Klinefelter syndrome derived hiPSCs show similar XCI behavior as female hPSCs
ABSTRACT: In this study, we reprogrammed fibroblasts from two azoospermic Klinefelter syndrome (KS) patients, and two healthy male and one healthy female donor with an efficient integration-free method using episomal plasmids and laminin-521 (LN521). Whole genome transcriptomics analysis showed differentially expressed genes between KS and healthy male donors with enrichment in gene ontology (GO) terms associated with fertility, cardiovascular development, ossification and brain development, for both KS hiPSCs and fibroblasts, correlating with the KS clinical phenotype. Thorough XCI analysis combining transcriptomics data, RNA FISH and H3K27me3 staining, revealed skewed XCI in one KS fibroblast line and variability in XCI state of KS hiPSCs similar to female hiPSCs, showing either XaXi or XaXe status. Furthermore, we found up-regulated X-linked genes involved in nervous system development, synaptic transmission as well as metabolic processes supporting the potential use of KS derived hiPSC as an in vitro model for KS.
Project description:We studied the variations of mRNA amounts after Evi1 knockdown or Flag-Evi1 overexpression in SKOV-3 cells. Despites Evi1 discovery in 1988, its recognized role as a dominant oncogene in myeloid leukemia and more recently in epithelial cancers, only a few target genes were known and it was not clear why Evi1 was involved in cancer progression. Here we obtained the genomic binding occupancy and expression data for Evi1 in human ovarian carcinoma cells. We identified numerous Evi1 target cancer genes and genes controlling cell migration and adhesion. Moreover, we characterized a transcriptional cooperation between AP1 and Evi1 that regulated proliferation and adhesion through a feed-forward loop. Furthermore, this study provides human genome-wide mapping and downstream analyses for Evi1 that will be useful for the research community. 16 samples were collected. Each condition was done in 4 replicates, collected 65 hours after transfection. Transfections with control siRNA or Flag-expressing vector were used as controls.
Project description:MEF from p8 knock-out mice were immortalized with rasV12/E1A and infected with an empty or a p8-overexpressing retroviral vector as described. Total RNA was isolated and gene expression profile was studied.
Project description:We analyzed the effects of cellular context on the function of the synovial sarcoma-specific fusion protein, SS18-SSX, using human pluripotent stem cells containing the drug-inducible SS18-SSX gene. To investigate the cell-type-dependent effecfts of SS18-SSX, we performed gene expression profiling experiments. Comparison of global gene expressions of hPSCs, hPSC-NCCs, and hPSC-MSCs with or without the inductuion of SS18-SSX2
Project description:X-chromosome inactivation (XCI) is the process that leads to silencing of one X-chromosome in female mammals. XCI is essential to peri-implantation development and is thought to be cell-autonomous, with all factors required to execute silencing being produced within each cell. Nevertheless, external cues to time XCI must exist in vivo, but such developmental signals have yet to be identified. Using multiple approaches to identify developmental regulators of XCI, we identify Indian Hedgehog (IHH) signaling as critical to this process. We demonstrate that IHH signaling keeps XCI in check in pluripotent cells. HH signal transduction through GLI transcription factors regulates XCI by directly binding control elements at the 5’ end of Tsix, the antisense repressor of XCI. GLI binding potentiates Tsix expression and thereby impedes XCI. In vivo, mutating Ihh results in a sex ratio bias against females. Importantly, this female-specific lethality is rescued by a second-site mutation in Tsix. We propose that XCI in the epiblast is regulated by IHH expressed from the visceral endoderm. Our data connect the HH and XCI pathways and support a role of cell-to-cell communication in the developmental timing of XCI. Overall design: Anti-FLAG ChIP-seq from female mouse ES cell lines bearing doxycycline inducible transgenes for GLI1-3xFLAG and GLI2-3xFLAG
Project description:The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them good sources of cells for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, to on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments in hEScs involving over 100 continuous passages, , we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher cell proliferation, and persistence of OCT4-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observedculture-dependent variations in global gene expression and DNA methylation. Verification of the negative effects of enzymatic passaging and feeder-free conditions was performed in hiPSC cultures. Our results highlight the need for careful assessment of effects of culture conditions on cells intended for clinical therapies. Three independent hiPSC clones reprogrammed from human fetal dermal fibroblasts: HDF51iPS1, HDF51iPS7, and HDF51iPS11.
Project description:Our purpose was to investigate genes and molecular mechanisms involved in patients with Leber congenital amaurosis (LCA). Fibroblasts from two unrelated clinically-identified patients (Coriell) were reprogrammed to pluripotency by retroviral transduction. These human induced Pluripotent Stem Cells (hiPSCs) were differentiated into neural stem cells (NSC) that mimicked the neural tube stage and retinal pigmented epithelial (RPE) cells that could be targeted by the disease. A genome wide transcriptome analysis was performed with Affymetrix Exon Array GeneChip®, comparing LCA-hiPSCs derivatives to controls. The aim was to identify differentially expressed genes which may be associated with early developmental defect before the establishment of mature retinal circuitry. We analyzed iPSC-derived retinal pigmented epithelial (RPE) cells from LCA patient's fibroblast (n=2) and iPSC-derivedretinal pigmented epithelial (RPE) cells from healthy people fibroblast (n=2). A total of 13 samples were analyzed : 9 RPE cells derived from iPSC LCA and 4 RPE cells derived from wild-type iPSC.
Project description:The reprogramming of human fibroblasts to generate induced pluripotent stem cells (hiPSCs) has been achieved through the expression of only a few exotic factors1-8, which is morphologically and molecularly verified in outer cellular states by characteristic markers, due to the remodeling of the somatic cell transcription programs in inner cellular states to the ES-like condition. Transcription factor-induced reprogramming to self-renewal and pluripotency raises the question as to how the exotic factors act to bring about these changes in the two cellular states9-11. Here, we applied RNA profiling to uncover gene expression changes and glycan profiling12 to survey structural changes in glycans, to compare hiPSCs and parental somatic cells, in total 51 cells, which were originally cultured and established, and the changes were analyzed by the combination of standard statistical techniques and a network approach13. We fist found a gene expression signature of 2502 genes with significant difference between iPSCs and SCs, and by the following network analysis by considering the expression signature, we found a network signature of 28 regulatory networks of 76 genes, which were related to the glycan biosynthetic pathways including 3 glycosyltransferase, in addition to well known signal pathways and cell-cell interaction pathways. Concomitantly, we found a glycan signature of six glycan structures characterized 16 lectins on lectin microarray by the correspondence with 12 glycosyltransferases in expression signature. In particular, the correspondence detected between the three expression signatures revealed 14 candidate glycosyltransferase, which are responsible for glycan transfer related to known epitopes for the differentiation such as SSEA epitope family in glycan biosynthesis pathway, based on characteristic changes in the cellular surface states of the hiPSCs. This sheds new light on a possible linkage between the inner and outer cellular states for reprogramming to self-renewal and pluripotency in hiPSC. Gene expressions in human induced pluripotent stem (iPS) cells and the provenance somatic cells . iPS cells were induced from four different somatic cells by infection of the retroviral vectors pMXs encoding OCT3/4, SOX2, KLF4 and c-MYC, simultaneously.
Project description:Kaposi's Sarcoma (KS) is a proliferation of aberrant vascular structures lined by spindle cells, and is caused by a gammaherpes virus (HHV8/KSHV). Its course is aggravated by co-infection with HIV-1, where the timing of infection with HIV-1 and HHV8 is important for the clinical outcome. METHODS: In order to better understand the pathogenesis of KS, we have analysed tissue from two AIDS-KS lesions, and from normal skin by serial analysis of gene expression (SAGE). Semi-quantitative RT-PCR was then used to validate the results. RESULTS: The expression profile of AIDS-related KS (AIDS-KS) reflects an active process in the skin. Transcripts of HHV8 were found to be very low, and HIV-1 mRNA was not detected by SAGE, although it could be found using RT-PCR. Comparing the expression profile of AIDS-KS tissue with publicly available SAGE libraries suggested that AIDS-KS mRNA levels are most similar to those in an artificially mixed library of endothelial cells and leukocytes, in line with the description of KS lesions as containing spindle cells with endothelial characteristics, and an inflammatory infiltrate. At least 64 transcripts were found to be significantly elevated, and 28 were statistically downregulated in AIDS-KS compared to normal skin. Five of the upregulated mRNAs, including Tie 1 and sialoadhesin/CD169, were confirmed by semi-quantitative PCR to be elevated in additional AIDS-KS biopsies. Antibodies to sialoadhesin/CD169, a known marker of activated macrophages, were shown to specifically label tumour macrophages. CONCLUSION: The expression profile of AIDS-KS showed 64 genes to be significantly upregulated, and 28 genes downregulated, compared with normal skin. One of the genes with increased expression was sialoadhesin (CD169). Antibodies to sialoadhesin/CD169 specifically labelled tumour-associated macrophages, suggesting that macrophages present in AIDS-KS lesions belong to a subset of human CD169+ macrophages. Keywords: other