Induced Pluripotent Stem Cells from the Pig Somatic Cells
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ABSTRACT:
ORGANISM(S): Sus scrofa
PROVIDER: GSE15472 | GEO | 2009/07/10
SECONDARY ACCESSION(S): PRJNA115913
REPOSITORIES: GEO
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Project description:We have derived induced porcine pluripotent stem cells (iPPSCs) from porcine fetal fibroblasts by lentiviral transduction of four human (h) reprogramming genes, hOCT4, hSOX2, hKLF4 and hc-MYC , the same combination of factors used for deriving induced pluripotent stem cell (iPSC) lines in both mouse and human. The obtained iPPSC lines resemble human embryonic stem cells (ESC) in their gross morphology and dependence on FGF2, on the other hand, the iPPSCs share characteristics like growth rate and cell surface markers with mESC . Additionally, the iPPSCs express pluripotency- associated genes similar to mouse and human iPSCs as well as ESC, along with the pig epiblast cells. Some of the iPPSC lines retained a stable karyotype and phenotype even in culture for a prolonged period of time (passage 39). The iPPSCs can be induced to differentiate along lineages representative of the three embryonic germ layers both in vitro and in vivo demonstrating the pluripotency of these cells. Experiment Overall Design: The open reading frames of the human (h) SOX2, hKLF4, and hc-MYC have been cloned into the BamHI and EcoRI sites of the lentiviral vector, FUGW and the hOCT4 cDNA into the pSIN18.cPPT.hEF1a.EGFP.WPRE vector at its BamHI and SalI sites. Pseudovirus was produced in human 293FT cells (Invitrogen) by transfection with each lentiviral vector (FUGW or pSIN18) along with VSV-G envelope vector (pMD2.G) and packaging vector (psPAX2) by using Lipofectamine-Plus reagents (Invitrogen). The target cells were porcine fibroblasts expressing enhanced green fluorescent protein (EGFP-PFF) derived at day 34 of pregnancy. Titered virus was used to infect the target cells (1 x 10^5 /35mm dish). On the second day following infection (day 2), the cells were dispersed with trypsin and transferred to 10 cm plates preseeded with irradiated mouse embryonic fibroblasts (MEF), and after day 3 the cells were maintained on a culture medium standardized for human ESC containing 4 ng/ml human FGF2.
2009-07-19 | E-GEOD-15472 | biostudies-arrayexpress
Project description:Chavez2009 - a core regulatory network of OCT4 in human embryonic stem cells
A core OCT4-regulated network has been identified as a test case, to analyase stem cell characteristics and cellular differentiation.
This model is described in the article:
In silico identification of a core regulatory network of OCT4 in human embryonic stem cells using an integrated approach.
Chavez L, Bais AS, Vingron M, Lehrach H, Adjaye J, Herwig R
BMC Genomics, 2009, 10:314
Abstract:
BACKGROUND: The transcription factor OCT4 is highly expressed in pluripotent embryonic stem cells which are derived from the inner cell mass of mammalian blastocysts. Pluripotency and self renewal are controlled by a transcription regulatory network governed by the transcription factors OCT4, SOX2 and NANOG. Recent studies on reprogramming somatic cells to induced pluripotent stem cells highlight OCT4 as a key regulator of pluripotency.
RESULTS: We have carried out an integrated analysis of high-throughput data (ChIP-on-chip and RNAi experiments along with promoter sequence analysis of putative target genes) and identified a core OCT4 regulatory network in human embryonic stem cells consisting of 33 target genes. Enrichment analysis with these target genes revealed that this integrative analysis increases the functional information content by factors of 1.3 - 4.7 compared to the individual studies. In order to identify potential regulatory co-factors of OCT4, we performed a de novo motif analysis. In addition to known validated OCT4 motifs we obtained binding sites similar to motifs recognized by further regulators of pluripotency and development; e.g. the heterodimer of the transcription factors C-MYC and MAX, a prerequisite for C-MYC transcriptional activity that leads to cell growth and proliferation.
CONCLUSION: Our analysis shows how heterogeneous functional information can be integrated in order to reconstruct gene regulatory networks. As a test case we identified a core OCT4-regulated network that is important for the analysis of stem cell characteristics and cellular differentiation. Functional information is largely enriched using different experimental results. The de novo motif discovery identified well-known regulators closely connected to the OCT4 network as well as potential new regulators of pluripotency and differentiation. These results provide the basis for further targeted functional studies.
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2005-01-01 | MODEL1305010000 | BioModels
Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naM-CM-/ve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. Transcript profiling utilized Affymetrix porcine microarrays were conducted to compare the gene expressions associated with pluripotency in the two LIF-dependent pESK lines (pESK I & II, passage 14 & 5, respectively) with a naM-CM-/ve phenotype and two porcine iPSC lines (ID4 & ID6, both passage 7, GSE15472) with a primed phenotype that re-programming of porcine fetal fibroblasts (EGFP-PFF, GSE15472). The pESK lines clustered separately from the porcine iPSC lines, EGFP-PFF and primary cultures established from explants of porine umbilical cord (PUC). Porcine pluripotent stem cells were derived from the inner cell mass with transduction with human KLF4 by lentiviral transduction.
2011-12-02 | E-GEOD-26369 | biostudies-arrayexpress
Project description:Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eightcell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.
2021-04-12 | GSE129760 | GEO
Project description:Human induced Pluripotent Stem cells (hiPSc) and their differentiated progeny have great potential for modelling disease. To realise this potential, robust protocols need to be developed for deriving authentic differentiated cell lineages and these lineages need to be rigorously characterised. We have generated hiPSc using retrovirus-mediated delivery of reprogramming factors, and have used them for characterising mid-brain dopaminergic neurons. hiPSc lines have been screened using SNP array to assess chromosomal stability, and validation of the pluripotency of the hiPSc lines is provided by Pluritest assessment of transcriptome datasets. A mature physiological cellular model of human dopaminergic neurons. human iPSc lines were derived from normal human dermal fibroblasts.
2013-01-31 | E-GEOD-43903 | biostudies-arrayexpress
Project description:Human induced Pluripotent Stem cells (hiPSc) and their differentiated progeny have great potential for modelling disease. To realise this potential, robust protocols need to be developed for deriving authentic differentiated cell lineages and these lineages need to be rigorously characterised. We have generated hiPSc using retrovirus-mediated delivery of reprogramming factors, and have used them for characterising mid-brain dopaminergic neurons. hiPSc lines have been screened using SNP array to assess chromosomal stability, and validation of the pluripotency of the hiPSc lines is provided by Pluritest assessment of transcriptome datasets. A mature physiological cellular model of human dopaminergic neurons. human iPSc lines were derived from normal human dermal fibroblasts.
2013-01-31 | E-GEOD-43902 | biostudies-arrayexpress
Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naïve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. Transcript profiling utilized Affymetrix porcine microarrays were conducted to compare the gene expressions associated with pluripotency in the two LIF-dependent pESK lines (pESK I & II, passage 14 & 5, respectively) with a naïve phenotype and two porcine iPSC lines (ID4 & ID6, both passage 7, GSE15472) with a primed phenotype that re-programming of porcine fetal fibroblasts (EGFP-PFF, GSE15472). The pESK lines clustered separately from the porcine iPSC lines, EGFP-PFF and primary cultures established from explants of porine umbilical cord (PUC).
2011-12-02 | GSE26369 | GEO
Project description:With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.
2024-03-26 | PXD045525 | Pride
Project description:With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.
2024-03-26 | PXD045347 | Pride
Project description:Understanding essential signaling network requirements and adjustment the culture conditions for porcine pluripotent stem cells (pPSC) that allow its full potential are necessary. Here we compared culture conditions of various cytokine and kinase inhibitor combinations by deriving porcine induced pluripotent stem cells (piPSC) and porcine embryonic stem cell (pESC)-like cells. We modified the naïve-type human PSC condition by replacing TGFB1 with TGFB inhibitor and developed a so-called FL6i condition, consisting of FGF2, LIF, p38i, JNKi, TGFBi, GSK3i, MEKi, and BMPi. In such conditions, the primed type lentiviral reprogrammed piPSC (Lv-piPSC) was converted into naïve-like state demonstrating increased expression of endogenous pluripotent genes. By using the FL6i condition, new piPSC lines were generated with non-integrative episomal plasmids (Epi-piPSC). While concurrently generated piPSC in condition with FGF2 and LIF only, and without inhibitors lost the pluripotency within a couple passages of the culture, piPSC in FL6i were successfully established stable lines (> 45 passages) with pluripotent phenotypes. The Epi-piPSC-FL6i lines expressed higher endogenous pPOU5F1, and pSOX2 expressions than Lv-piPSC, however, variable transgene expressions were detected with consistent presence of POU5F1. The teratomas generated from Epi-piPSC-FL6i that developed fully differentiated teratomas while those from Lv-piPSC lines and Epi-piPSC derived in a different condition were limited in differentiation range, suggesting the beneficial role of the FL6i condition supporting elevated pluripotent phenotypes. FL6i conditions were also able to maintain the embryo derived cells with dome-shaped phenotype and extended the culture period that cells showed undifferentiated phenotype than the ones cultured with FGF2 alone. But neither conditions established stable pESC lines. RNAseq analysis with the piPSC and pESC-like cells revealed elevated expressions of early differentiation markers in the lines derived without inhibitors. The pathway analysis suggested that the activation of TGFB and BMP signaling pathways may be responsible for losing pluripotent state in porcine cells.
2019-08-31 | GSE126150 | GEO