Expression data from induced pluripotent stem cells (iPSCs), mouse embryonic stem cells (mESCs), and mouse embryonic fibroblast (MEFs)
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ABSTRACT: During reprogramming of mouse embryonic fibroblast, pluripotent genes are up-regulated. Once iPSCs are successfully reprogrammed, the global gene profiles of iPSCs are comparable to mouse ESC. We used microarrays to detail the global programme of gene expression in iPSCs, mESCs, MEFs.
Project description:Leptin receptors (Lepr) are expressed by various types of stem cells including mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, and induced pluripotent stem cells. Leptin/lepr signaling is also a central regulator of metabolism. However, the role of Lepr in pluripotency, metabolic disease progression and growth development is still controversial and poorly understood. In the present study, we explored the Lepr function in disease progression, pluripotency and metabolism using day 14.5 mouse embryonic fibroblasts (MEFs) and their reprogrammed induced pluripotent stem cells (iPSCs) as model system. We successfully reprogrammed mouse embryonic fibroblasts into iPSCs from control and db/db (Lepr deficient) mice. Using a global quantitative proteomic approach, we identified key pathways regulating pluripotency, metabolic homeostasis and protein synthesis during fetal growth and development. The Lepr MEFs show abnormal metabolic abnormalities and mitochondrial dysfunction as compared to control MEFs, while Lepr iPSCs show upregulated elongated factor 4 e (eIF4e) protein synthesis pathway and altered Oct4 and Stat3 pathways which are involved in normal fetal growth development. Furthermore, chip analysis revealed that higher Stat3 binding on the promoter of eIF4e in Lepr iPSCs leads to higher protein synthesis in these cell types as compared to control iPSCs. Finally, point mutation corrected Lepr iPSCs using CRISPR/Cas9 gene editing method showed recovered pluripotency, metabolic and protein synthesis pathways. In conclusion, we have shown that Lepr signaling is involved in the regulation of the metabolic properties and key developmental pathways in MEFs and stemness of pluripotent stem cells. Disruption of Lepr signaling has been shown to involve in the pathophysiology of various diseases including obesity and diabetes. The generated MEFs and iPSCs in this present study provide valuable tools to explore the role of Lepr in the progression of obesity, diabetes and metabolic abnormalities, and to find the putative targets of Lepr signaling during the development of these diseases.
Project description:Genome-wide occupancy of the circadian clock Cryptochrome1 (CRY1) was compared between somatic differentiated mouse embryonic fibroblasts (MEFs) and pluripotent stem cells including precursor-induced pluripotent stem cells (iPSCs), iPSCs, and embryonic stem cells (ESCs)
Project description:We used RRBS to analyze DNA methylation in mESC lines deficient for maternal Dnmt3L (Dnmt3L mKO), zygotic Dnmt3L (Dnmt3L KO), and both maternal and zygotic Dnmt3L (Dnmt3L mzKO). Compared to wild-type (WT) mESCs, Dnmt3L mKO mESCs exhibit severe loss of methylation at imprinted loci but no changes in global DNA methylation, Dnmt3L KO mESCs exhibit moderate loss of methylation at many Dnmt3a target regions but do not affect methylation at imprinted loci, and Dnmt3L mzKO mESCs exhibit combined changes of mKO and KO cells, with severe loss of methylation at imprinted loci and moderate loss of methylation at Dnmt3a target regions.
Project description:The generation of induced pluripotent stem cells (iPSCs) involves activation of the endogenous pluripotency circuitry and global DNA demethylation late in reprogramming, but temporal resolution of these events are insufficient using existing stage markers. Here, we generated murine transgenic lines harboring dual fluorescent reporters reflecting cell-state specific expression of the master pluripotency factor Oct4 and the 5-methylcytosine dioxygenase Tet1. By assessing reprogramming intermediates based on dual reporter patterns, we identified a sequential order of Tet1 and Oct4 gene activation at proximal and distal regulatory elements following pluripotency entry. A transient phase of global gene repression accompanies full activation of Tet1, precedes activation of meiotic and gametogenesis genes, and distinguishes phases of global DNA demethylation reminiscent of germ-line reprogramming. Loss of Tet1 is compatible with reprogramming towards full Oct4 gene activation, but generates iPSCs with epigenetic defects. Therefore, the transcriptional logic of Tet1 expression signals an epigenetic roadmap towards efficient reprogramming.
Project description:This experiment is to compare derived iPSCs with original MEFs and real mES to test the overall quality of iPSCs and whether they are similar to mES on global mRNA expressions
Project description:Here, we focused on the intermediate stages of SCR by comparing the somatic cell line induced by OCT4, SOX2, and KLF4 (OSK) for 7 days with mouse embryonic fibroblasts (MEFs), iPSCs, and embryonic stem cells (ESCs). Transcriptional profiles of these four cell lines were analyzed by microarray, and we found that the transition process from day 7 to the formation of iPSCs is crucial for SCR and that the reverse expression patterns can provide more candidate markers to distinguish ESCs and somatic cells iPSC. Data confirmed that the viral infection results in defense innate immunity, DNA damage, and apoptosis in MEFs, which slows down cell proliferation and immortalization to inhibit SCR. Although SCR is initiated by OSK, the p53 signaling pathway can affect the transcriptional regulatory networks through cell cycle and genomic instability as a powerful core node. MEFs were derived from embryonic day 13.5 C57BL6 mice embryos. Cell line day7 is MEF induced by OCT4, SOX2, and KLF4 (OSK) for 7 days with mouse embryonic fibroblasts (MEFs), iPSCs, and embryonic stem cells (ESCs).
Project description:The generation of induced pluripotent stem cells (iPSCs) involves activation of the endogenous pluripotency circuitry and global DNA demethylation late in reprogramming, but temporal resolution of these events are insufficient using existing stage markers. Here, we generated murine transgenic lines harboring dual fluorescent reporters reflecting cell-state specific expression of the master pluripotency factor Oct4 and the 5-methylcytosine dioxygenase Tet1. By assessing reprogramming intermediates based on dual reporter patterns, we identified a sequential order of Tet1 and Oct4 gene activation at proximal and distal regulatory elements following pluripotency entry. A transient phase of global gene repression accompanies full activation of Tet1, precedes activation of meiotic and gametogenesis genes, and distinguishes phases of global DNA demethylation reminiscent of germ-line reprogramming. Loss of Tet1 is compatible with reprogramming towards full Oct4 gene activation, but generates iPSCs with epigenetic defects. Therefore, the transcriptional logic of Tet1 expression signals an epigenetic roadmap towards efficient reprogramming.
Project description:Global heterochromatin reduction, which is one of the hallmarks of aging cells, is associated with reduced transposable element repression and increased risk of chromatin instability. To ensure genomic integrity, the irreparable cells in a population exit permanently from the cell cycle, and this process is termed “senescence”. However, senescence only blocks the expansion of unwanted cells, and the aberrant chromatin of senescent cells remains unstable. Serendipitously, we found that the transient ectopic expression of a repressive epigenetic modulator, DNA methyltransferase 3-like (DNMT3L) was sufficient to delay the premature senescence progression of late-passage mouse embryonic fibroblasts (MEFs) associated with a tightened global chromatin structure. DNMT3L induces more repressive H3K9 methylation on endogenous retroviruses and downregulates the derepressed transposons in aging MEFs. In addition, we found that a pulse of ectopic DNMT3L resulted in the reestablishment of H3K27me3 on polycomb repressive complex 2 (PRC2)-target genes that were derepressed in old MEFs. We demonstrated that ectopic DNMT3L interacted with PRC2 in MEFs. Our data also suggested that ectopic DNMT3L might guide PRC2 to redress deregulated chromatin regions in aging cells. This study might lead to an epigenetic reinforcement strategy for overcoming aging-associated epimutation and senescence.