Project description:The wide application of pig disease model has caused a surge of interest in the study of derivation of pig induced pluripotent cells (iPSCs). Here we performed genome-wide analysis of gene expression profiling by RNA-seq and small RNA-seq and DNA methylation profile by MeDIP-seq in pig iPSCs through comparison with somatic cells. We identified mRNA and microRNA transcripts that were specifically expressed in pig iPSCs. Our analysis identifies the genes up-regulated in pig iPS compared with somatic cells and also the differentially expressed genes between pig iPSCs under different culture medium. We then pursued comprehensive bioinformatics analyses, including functional annotation of the generated data within the context of biological pathways, to uncover novel biological functions associated with maintenance of pluripotency in pig. This result supports that pig iPS have transcript profiles linked to “ribosome”, “chromatin remodeling”, and genes involved in “cell cycle “that may be critical to maintain their pluripotency, plasticity, and stem cell function. Our analysis demonstrates the key role of RNA splicing in regulating the pluripotency phenotype of pig cells. Specifically, the data indicate distinctive expression patterns for SALL4 spliced variants in different pig cell types and highlight the necessity of defining the type of SALL4 when addressing the expression of this gene in pig cells. MeDIP-seq data revealed that the distribution patterns of methylation signals in pig iPS and somatic cells along the genome. We identify 25 novel porcine miRNA, including pluripotency-related miR-302/367cluster up-regulated in pig iPSCs. At last, we profile the dynamic gene expression signature of pluripotent genes in the preimplantation development embryo of pig. The resulting comprehensive data allowed us to compare various different subsets of pig pluripotent cell. This information provided by our analysis will ultimately advance the efforts at generating stable naïve pluripotency in pig cells.

Project description:The wide application of pig disease model has caused a surge of interest in the study ofderivation of pig induced pluripotent cells (iPSCs). Here we performed genome-wide analysis of gene expression profiling by RNA-seq and small RNA-seq and DNA methylation profile by MeDIP-seq in pig iPSCs through comparison with somatic cells. We identified mRNA and microRNA transcripts that were specifically expressed in pig iPSCs. Our analysis identifies the genes up-regulated in pig iPS compared with somatic cells and also the differentially expressed genes between pig iPSCs under different culture medium. We then pursued comprehensive bioinformatics analyses, including functional annotation of the generated data within the context of biological pathways, to uncover novel biological functions associated with maintenance of pluripotency in pig. This result supports that pig iPS have transcript profiles linked to M-bM-^@M-^\ribosomeM-bM-^@M-^], M-bM-^@M-^\chromatin remodelingM-bM-^@M-^], and genes involved in M-bM-^@M-^\cell cycle M-bM-^@M-^\that may be critical to maintain their pluripotency, plasticity, and stem cell function. Our analysis demonstrates the key role of RNA splicing in regulating the pluripotency phenotype of pig cells. Specifically, the data indicate distinctive expression patterns for SALL4 spliced variants in different pig cell types and highlight the necessity of defining the type of SALL4 when addressing the expression of this gene in pig cells. MeDIP-seq data revealed that the distribution patterns of methylation signals in pig iPS and somatic cells along the genome. We identify 25 novel porcine miRNA, including pluripotency-related miR-302/367cluster up-regulated in pig iPSCs. At last, we profile the dynamic gene expression signature of pluripotent genes in the preimplantation development embryo of pig. The resulting comprehensive data allowed us to compare various different subsets of pig pluripotent cell. This information provided by our analysis will ultimately advance the efforts at generating stable naM-CM-/ve pluripotency in pig cells.

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.

Dataset Information

MeDIP-seq of pig induced pluripotent stem cells

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