Cellular plasticity in reprogramming, rejuvenation and tumorigenesis: a pioneer TF perspective.

The multistep process of in vivo reprogramming, mediated by the transcription factors (TFs) Oct4, Sox2, Klf4, and c-Myc (OSKM), holds great promise for the development of rejuvenating and regenerative strategies. However, most of the approaches developed so far are accompanied by a persistent risk of tumorigenicity. Here, we review the groundbreaking effects of in vivo reprogramming with a particular focus on rejuvenation and regeneration. We discuss how the activity of pioneer TFs generates cellular plasticity that may be critical for inducing not only reprogramming and regeneration, but also cancer initiation. Finally, we highlight how a better understanding of the uncoupled control of cellular identity, plasticity, and aging during reprogramming might pave the way to the development of rejuvenating/regenerating strategies in a nontumorigenic manner.

Molecular versatility during pluripotency progression.

A challenge during development is to ensure lineage segregation while preserving plasticity. Using pluripotency progression as a paradigm, we review how developmental transitions are coordinated by redeployments, rather than global resettings, of cellular components. We highlight how changes in response to extrinsic cues (FGF, WNT, Activin/Nodal, Netrin-1), context- and stoichiometry-dependent action of transcription factors (Oct4, Nanog) and reconfigurations of epigenetic regulators (enhancers, promoters, TrxG, PRC) may confer robustness to naïve to primed pluripotency transition. We propose the notion of Molecular Versatility to regroup mechanisms by which molecules are repurposed to exert different, sometimes opposite, functions in close stem cell configurations.

RSL24D1 sustains steady-state ribosome biogenesis and pluripotency translational programs in embryonic stem cells.

Embryonic stem cell (ESC) fate decisions are regulated by a complex circuitry that coordinates gene expression at multiple levels from chromatin to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key pathways that efficiently control stem cell homeostasis, yet the underlying molecular mechanisms remain largely unknown. Here, we identified RSL24D1 as highly expressed in both mouse and human pluripotent stem cells. RSL24D1 is associated with nuclear pre-ribosomes and is required for the biogenesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors and of components from the Polycomb Repressive Complex 2 (PRC2). While having a moderate impact on differentiation, RSL24D1 depletion significantly alters ESC self-renewal and lineage commitment choices. Altogether, these results demonstrate that RSL24D1-dependant ribosome biogenesis is both required to sustain the expression of pluripotent transcriptional programs and to silence PRC2-regulated developmental programs, which concertedly dictate ESC homeostasis.

Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/ß and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/ß and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/ß and stabilize ß-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.

Cellular Pliancy and the Multistep Process of Tumorigenesis.

Completion of early stages of tumorigenesis relies on the dynamic interplay between the initiating oncogenic event and the cellular context. Here, we review recent findings indicating that each differentiation stage within a defined cellular lineage is associated with a unique susceptibility to malignant transformation when subjected to a specific oncogenic insult. This emerging notion, named cellular pliancy, provides a rationale for the short delay in the development of pediatric cancers of prenatal origin. It also highlights the critical role of cellular reprogramming in early steps of malignant transformation of adult differentiated cells and its impact on the natural history of tumorigenesis.

Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance.

The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1's function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells.

Regulation by miR181 family of the dependence receptor CDON tumor suppressive activity in neuroblastoma.

BACKGROUND: The Sonic Hedgehog (SHH) signaling pathway plays an important role in neural crest cell fate during embryonic development and has been implicated in the progression of multiple cancers that include neuroblastoma, a neural crest cell-derived disease. While most of the SHH signaling is mediated by the well-described canonical pathway leading to the activation of Smoothened and Gli, it has recently been shown that cell-adhesion molecule-related/downregulated by oncogenes (CDON) serves as a receptor for SHH and contributes to SHH-induced signaling. CDON has also been recently described as a dependence receptor, triggering apoptosis in the absence of SHH. This CDON proapoptotic activity has been suggested to constrain tumor progression. METHODS: CDON expression was analyzed by quantitative-reverse transcription-polymerase chain reaction in a panel of 226 neuroblastoma patients and associated with stages, overall survival, and expression of miR181 family members using Kaplan Meier and Pearson correlation methods. Cell death assays were performed in neuroblastoma cell lines and tumor growth was investigated in the chick chorioallantoic model. All statistical tests were two-sided. RESULTS: CDON expression was inversely associated with neuroblastoma aggressiveness (P < .001). Moreover, re-expression of CDON in neuroblastoma cell lines was associated with apoptosis in vitro and tumor growth inhibition in vivo. We show that CDON expression is regulated by the miR181 miRNA family, whose expression is directly associated with neuroblastoma aggressiveness (survival: high miR181-b 73.2% vs low miR181-b 54.6%; P = .03). CONCLUSIONS: Together, these data support the view that CDON acts as a tumor suppressor in neuroblastomas, and that CDON is tightly regulated by miRNAs.

Dependence receptor TrkC is a putative colon cancer tumor suppressor.

The TrkC neurotrophin receptor belongs to the functional dependence receptor family, members of which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer tumor-suppressor activity. Indeed, cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC is a classic tyrosine kinase receptor and therefore generally considered to be a proto-oncogene. We show here that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Finally, we provide evidence that a mutation of TrkC detected in a sporadic cancer is a loss-of-proapoptotic function mutation. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor.

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.

Ncoa3 functions as an essential Esrrb coactivator to sustain embryonic stem cell self-renewal and reprogramming.

Embryonic stem cell (ESC) pluripotency depends on a well-characterized gene regulatory network centered on Oct4, Sox2, and Nanog. In contrast, little is known about the identity of the key coregulators and the mechanisms by which they may potentiate transcription in ESCs. Alongside core transcription factors, the orphan nuclear receptor Esrrb (estrogen-related receptor ß) is vital for the maintenance of ESC identity and furthermore is uniquely associated with the basal transcription machinery. Here, we show that Ncoa3, an essential coactivator, is required to mediate Esrrb function in ESCs. Ncoa3 interacts with Esrrb via its ligand-binding domain and bridges Esrrb to RNA polymerase II complexes. Functionally, Ncoa3 is critical for both the induction and maintenance of pluripotency. Through chromatin immunoprecipitation (ChIP) sequencing and microarray experiments, we further demonstrate that Ncoa3 shares overlapping gene regulatory functions with Esrrb and cooperates genome-wide with the Oct4-Sox2-Nanog circuitry at active enhancers to up-regulate genes involved in self-renewal and pluripotency. We propose an integrated model of transcriptional and coactivator control, mediated by Ncoa3, for the maintenance of ESC self-renewal and somatic cell reprogramming.

Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development.

The transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner.

MicroRNA regulation of Cbx7 mediates a switch of Polycomb orthologs during ESC differentiation.

The Polycomb Group (PcG) of chromatin modifiers regulates pluripotency and differentiation. Mammalian genomes encode multiple homologs of the Polycomb repressive complex 1 (PRC1) components, including five orthologs of the Drosophila Polycomb protein (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8). We have identified Cbx7 as the primary Polycomb ortholog of PRC1 complexes in embryonic stem cells (ESCs). The expression of Cbx7 is downregulated during ESC differentiation, preceding the upregulation of Cbx2, Cbx4, and Cbx8, which are directly repressed by Cbx7. Ectopic expression of Cbx7 inhibits differentiation and X chromosome inactivation and enhances ESC self-renewal. Conversely, Cbx7 knockdown induces differentiation and derepresses lineage-specific markers. In a functional screen, we identified the miR-125 and miR-181 families as regulators of Cbx7 that are induced during ESC differentiation. Ectopic expression of these miRNAs accelerates ESC differentiation via regulation of Cbx7. These observations establish a critical role for Cbx7 and its regulatory miRNAs in determining pluripotency.

Astacin-like metallo-endopeptidase is dynamically expressed in embryonic stem cells and embryonic epithelium during morphogenesis.

BACKGROUND: Astacin-like metallo-proteases are zinc endopeptidases conserved among vertebrates and invertebrates. First described as hatching gland enzymes, many members of the family possess other functions during embryonic development. In the chick, however, functions of Astacin-like proteins remain elusive. RESULTS: We report here that Astacin-like (ASTL) is strongly expressed in mouse and chicken embryonic stem (ES) cells and exhibits a very dynamic expression pattern during embryogenesis and organogenesis, mostly in remodeled epithelia. Consistent with its expression in ES cells, chick ASTL is detected in vivo in the pluripotent cells of the epiblast and then disappears from the newly induced neural plate. ASTL expression remains at the junction of non-neural and neural ectoderm, just before neural tube closure. At later stages, chick ASTL is detected in the ventral epidermis before ventral closure, in the intermediate mesoderm, in the gonads and in the forming nephric duct and tubules of the mesonephros and metanephros. CONCLUSIONS: ASTL is dynamically expressed in the embryonic epithelium and in embryonic stem cells, suggesting an important function for the control of epithelial cell behavior during early development.

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.

Ring1B and Suv39h1 delineate distinct chromatin states at bivalent genes during early mouse lineage commitment.

Pluripotent cells develop within the inner cell mass of blastocysts, a mosaic of cells surrounded by an extra-embryonic layer, the trophectoderm. We show that a set of somatic lineage regulators (including Hox, Gata and Sox factors) that carry bivalent chromatin enriched in H3K27me3 and H3K4me2 are selectively targeted by Suv39h1-mediated H3K9me3 and de novo DNA methylation in extra-embryonic versus embryonic (pluripotent) lineages, as assessed both in blastocyst-derived stem cells and in vivo. This stably repressed state is linked with a loss of gene priming for transcription through the exclusion of PRC1 (Ring1B) and RNA polymerase II complexes at bivalent, lineage-inappropriate genes upon trophoblast lineage commitment. Collectively, our results suggest a mutually exclusive role for Ring1B and Suv39h1 in regulating distinct chromatin states at key developmental genes and propose a novel mechanism by which lineage specification can be reinforced during early development.

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.

Chicken embryonic stem cells as a non-mammalian embryonic stem cell model.

Embryonic stem cells (ESCs) were isolated in the early 1980s from mouse and in the late 1990s from primate and human. These cells present the unique property of self-renewal and the ability to generate differentiated progeny in all embryonic lineages both in vitro and in vivo. The mESCs (mouse embryonic stem cells) can contribute to both somatic and germinal lineages once re-injected into a recipient embryo at the blastocyst stage. In avian species, chicken embryonic stem cells (cESCs) have been isolated from the in vitro culture of early chicken blastodermal cells (cBCs) taken from stage X embryo (EG&K) These cESCs can be maintained under specific culture conditions and have been characterized on the basis of their morphology, biochemical features, in vitro differentiation potentialities and in vivo morphogenetic properties. The relationship between these cESCs and some of the chicken germ cells identified and grown under specific culture conditions are still under debate, in particular with the identification of the Cvh gene as a key factor for germ cell determination. Moreover, by cloning the avian homologue of the Oct4 mammalian gene, we have demonstrated that this gene, as well as the chicken Nanog gene, was involved in the characterization and maintenance of the chicken pluripotency. These first steps toward the understanding of pluripotency control in a non-mammalian species opens the way for the development and characterization of putative new cell types such as chicken EpiSC and raises the question of the existence of reprogramming in avian species. These different points are discussed.

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.

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.