Characterization of the gene expression profile of porcine iPS-like cells derived by two reprogramming techniques from translocated t(Y;14) fibroblasts
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ABSTRACT:
ORGANISM(S): Sus scrofa
PROVIDER: GSE71792 | GEO | 2016/01/01
SECONDARY ACCESSION(S): PRJNA292094
REPOSITORIES: GEO
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Project description:In the present study we compare the effect of integrative and non-integrative strategies on pluripotency during pig somatic cells reprogramming to pluripotency. Microarray analysis was used to compare the gene expression profile of non reprogrammed porcine embryonic fibroblasts (PEF) and of iPS-like cells obtained by cell reprogramming of control amniocytes (PB20) or fibroblasts from animals carrying a t(Y;14) translocation (I, NI). We investigate the impact of different reprogramming techniques by using a first integrative method based on viral infection (PB20, I) and a second non-integrative method based on the use of Sendaï viruses (NI). Moreover, we analyzed the changes in gene expression profile upon embryoïd formation in the NI-iPS-like cells (EB). We cultured one cell line of porcine embryonic fibroblast, one cell line of iPS-like cells derived from normal amniocytes (PB20), 2 cell lines derived from t(Y;14) fibroblast by the integrative technique (I3 and I4) both at early (ep) and late passages (lp), and three cell lines derived from t(Y;14) fibroblasts by the non-integrative method (NI12, NI13 and NI20). Embryoïd bodies from NI cell lines were obtained by 10 days of floating culture in a specific medium under low agitation (EB12, EB13 and EB20). Total RNA from cell pellets of each sample was extracted in triplicate using the Nucleospin RNA kit (Macherey-Nagel) and checked for quantity and quality. After the microarrays experimentation, quality control and normalization, 34 over 36 samples were conserved (represented here).
2016-07-03 | E-GEOD-71792 | biostudies-arrayexpress
Project description:In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1-11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming the cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed that the transcriptional regulators Nr0b1 and Etv5 are specifically expressed in this early reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study shows an ordered sequence of transitions during the earliest steps of iPS cell reprogramming that deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. Samples for poised (CD73+ or CD49d+) and non-poised (CD73-) reprogramming samples were FACS sorted 6 and 9 days after induction of Klf4, Oct4, Sox2 and cMyc in Rosa-rtTA +/- mouse embryonic fibroblasts (MEFs). 'Total' populations are expression analyses for unsorted populations analyzed at the same time points. Control populations were also sampled: mouse embryonic fibroblasts (MEFs), partially reprogrammed cells (SC4) and mouse embryonic stem cell (ESC).
2015-01-31 | E-GEOD-62957 | biostudies-arrayexpress
Project description:Forced expression of four transcription factors Oct4,Sox2, Klf4 and Myc (OSKM) induces somatic cell reprogramming towards pluripotency. Major efforts have been made to characterize the molecular events involved in this process. Yet, it remains elusive how gene expression change, epigenetic landscape remodelling and cell fate conversion are triggered by expression of these Yamanaka factors.To address this gap,we utilized a secondary inducible reprogramming system and performed genome-wide profilings of Oct4 binding, histone modification(H3K4me3/H3K27me3/H3K4me1/H3K27ac), and gene expression analysis during this process. Through integrative analysis, we revealed stage-specific Oct4 binding and enhancer signatures in consistence with gene expression changes,in which the initial regression of somatic program is followed by the gradual acquisition of pluripotent program. Oct4 preferatially binds to H3K4me1 marked enhancer regions and Oct4 binding is positively correlated with active mark H3K27ac. Moreover,we observed significant enhancer activation of epigenetic related genes, especially acetylation associated genes, prior to pluripotency network activation, suggesting a pivotal role of epigenetic remodelling in the process of pluripotency acquisition and maintenance. We used microarrays to explore the global changes of gene expression during OSKM-mediated somatic cell reprogramming. time series design with bulk population samples collected at different time point of 2nd reprogramming as well as a corresponding iPS cell line. Each sample include two replicates. 2nd mouse embryonic fibroblasts(sample day0) were treated with ES medium supplemented with 1µg/ml Doxcycline and 50µg/ml Vitamin C for 15 days (sample day1-day15); 72 hours after dox and Vc withdrawl, dox-independent iPS cells are collected(sample day18).
2015-12-31 | E-GEOD-67462 | biostudies-arrayexpress
Project description:Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. However, microarray analysis showed genes differentially expressed in ES and iPS cell lines according to their hematopoietic potential. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.
2015-11-17 | GSE75051 | GEO
Project description:We used heterokaryon cell fusion based reprogramming and identified the cytokine IL6 as a potential regulator of reprogramming to pluripotency. We generated iPS clones using the four reprogramming factors (4F) Oct4, Klf4, Sox2, and c-Myc. In addition, iPS clones were generated using only three factors (3F: Oct4, Klf4, amd Sox2) with the addition of the cytokine IL6 to reprogramming culture conditions. Global RNA-Seq of the 3F + IL6 derived iPS clones was done for comparison with 4F-derived iPS clones, mouse embryonic stem cells and mouse embryonic fibroblasts.
2013-08-28 | GSE46104 | GEO
Project description:In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1-11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming the cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed that the transcriptional regulators Nr0b1 and Etv5 are specifically expressed in this early reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study shows an ordered sequence of transitions during the earliest steps of iPS cell reprogramming that deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.
2015-01-31 | GSE62957 | GEO
Project description:Somatic cells gain pluripotency after transfection of Oct4, Sox2, Klf4, and cMyc (OSKM), and chromatin remodeling has been proved to be involved in this process. To date, chromatin accessibility in porcine induced pluripotency stem cells (piPSCs) was less researched. Here, we explore the connection of chromatin accessibility and transcriptome between genetically matched PEF and piPSCs with high throughput sequencing (ATAC-seq and RNA-seq) approach. In addition, we acquire parent-of-origin chromatin loci and genes after blasting ATAC-seq and RNA-seq data with polymorphic SNP loci which derive from two parental genomes. As a result, we find 4,373 differentially expressed genes (DEGs), and 10,883 differential chromatin locus. Compared with PEF, the closed chromatin loci in piPSCs are tightly connected with the down-expressed genes, while the opened chromatin plays a limited role in the up-regulation of genes. Potential mechanisms of transcription factors (TF) in piPSCs reprogramming have been identified. Moreover, we find 77 parent-of-origin genes and 7 parent-of-origin chromatin loci commonly existed in PEF and piPSC; parent-of-origin chromatin accessibility is not related to the bias expression of RNA. These data reveal regulatory association between global or parent-of-origin chromatin accessibility and transcriptome, which provides new sight to research porcine iPSC reprogramming and genome imprinting.
2022-10-19 | GSE173304 | GEO
Project description:Somatic cells gain pluripotency after transfection of Oct4, Sox2, Klf4, and cMyc (OSKM), and chromatin remodeling has been proved to be involved in this process. To date, chromatin accessibility in porcine induced pluripotency stem cells (piPSCs) was less researched. Here, we explore the connection of chromatin accessibility and transcriptome between genetically matched PEF and piPSCs with high throughput sequencing (ATAC-seq and RNA-seq) approach. In addition, we acquire parent-of-origin chromatin loci and genes after blasting ATAC-seq and RNA-seq data with polymorphic SNP loci which derive from two parental genomes. As a result, we find 4,373 differentially expressed genes (DEGs), and 10,883 differential chromatin locus. Compared with PEF, the closed chromatin loci in piPSCs are tightly connected with the down-expressed genes, while the opened chromatin plays a limited role in the up-regulation of genes. Potential mechanisms of transcription factors (TF) in piPSCs reprogramming have been identified. Moreover, we find 77 parent-of-origin genes and 7 parent-of-origin chromatin loci commonly existed in PEF and piPSC; parent-of-origin chromatin accessibility is not related to the bias expression of RNA. These data reveal regulatory association between global or parent-of-origin chromatin accessibility and transcriptome, which provides new sight to research porcine iPSC reprogramming and genome imprinting.
2022-10-19 | GSE173275 | GEO
Project description:Pluripotent stem cells are derived from culture of early embryos or the germline, and can be induced by reprogramming of somatic cells. Barriers to reprogramming are expected to exist that stabilize the differentiated state and have tumor suppression functions. However, we have a limited understanding of what such barriers might be. To find novel barriers to reprogramming to pluripotency, we compared the transcriptional profiles of the mouse germline to pluripotent and somatic cells, in vivo and in vitro. There is a remarkable global expression of the transcriptional program for pluripotency in Primordial Germ Cells (PGCs). We identify parallels between PGCs reprogramming to pluripotency and human germ cell tumorigenesis, including the loss of LATS2, a tumor suppressor kinase of the Hippo pathway. We show that knockdown of LATS2 increases the efficiency of induction of pluripotency in human cells. LATS2 RNAi, unlike p53 RNAi, specifically enhances the generation of fully reprogrammed iPS cells without accelerating cell proliferation. We further show that LATS2 represses reprogramming in human cells by post-transcriptionally antagonizing TAZ but not YAP, two downstream effectors of the Hippo pathway. These results reveal transcriptional parallels between germ cell transformation and the generation of iPS cells, and indicate that the Hippo pathway constitutes a barrier to cellular reprogramming. Mouse pluripotent cells isolated directly from embryos or cultured in vitro as stem cells were analyzed using Affymetrix expression microarrays, together with several non-pluripotent cell controls, in 2-6 replicates per sample.
2012-01-31 | E-GEOD-35416 | biostudies-arrayexpress
Project description:Clinical application of induced pluripotent stem (iPS) cells is limited by low efficiency of iPS derivation, and protocols that permanently modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-mutagenic strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem (RiPS) cells into terminally differentiated myogenic cells. Our method represents a safe, efficient strategy for somatic cell reprogramming and directing cell fates that has broad applicability for basic research, disease modeling and regenerative medicine. We isolated RNA from human RNA derived iPS cells, viral derived iPS cells, different human fibroblasts and human embryonic stem cells for hybridization to the Affymetrix gene expression microarrays.
2010-09-30 | E-GEOD-23583 | biostudies-arrayexpress