Bertrand Pain

Role of miR-34c microRNA in the late steps of spermatogenesis

Spermatogenesis is a cyclic process in which diploid spermatogonia differentiate into haploid spermatozoa. This process is highly regulated, notably at the post-transcriptional level. MicroRNAs (miRNAs), single-stranded noncoding RNA molecules of about 20-25 nucleotides, are implicated in the regulation of many important biological pathways such as proliferation, apoptosis, and differentiation. We wondered whether miRNAs could play a role during spermatogenesis. The miRNA expression repertoire was tested in germ cells, and we present data showing that miR-34c was highly expressed only in these cells. Furthermore, our findings indicate that in male gonads, miR-34c expression is largely p53 independent in contrast to previous results showing a direct link in somatic cells between the miR-34 family and this tumor suppressor protein. In order to identify target genes involved in germinal lineage differentiation, we overexpressed miR-34c in HeLa cells, analyzed the transcriptome of these modified cells, and noticed a shift of the expression profile toward the germinal lineage. Recently, it has been shown that exogenous expression of Ddx4/Vasa in embryonic chicken stem cells (cESC) induces cESC reprogramming toward a germ cell fate. When we simultaneously expressed miR-34c in such cells, we could detect an up-regulation of germ cell-specific genes whereas the expression of other lineage specific markers remained unchanged. These data suggest that miR-34c could play a role by enhancing the germinal phenotype of cells already committed to this lineage.

The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells

Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.

Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency

Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.

Reprogramming capacity of Nanog is functionally conserved in vertebrates and resides in a unique homeodomain.

Pluripotency is a developmental ground state that can be recreated by direct reprogramming. Establishment of pluripotency is crucially dependent on the homeodomain-containing transcription factor Nanog. Compared with other pluripotency-associated genes, however, Nanog shows relatively low sequence conservation. Here, we investigated whether Nanog orthologs have the capacity to orchestrate establishment of pluripotency in Nanog–/– somatic cells. Mammalian, avian and teleost orthologs of Nanog enabled efficient reprogramming to full pluripotency, despite sharing as little as 13% sequence identity with mouse Nanog. Nanog orthologs supported self-renewal of pluripotent cells in the absence of leukemia inhibitory factor, and directly regulated mouse Nanog target genes. Related homeodomain transcription factors showed no reprogramming activity. Nanog is distinguished by the presence of two unique residues in the DNA recognition helix of its homeodomain, and mutations in these positions impaired reprogramming. On the basis of genome analysis and homeodomain identity, we propose that Nanog is a vertebrate innovation, which shared an ancestor with the Bsx gene family prior to the vertebrate radiation. However, cephalochordate Bsx did not have the capacity to replace mouse Nanog in reprogramming. Surprisingly, the Nanog homeodomain, a short sequence that contains the only recognizable conservation between Nanog orthologs, was sufficient to induce naive pluripotency in Nanog–/– somatic cells. This shows that control of the pluripotent state resides within a unique DNA-binding domain, which appeared at least 450 million years ago in a common ancestor of vertebrates. Our results support the hypothesis that naive pluripotency is a generic feature of vertebrate development.

Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate

When they are derived from blastodermal cells of the pre-primitive streak in vitro, the pluripotency of Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These cESC can differentiate into derivatives of the three germ layers both in vitro and in vivo, but they only weakly colonize the gonads of host embryos. By contrast, non-cultured blastodermal cells and long-term cultured chicken primordial germ cells maintain full germline competence. This restriction in the germline potential of the cESC may result from either early germline determination in the donor embryos or it may occur as a result of in vitro culture. We are interested in understanding the genetic determinants of germline programming. The RNA binding protein Cvh (Chicken Vasa Homologue) is considered as one such determinant, although its role in germ cell physiology is still unclear. Here we show that the exogenous expression of Cvh, combined with appropriate culture conditions, induces cESC reprogramming towards a germ cell fate. Indeed, these cells express the Dazl, Tudor and Sycp3 germline markers, and they display improved germline colonization and adopt a germ cell fate when injected into recipient embryos. Thus, our results demonstrate that Vasa can drive ES cell differentiation towards the germ cell lineage, both in vitro and in vivo.

Transcriptome analysis of chicken ES, blastodermal and germ cells reveals that chick ES cells are equivalent to mouse ES cells rather than EpiSC

Pluripotent Embryonic Stem cell (ESC) lines can be derived from a variety of sources. Mouse lines derived from the early blastocyst and from primordial germ cells (PGCs) can contribute to all somatic lineages and to the germ line, whereas cells from slightly later embryos (EpiSC) no longer contribute to the germ line. In chick, pluripotent ESCs can be obtained from PGCs and from early blastoderms. Established PGC lines and freshly isolated blastodermal cells (cBC) can contribute to both germinal and somatic lineages but established lines from the former (cESC) can only produce somatic cell types. For this reason, cESCs are often considered to be equivalent to mouse EpiSC. To define these cell types more rigorously, we have performed comparative microarray analysis to describe a transcriptomic profile specific for each cell type. This is validated by real time RT-PCR and in situ hybridisation. We find that both cES and cBC cells express classic pluripotency-related genes (including cPOUV/OCT4, NANOG, SOX2/3, KLF2 and SALL4), whereas expression of DAZL, DND1, DDX4 and PIWIL1 defines a molecular signature for germ cells. Surprisingly, contrary to the prevailing view, our results also suggest that cES cells resemble mouse ES cells more closely than mouse EpiSC.

Pluripotent genes in avian stem cells

Embryonic stem (ES) cells were first isolated in 1981 in the mouse from the in vitro proliferation of the inner cell mass of a 3.5 days post‐coitum (dpc) blastocyst. Later on, epiblast stem cells (EpiSC) were identified from in vitro culture of the epiblast of a 6.5 dpc mouse embryo, leading to the concept of naïve and primed stem cells. Among non‐mammalian species, ES cells have been characterized both in birds and fish; here, we focus on cells derived from chicken and the pluripotent associated markers such as OCT4, SOX2, NANOG, and KLF, previously identified in mammalian cells. In this review, we present both published and original data regarding the involvement of those pluripotent associated genes in the ES cells and early embryo of chicken.

Chicken embryonic stem cells: establishment and characterization.

Embryonic stem (ES) cells are unique models for investigating early development and cell differentiation. First identified in mouse and later in other mammals, these cells have also been isolated in avian species. Here, using chicken as a model, we describe a set of protocols allowing the isolation, maintenance, genetic modification, differentiation, and injection of the chicken embryonic stem (cES) cells into embryos for obtaining chimeric animals.

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.

In vitro generation and characterization of chicken long-term germ cells from different embryonic origins

Primordial germ cells (PGCs) are the precursors of differentiated germ cells. Located in the epiblast of a stage X (EG&K) embryo, the PGCs translocate anteriorly to the germinal crescent and migrate, within 48 to 56 hours of development, through the blood vascular system to the germinal ridges where they become the gonadal germ cells (GGCs). We aim to generate, compare, and determine the basic characters of the in vitro long-term cultured PGCs derived from (1) the chicken blastodermal cells (at stages IX-XII); (2) the chicken blood of a 2-day old embryo (stages 14-17 Hamburger Hamilton [HH]); and (3) the long-term cultured gonocytes taken from male gonads of a 5- to 6-day-old embryo (stages 29-30 HH). In presence of fibroblast growth factor, chicken blastodermal cells are able to long-term proliferate and generate small, round, alkaline phosphatase-positive cell clusters. Molecular characterization shows that these selected and amplified clusters show a PGC-like cell profile, as they express cPOUV (a pluripotent-associated marker), NR6A1/GCNF and DDX4/CVH (germ cell-specific genes). Both chicken PGCs and GGCs, obtained from embryonic blood and gonads, at 14 to 17 HH and 29 to 30 HH, respectively, generate long-term germ cell cultures and positively react in vitro to periodic acid-Schiff. Immunochemical analyses reveal that these cell lines are specifically recognized by anti-SSEA-1, anti-EMA-1, anti-CVH, anti-β1-integrin, and anti-CEACAM antibodies. The presence of surrounding cells may suggest a stronger dependency toward the niche process for the 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 morphologic analysis specific for chicken PGCs and GGCs will further contribute to quick and reliable characterization of long-term cultured in vitro chicken germ cells.