Long-term cultures of chicken embryonic primordial and gonadal germ cells.

National audiencePrimordial germ cells (PGCs) are the precursors of germ cell lineage. Located in the epiblast at stage X embryo (EG&K), the PGC translocate anteriorly to the germinal crescent and migrate through the blood vascular system to the germinal ridges within 48-56 hours of development, where they become the gonadal germ cells. We aim to compare the in vitro long-term cultured PGCs derived from the chicken blood of a 2 days old embryo (stages 14 to 17 HH) with the long-term cultured gonocytes taken from male gonads of a 5-6 days old embryo (stages 29 to 30 HH) and determined their basic characters. The results of immunostaining demonstrated that anti-SSEA-1, anti-EMA-1, anti-CVH, anti-integrin β1 and anti-CEACAM, together with PAS stain, bound specifically to chicken PGCs and GGCs. The reactivity of chicken embryonic germ cells obtained from the two different sources to the specific markers used in this study was not altered through the culture. In conclusion, the morphological analysis specific for chicken PGCs and GGCs presented in this study, will further contribute to quick and reliable characterization of these cell types in vitro

Epigenetic landscape of avian stem cells

International audienc

Deciphering the role of key epigenetic actors in chicken ESC and PGC physiology

International audienc

Marques épigénétiques des cellules pluripotentes de poulet

National audienc

Chicken embryonic stem cells and primordial germ cells display different heterochromatic histone marks than their mammalian counterparts

International audienceAbstractBackgroundChromatin epigenetics participate in control of gene expression during metazoan development. DNA methylation and post-translational modifications (PTMs) of histones have been extensively characterised in cell types present in, or derived from, mouse embryos. In embryonic stem cells (ESCs) derived from blastocysts, factors involved in deposition of epigenetic marks regulate properties related to self-renewal and pluripotency. In the germ lineage, changes in histone PTMs and DNA demethylation occur during formation of the primordial germ cells (PGCs) to reset the epigenome of the future gametes. Trimethylation of histone H3 on lysine 27 (H3K27me3) by Polycomb group proteins is involved in several epigenome-remodelling steps, but it remains unclear whether these epigenetic features are conserved in non-mammalian vertebrates. To investigate this question, we compared the abundance and nuclear distribution of the main histone PTMs, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in chicken ESCs, PGCs and blastodermal cells (BCs) with differentiated chicken ESCs and embryonic fibroblasts. In addition, we analysed the expression of chromatin modifier genes to better understand the establishment and dynamics of chromatin epigenetic profiles.ResultsThe nuclear distributions of most PTMs and 5hmC in chicken stem cells were similar to what has been described for mammalian cells. However, unlike mouse pericentric heterochromatin (PCH), chicken ESC PCH contained high levels of trimethylated histone H3 on lysine 27 (H3K27me3). In differentiated chicken cells, PCH was less enriched in H3K27me3 relative to chromatin overall. In PGCs, the H3K27me3 global level was greatly reduced, whereas the H3K9me3 level was elevated. Most chromatin modifier genes known in mammals were expressed in chicken ESCs, PGCs and BCs. Genes presumably involved in de novo DNA methylation were very highly expressed. DNMT3B and HELLS/SMARCA6 were highly expressed in chicken ESCs, PGCs and BCs compared to differentiated chicken ESCs and embryonic fibroblasts, and DNMT3A was strongly expressed in ESCs, differentiated ESCs and BCs.ConclusionsChicken ESCs and PGCs differ from their mammalian counterparts with respect to H3K27 methylation. High enrichment of H3K27me3 at PCH is specific to pluripotent cells in chicken. Our results demonstrate that the dynamics in chromatin constitution described during mouse development is not universal to all vertebrate species

Epigenetic marks in chicken pluripotent cells and embryo

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NANOG Is Required for the Long-Term Establishment of Avian Somatic Reprogrammed Cells

Summary: Somatic reprogramming, which was first identified in rodents, remains poorly described in non-mammalian species. Here, we generated avian reprogrammed cells by reprogramming of chicken and duck primary embryonic fibroblasts. The efficient generation of long-term proliferating cells depends on the method of delivery of reprogramming factors and the addition of NANOG and LIN28 to the canonical OCT4, SOX2, KLF4, and c-MYC gene combination. The reprogrammed cells were positive for several key pluripotency-associated markers including alkaline phosphatase activity, telomerase activity, SSEA1 expression, and specific cell cycle and epigenetic markers. Upregulated endogenous pluripotency-associated genes included POU5F3 (POUV) and KLF4, whereas cells failed to upregulate NANOG and LIN28A. However, cells showed a tumorigenic propensity when injected into recipient embryos. In conclusion, although the somatic reprogramming process is active in avian primary cells, it needs to be optimized to obtain fully reprogrammed cells with similar properties to those of chicken embryonic stem cells. : In this article, the authors show that the NANOG gene plays a crucial role in the long-term establishment of avian reprogrammed cells that have some of the specific markers of stem cells but that do not present all the developmental properties of induced pluripotent stem cells such as described in mammals. Keywords: avian species, somatic reprogramming, embryonic stem cells, NANOG, pluripotency, chicken, duck, embryos, blastoderm, phylogenic compariso

Transcriptomic analysis of MDV permissive chicken embryonic stem (ES)- derived cells

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ESCDL-1 cell-line derived from chicken ES cells is fully permissive to Mardiviruses

National audienc

ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses.

Marek's disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek's disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies