' acronym: V, VPA; C, CHIR; 6, 616452; T, tranylcypromine; F, FSK; Z, DZNep; P, PGE2; R, RG108; S, SRT1720; M, 2-Me-5HT; D, D4476; B, Sodium butyrate. mRNA expression analysis of mouse embryonic fibroblasts (MEFs), GFP- cells, GFP+ clusters,GFP+ colonies, embryonic stem cells (ESCs) and CiPSCs by RNA sequencing.
2013-07-22 | E-GEOD-48252 | biostudies-arrayexpress
Project description:Telomere length heterogeneity in various cell types including stem cells and cancer cells has been recognized. Cell heterogeneity also is found in pluripotent stem cells such as embryonic stem cells (ESCs). The implication and mechanisms underlying the heterogeneity remain to be defined. We have optimized a robust method that can simultaneously measure telomere length coupled with RNA-sequencing analysis (scT&R-seq) in the same human ES cell. Using this method, we show that telomere length varies with pluripotency state. Long telomere hESCs highly express TERF1/TRF1 as well as ZFP42/REX1, PRDM14 and NANOG for naïve pluripotency, in contrast to short telomere hESCs. hESCs express high telomerase activity as expected, and ubiquitously express NOP10 and DKC1, stabilizing components of telomerase complexes, regardless of telomere lengths. Moreover, new candidate genes such as MELK, MSH6 and UBQLN1 cluster with long telomeres and pluripotency network. Notably, short telomere hESCs exhibit higher oxidative phosphorylation primed for lineage differentiation, whereas long telomere hESCs show elevated glycolysis, another key feature for naïve pluripotency. Our data further suggest that telomere length is implicated in metabolism activity and pluripotency state of hESCs. Single cell analysis of telomere and RNA-sequencing can be exploited to further understand the molecular mechanisms of telomere heterogeneity.
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Project description:Pluripotency confers Embryonic Stem Cells (ESCs) the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. Although the majority of ESCs divide without losing pluripotency, it has become evident that ESCs culture consists of multiple cell populations with different degrees of potency that are spontaneously induced in regular ESC culture conditions. Zscan4, a key pluripotency factor, marks ESC subpopulation that is referred to as high-level of pluripotency metastate. Here, we report that in ESC cultures treated with retinoic acid (RA), Zscan4 ESCs metastate is strongly enhanced. In particular, we found that induction of Zscan4 metastate is mediated via RA receptors (RAR-alpha, RAR-beta, and RAR-gamma), and it is dependent on phosphoinositide-3-kinase (PI3K) signaling. Remarkably, Zscan4 metastate induced by RA lacks canonical pluripotency genes Oct3/4 and Nanog but retained both self-renewal and pluripotency capabilities. Finally we demonstrated that the conditional ablation of Zscan4 subpopulation is dispensable for both endoderm and mesoderm but is required for ectoderm lineage. In conclusion, our research provides new insights about the role of RA signaling during ESCs high pluripotency metastate fluctuation.
2017-02-13 | GSE75977 | GEO
Project description:Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and causing malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of specific human diseases. We derived induced pluripotent stem cells (iPSCs) from patients with mutations in the TERT and TERC telomerase genes. Telomerase-mutant iPSCs elongated telomeres, but at a lower rate than healthy iPSCs, and the magnitude of the elongation deficit correlated with the specific mutation’s impact on telomerase activity. However, elongation significantly varied among iPSC clones harboring the same mutation, and was affected by genetic and environmental factors. iPSCs cultured in hypoxia showed increased telomere length. Potential influence of residual expression of reprogramming factors on telomerase regulation and telomere length was ruled out by excising the transgenes after successful reprogramming. Evidence for telomerase-independent telomere elongation was not observed in these cells. We demonstrate that telomerase is required for telomere elongation in iPSCs and uncover heterogeneity in telomere maintenance even between clones derived from individual patients or siblings with the same mutation, indicating that telomere phenotype may be influenced by acquired and environmental agents. Our data underscore the necessity of studying multiple clones when using iPSCs to model disease. The exon array were done to validate the pluripotent phenotype of the derived normal and telomerase mutant iPSC and to potentially identify differentially expressed genes in mutant iPSC. Objective: confirming pluripotency by comparing telomerase mutated-, control-iPSC to human ESC and to their parental somatic cells (fibroblast used for iPSC derivation) 20 samples total, 5 different fibroblast cells, 13 iPSC lines, 1 ES line (H1) from different passages
2013-02-11 | E-GEOD-42869 | biostudies-arrayexpress