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A survey of imprinted gene expression in mouse trophoblast stem cells.

ABSTRACT: Several hundred mammalian genes are expressed preferentially from one parental allele as the result of a process called genomic imprinting. Genomic imprinting is prevalent in extra-embryonic tissue, where it plays an essential role during development. Here, we profiled imprinted gene expression via RNA-Seq in a panel of six mouse trophoblast stem lines, which are ex vivo derivatives of a progenitor population that gives rise to the placental tissue of the mouse. We found evidence of imprinted expression for 48 genes, 31 of which had been described previously as imprinted and 17 of which we suggest as candidate imprinted genes. An equal number of maternally and paternally biased genes were detected. On average, candidate imprinted genes were more lowly expressed and had weaker parent-of-origin biases than known imprinted genes. Several known and candidate imprinted genes showed variability in parent-of-origin expression bias between the six trophoblast stem cell lines. Sixteen of the 48 known and candidate imprinted genes were previously or newly annotated noncoding RNAs and six encoded for a total of 60 annotated microRNAs. Pyrosequencing across our panel of trophoblast stem cell lines returned levels of imprinted expression that were concordant with RNA-Seq measurements for all eight genes examined. Our results solidify trophoblast stem cells as a cell culture-based experimental model to study genomic imprinting, and provide a quantitative foundation upon which to delineate mechanisms by which the process is maintained in the mouse.

SUBMITTER: Calabrese JM

PROVIDER: S-EPMC4426363 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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A survey of imprinted gene expression in mouse trophoblast stem cells.

Calabrese J Mauro JM Starmer Joshua J Schertzer Megan D MD Yee Della D Magnuson Terry T

G3 (Bethesda, Md.) 20150223 5

Several hundred mammalian genes are expressed preferentially from one parental allele as the result of a process called genomic imprinting. Genomic imprinting is prevalent in extra-embryonic tissue, where it plays an essential role during development. Here, we profiled imprinted gene expression via RNA-Seq in a panel of six mouse trophoblast stem lines, which are ex vivo derivatives of a progenitor population that gives rise to the placental tissue of the mouse. We found evidence of imprinted ex ...[more]

PMID: 25711832

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Project description:Trophoblast stem (TS) cells were first isolated from the mouse placenta; however, little is known about their maintenance and niche in vivo. TS cells, like other stem cells, have a unique microenvironment in which the extracellular matrix (ECM) is a component. Placental pathology is associated with ECM change. However, how these changes and the individual ECM components impact the maintenance or differentiation of TS cells has not been established. This study identified which ECM component(s) maintain the greatest expression of markers associated with undifferentiated mouse trophoblast stem (mTS) cells and which alter the profile of markers of differentiation based on mRNA analysis. mTS cells cultured on individual ECM components and subsequent quantitative polymerase chain reaction analysis revealed that laminin promoted the expression of markers associated with undifferentiated TS cells, fibronectin promoted gene expression associated with syncytiotrophoblast (SynT) layer II cells, and collagen IV promoted the expression of genes associated with differentiated trophoblast. To investigate whether pathological placental ECM influenced the expression of genes associated with different trophoblast subtypes, the mouse model of streptozotocin (STZ)-induced pancreatic β cell ablation and diabetes was used. Female mice administered STZ (blood glucose ≥300 mg/dL) or control (blood glucose ≤150 mg/dL) were mated. Placental pathology at embryonic day (E)14.5 was confirmed with reduced fetal blood space area, reduced expression of the pericyte marker αSMA, and decreased expression of ECM proteins. mTS cells cultured on ECM isolated from STZ placenta were associated with reduced expression of undifferentiated mTS markers and increased expression of genes associated with terminally differentiated trophoblast [Gcm-1 and SynA (SynT) and junctional zone Tpbpa and Prl2c2]. Altogether, these results support the value of using ECM isolated from the placenta as a tool for understanding trophoblast contribution to placental pathology.

Project description:BackgroundIn a number of disease processes, the body is unable to repair injured tissue, promoting the need to develop strategies for tissue repair and regeneration, including the use of cellular therapeutics. Trophoblast stem cells (TSCs) are considered putative stem cells as they differentiate into other subtypes of trophoblast cells. To identify cells for future therapeutic strategies, we investigated whether TSCs have properties of stem/progenitor cells including self-renewal and the capacity to differentiate into parenchymal cells of fetal organs, in vitro and in vivo.MethodsTSCs were isolated using anti-CD117 micro-beads, from embryonic day 18.5 placentas. In vitro, CD117+ TSCs were cultured, at a limiting dilution in growth medium for the development of multicellular clones and in specialized medium for differentiation into lung epithelial cells, cardiomyocytes, and retinal photoreceptor cells. CD117+ TSCs were also injected in utero into lung, heart, and the sub-retinal space of embryonic day 13.5 fetuses, and the organs were harvested for histological assessment after a natural delivery.ResultsWe first identified CD117+ cells within the labyrinth zone and chorionic basal plate of murine placentas in late pregnancy, embryonic day 18.5. CD117+ TSCs formed multicellular clones that remained positive for CD117 in vitro, consistent with self-renewal properties. The clonal cells demonstrated multipotency, capable of differentiating into lung epithelial cells (endoderm), cardiomyocytes (mesoderm), and retinal photoreceptor cells (ectoderm). Finally, injection of CD117+ TSCs in utero into lungs, hearts, and the sub-retinal spaces of fetuses resulted in their engraftment on day 1 after birth, and the CD117+ TSCs differentiated into lung alveolar epithelial cells, heart cardiomyocytes, and retina photoreceptor cells, corresponding with the organs in which they were injected.ConclusionsOur findings demonstrate that CD117+ TSCs have the properties of stem cells including clonogenicity, self-renewal, and multipotency. In utero administration of CD117+ TSCs engraft and differentiate into resident cells of the lung, heart, and retina during mouse development.

Project description:BackgroundGenomic imprinting of the largest known cluster, the Kcnq1/KCNQ1 domain on mChr7/hChr11, displays significant differences between mouse and man. Of the fourteen transcripts in this cluster, imprinting of six is ubiquitous in mice and humans, however, imprinted expression of the other eight transcripts is only found in the mouse placenta. The human orthologues of the latter eight transcripts are biallelically expressed, at least from the first trimester onwards. However, as early development is less divergent between species, placental specific imprinting may be present in very early gestation in both mice and humans.Methodology/principal findingsHuman embryonic stem (hES) cells can be differentiated to embryoid bodies and then to trophoblast stem (EB-TS) cells. Using EB-TS cells as a model of post-implantation invading cytotrophoblast, we analysed allelic expression of two telomeric transcripts whose imprinting is placental specific in the mouse, as well as the ncRNA KCNQ1OT1, whose imprinted expression is ubiquitous in early human and mouse development. KCNQ1OT1 expression was monoallelic in all samples but OSBPL5 and NAP1L4 expression was biallelic in EB-TS cells, as well as undifferentiated hES cells and first trimester human fetal placenta. DCN on hChr12, another gene imprinted in the mouse placenta only, was also biallelically expressed in EB-TS cells. The germline maternal methylation imprint at the KvDMR was maintained in both undifferentiated hES cells and EB-TS cells.Conclusions/significanceThe question of placental specific imprinting in the human has not been answered fully. Using a model of human trophoblast very early in gestation we show a lack of imprinting of two telomeric genes in the KCNQ1 region and of DCN, whose imprinted expression is placental specific in mice, providing further evidence to suggest that humans do not exhibit placental specific imprinting. The maintenance of both differential methylation of the KvDMR and monoallelic expression of KCNQ1OT1 indicates that the region is appropriately regulated epigenetically in vitro. Human gestational load is less than in the mouse, resulting in reduced need for maternal resource competition, and therefore maybe also a lack of placental specific imprinting. If genomic imprinting exists to control fetal acquisition of maternal resources driven by the placenta, placenta-specific imprinting may be less important in the human than the mouse.

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A survey of imprinted gene expression in mouse trophoblast stem cells.

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A survey of imprinted gene expression in mouse trophoblast stem cells.

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