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International audience; Generating patient-specific stem cells representing the onset of development has become possible since the discovery of somatic cell reprogramming into induced pluripotent stem cells. However, human pluripotent stem cells are generally cultured in a primed pluripotent state: they are poised for differentiation and represent a stage of development corresponding to post-implantation epiblast. Here, we describe a protocol to reprogram human fibroblasts into naive pluripotent stem cells by overexpressing the transcription factors OCT4, SOX2, KLF4, and c-MYC using Sendai viruses. The resulting cells represent an earlier stage of development that corresponds to pre-implantation epiblast. We also discuss validation methods for human naive pluripotent stem cells.

Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX genes within axial stem cells that fuel axial embryo elongation. Whether the pace of sequential activation of HOX genes, the 'HOX clock', is controlled by intrinsic chromatin-based timing mechanisms or by temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in human pluripotent stem cell-derived axial progenitors differentiating into diverse spinal cord motor neuron subtypes. We show that the progressive activation of caudal HOX genes is controlled by a dynamic increase in FGF signaling. Blocking the FGF pathway stalled induction of HOX genes, while a precocious increase of FGF, alone or with GDF11 ligand, accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The pacing of the HOX clock is thus dynamically regulated by exposure to secreted cues. Its manipulation by extrinsic factors provides synchronized access to multiple human neuronal subtypes of distinct rostro-caudal identities for basic and translational applications. This article has an associated ‘The people behind the papers’ interview.

Pluripotent stem cells (PSCs) transition between cell states in vitro, reflecting developmental changes in the early embryo. PSCs can be stabilized in the naive state by blocking extracellular differentiation stimuli, particularly FGF–MEK signalling. Here, we report that multiple features of the naive state in human and mouse PSCs can be recapitulated without affecting FGF–MEK signalling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 (hereafter CDK8/19) kinases removes their ability to repress the Mediator complex at enhancers. CDK8/19 inhibition therefore increases Mediator-driven recruitment of RNA polymerase II (RNA Pol II) to promoters and enhancers. This efficiently stabilizes the naive transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naive pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naive pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity., M.N.S. was funded by a Leverhulme Trust early career fellowship. Work in the laboratory of M.Z.-G. was funded by the Wellcome Trust (098287/Z/12/Z) and the European Research Council (ERC) (669198). I.C. was funded by the Secretaria d’Universitats i Recerca de la Generalitat de Catalunya and European Social Fund. I.A. and P.S. were supported by the Fondation pour la Recherche Medicale (DEQ20170336757), Infrastructure Nationale en Biologie et Santé INGESTEM (ANR-11-INBS-0009), IHU-B CESAME (ANR-10-IBHU-003), LabEx REVIVE (ANR-10-LABX-73), LabEx DEVweCAN (ANR-10-LABX-0061) and LabEx CORTEX (ANR-11-LABX-0042) of University of Lyon within the programme ‘Investissements d’Avenir’ (ANR-11-IDEX-0007). Research by J.P., S.M. and C.B.-A. was supported in part by a grant from the Spanish Ministry of Economy and Competitiveness (SAF2013-44267-R) and by the CNIO. Work in the laboratory of D.F. was funded by the Institut National du Cancer (PLBIO10-068 and PLBIO15-005) and the Ligue National Contre le Cancer (EL2018.LNCC/DF). Work in the laboratory of N.M. was funded by the ERC, under the European Union Horizon 2020 research and innovation programme (StG-2014–640525_REGMAMKID), the Spanish Association Against Cancer (AECC/LABAE16006), Carlos III Health Institute (Red TerCel, CardioCel, RD16/0011/0027), Ministry of Economy and Competitiveness (MINECO) projects SAF2017–89782-R, SAF2015–72617-EXP and RYC-2014–16242, and the CERCA/Government of Catalonia (2017 SGR 1306). Work in the laboratory of S.O. was funded by SAF2013–44866-R from MINECO Spain. Work in the laboratory of M.F.F. was funded by Plan Nacional de I+D+I 2013–2016/FEDER (PI15/00892, to M.F.F. and A.F.F.); the ISCIII-Subdireccion General de Evaluación y Fomento de la Investigación and Plan Nacional de I+D+I 2008–2011/FEDER (CP11/00131, to A.F.F.); IUOPA (to M.I.S.); and the Asturias Regional Government (GRUPIN14–052, to M.F.F.). The IUOPA is supported by the Obra Social Liberbank-Cajastur, Spain. Work in the laboratory of M.S. was funded by the CNIO, the IRB and by grants from Spanish Ministry of Economy co-funded by the European Regional Development Fund (SAF2017-82613-R), ERC (ERC-2014-AdG/669622), Botin Foundation, Banco Santander (Santander Universities Global Division), laCaixa Foundation and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282).

International audience; Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).

International audience; The leukemia inhibitory factor (LIF)/glycoprotein (GP) 130/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 signaling pathway is a hallmark of naïve pluripotency in mice. In this review, we discuss the role of the LIF/GP130/JAK/STAT3 pathway in supporting the naïve-state pluripotency in human, monkey, and pig pluripotent stem cells (PSCs). We highlight the role of LIF/GP130/JAK/STAT3 signaling in reprogramming conventional human and monkey PSCs to naïve-like pluripotency. We analyze published single-cell RNA sequencing datasets of human and monkey embryos and note that the main components of the GP130/JAK/STAT3 pathway are expressed in pluripotent cells at preimplantation stages. We conclude that there is a growing body of evidence supporting the involvement of GP130/JAK/STAT3 in the regulation of naïve pluripotency in non-rodent species including humans, monkeys, and pigs.

International audience; The study of molecular mechanism driving osteoarticular diseases like osteoarthritis or osteoporosis is impaired by the low accessibility to mesenchymal stem cells (MSC) from healthy donors (HD) for differential multi-omics analysis. Advances in cell reprogramming have, however, provided both a new source of human cells for laboratory research and a strategy to erase epigenetic marks involved in cell identity and the development of diseases. To unravel the pathological signatures on the MSC at the origin of cellular drifts during the formation of bone and cartilage, we previously developed iPSC from MSC of osteoarthritis donors. Here we present the derivation of three iPSCs from healthy age matched donors to model the disease and further identify (epi)genomic signatures of the pathology.

Summary Genomic integrity of human pluripotent stem cells (hPSCs) is essential for research and clinical applications. However, genetic abnormalities can accumulate during hPSC generation and routine culture and following gene editing. Their occurrence should be regularly monitored, but the current assays to assess hPSC genomic integrity are not fully suitable for such regular screening. To address this issue, we first carried out a large meta-analysis of all hPSC genetic abnormalities reported in more than 100 publications and identified 738 recurrent genetic abnormalities (i.e., overlapping abnormalities found in at least five distinct scientific publications). We then developed a test based on the droplet digital PCR technology that can potentially detect more than 90% of these hPSC recurrent genetic abnormalities in DNA extracted from culture supernatant samples. This test can be used to routinely screen genomic integrity in hPSCs., Graphical Abstract, Highlights • A meta-analysis was carried out to list genetic abnormalities detected in hPSCs • Recurrent genetic abnormalities in hPSCs were precisely defined • ddPCR is a robust and sensitive approach to screen these recurrent abnormalities • In culture supernatant digital test can be used to rapidly screen iPSC clones, In this article, De Vos and colleagues performed a comprehensive meta-analysis of genetic abnormalities detected in hPSC lines. They then used this information to devise a ddPCR test targeting the majority of recurrent abnormalities and show that this test can be applied to the cell culture supernatant. Finally, they illustrate the interest of this test for routine hPSC screening.

Summary The transplantation of pluripotent stem-cell-derived neurons constitutes a promising avenue for the treatment of several brain diseases. However, their potential for the repair of the cerebral cortex remains unclear, given its complexity and neuronal diversity. Here, we show that human visual cortical cells differentiated from embryonic stem cells can be transplanted and can integrate successfully into the lesioned mouse adult visual cortex. The transplanted human neurons expressed the appropriate repertoire of markers of six cortical layers, projected axons to specific visual cortical targets, and were synaptically active within the adult brain. Moreover, transplant maturation and integration were much less efficient following transplantation into the lesioned motor cortex, as previously observed for transplanted mouse cortical neurons. These data constitute an important milestone for the potential use of human PSC-derived cortical cells for the reassembly of cortical circuits and emphasize the importance of cortical areal identity for successful transplantation., Graphical Abstract, Highlights • Human PSC-derived cortical neurons efficiently integrate into the adult mouse brain • PSC-derived human neurons reestablish axonal pathways in the lesioned adult cortex • Restoration of cortical pathways requires a donor and recipient area-identity match, Espuny-Camacho et al. show that transplanted ESC-derived human cortical neurons integrate functionally into the lesioned adult mouse brain. Transplanted neurons display visual cortical identity and show specific restoration of damaged cortical pathways following transplantation into the visual but not the motor cortex, suggesting the importance of areal-identity match for successful cortical repair.

The work described in this article is partially covered by patent application PCT/EP2012/066793 (publication number WO2013030243 A1). O.P., J.C. and M.K. are co-founders and shareholders of Anagenesis Biotechnologies, a start-up company specializing in the production of muscle cells in vitro for cell therapy and drug screenin; International audience; Body skeletal muscles derive from the paraxial mesoderm, which forms in the posterior region of the embryo. Using microarrays, we characterize novel mouse presomitic mesoderm (PSM) markers and show that, unlike the abrupt transcriptome reorganization of the PSM, neural tube differentiation is accompanied by progressive transcriptome changes. The early paraxial mesoderm differentiation stages can be efficiently recapitulated in vitro using mouse and human pluripotent stem cells. While Wnt activation alone can induce posterior PSM markers, acquisition of a committed PSM fate and efficient differentiation into anterior PSM Pax3+ identity further requires BMP inhibition to prevent progenitors from drifting to a lateral plate mesoderm fate. When transplanted into injured adult muscle, these precursors generated large numbers of immature muscle fibers. Furthermore, exposing these mouse PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program to generate myotubes and associated Pax7+ cells. This protocol results in improved in vitro differentiation and maturation of mouse muscle fibers over serum-free protocols and enables the study of myogenic cell fusion and satellite cell differentiation.

Autism spectrum disorders affect millions of individuals worldwide, but their heterogeneity complicates therapeutic intervention that is essentially symptomatic. A versatile yet relevant model to rationally screen among hundreds of therapeutic options would help improving clinical practice. Here we investigated whether neurons differentiated from pluripotent stem cells can provide such a tool using SHANK3 haploinsufficiency as a proof of principle. A library of compounds was screened for potential to increase SHANK3 mRNA content in neurons differentiated from control human embryonic stem cells. Using induced pluripotent stem cell technology, active compounds were then evaluated for efficacy in correcting dysfunctional networks of neurons differentiated from individuals with deleterious point mutations of SHANK3. Among 202 compounds tested, lithium and valproic acid showed the best efficacy at corrected SHANK3 haploinsufficiency associated phenotypes in cellulo. Lithium pharmacotherapy was subsequently provided to one patient and, after one year, an encouraging decrease in autism severity was observed. This demonstrated that pluripotent stem cell-derived neurons provide a novel cellular paradigm exploitable in the search for specific disease-modifying treatments., Highlights • Human neurons were used to screen for compounds correcting symptoms associated with SHANK3 haploinsufficiency syndrome. • Screening criteria were the ability to increase SHANK3 expression and to increase glutamatergic transmission. • Selected hit compounds were then validated using neurons differentiated from individuals with SHANK3 disrupting mutations. • Lithium was selected and delivered to one of SHANK3 patient showing encouraging positive clinical outcomes after one year. The clinical heterogeneity between individuals affected by autism makes it difficult to anticipate the effectiveness of a treatment. Furthermore, clinical practice lacks biological tools to help make such decisions. Here we use neurons, produced from pluripotent stem cells derived from patients affected by SHANK3 haploinsufficiency syndrome, to test the efficiency of therapeutic compounds. We screened the biological activity of more than 200 compounds on SHANK3 expression. Lithium was ultimately selected and delivered to one patient with a SHANK3-disruptive mutation. This resulted in a positive outcome, as determined by improved autistic core symptoms, thus supporting the usefulness of this type of predictive approach.

Summary The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives., Graphical Abstract Image 1, Highlights • GAPTrap vector system targets transgenes to the ubiquitously expressed GAPDH locus • Targeting transgenes to the GAPDH locus yields reliable transgene expression • Transgenes at this locus are robustly expressed in differentiated cells • Generation of GAPTrap targeted human PSC lines is simple and efficient, In this article Stanley, Elefanty, and colleagues describe a simple vector system, GAPTrap, for constitutively expressing transgenes in pluripotent stem cells and their differentiated derivatives. GAPTrap vectors target transgenes to the GAPDH locus, ensuring robust and reliable expression in a wide variety of cell types, and providing an ideal base for cell tagging and gene overexpression experiments.

Le rat et la souris sont, depuis deja longtemps, des modeles de choix pour l’etude de la biologie des mammiferes : faciles d’entretien, ils offrent aussi toute une panoplie de lignees genetiquement pures et de mutants bien caracterises. Bien que longtemps prefere a la souris en raison de sa taille et de sa physiologie, le rat a progressivement ete delaisse au profit de la souris, espece chez laquelle les techniques de mutagenese dirigee alimentent depuis plus de vingt ans le repertoire des modeles d’etudes. Aujourd’hui, deux avancees techniques devraient changer la donne : l’utilisation de nucleases artificielles a doigts de zinc pour inactiver un gene d’interet des les premiers stades du developpement et la derivation et la manipulation genetique de cellules ES (embryonic stem cells ) de rat. Nul doute que les annees qui viennent verront se multiplier les modeles de maladies humaines etablies chez le rat.

The therapeutical interest of pluripotent cells and ethical issues related to the establishment of human embryonic stem cell (ESC) or embryonic germ cell (EGC) lines raise the understanding of the mechanism underlying pluripotency to a fundamental issue. Establishing a protein pluripotency signature for these cells can be complicated by the presence of unrelated proteins produced by the culture environment. Here, we have analyzed the environment supporting ESC and EGC growth, and established 2-D reference maps for each constituent present in this culture environment: mouse embryonic fibroblast feeder cells, culture medium (CM) and gelatin. The establishment of these reference maps is essential prior to the study of ESC and EGC specific proteomes. Indeed, these maps can be subtracted from ESC or EGC maps to allow focusing on spots specific for ESCs or EGCs. Our study led to the identification of 110 unique proteins from fibroblast feeder cells and 23 unique proteins from the CM, which represent major contaminants of ESC and EGC proteomes. For gelatin, no collagen-specific proteins were identified, most likely due to difficulties in resolution and low quantities. Furthermore, no differences were observed between naive and conditioned CM. Finally, we compared these reference maps to ESC 2-D gels and isolated 17 ESC specific spots. Among these spots, proteins that had already been identified in previous human and mouse ESC proteomes were identified but no apparent ESC-specific pluripotency marker could be identified. This work represents an essential step in furthering the knowledge of environmental factors supporting ESC and EGC growth.

International audience; Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis (1). The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

International audience; Pluripotent stem-cell-derived neurons constitute an attractive source for replacement therapies, but their utility remains unclear for cortical diseases. Here, we show that neurons of visual cortex identity, differentiated in vitro from mouse embryonic stem cells (ESCs), can be transplanted successfully following a lesion of the adult mouse visual cortex. Reestablishment of the damaged pathways included long-range and reciprocal axonal projections and synaptic connections with targets of the damaged cortex. Electrophysiological recordings revealed that some grafted neurons were functional and responsive to visual stimuli. No significant integration was observed following grafting of the same neurons in motor cortex, or transplantation of embryonic motor cortex in visual cortex, indicating that successful transplantation required a match in the areal identity of grafted and lesioned neurons. These findings demonstrate that transplantation of mouse ESC-derived neurons of appropriate cortical areal identity can contribute to the reconstruction of an adult damaged cortical circuit.

Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cell pluripotency is being investigated increasingly to obtain specific cell lineages for therapeutic treatments and tissue engineering. Type II alveolar epithelial cells have been derived from murine ES cells, but the capacity of the latter to generate differentiated airway epithelial tissue has never been reported. Herein, we show by RT-PCR and immunocytochemistry that murine ES cells are able to differentiate into nonciliated secretory Clara cells, and that type I collagen induces this commitment. Moreover, when cultured at the air-liquid interface, ES cells give rise to a fully differentiated airway epithelium. By quantitative histologic examination, immunohistochemistry, and scanning electron microscopy, we show that the bioengineered epithelium is composed of basal, ciliated, intermediate, and Clara cells, similar to those of native tracheobronchial airway epithelium. Transmission electron microscopy and Western blotting reveal that the generated epithelium also exhibits the ultrastructural features and secretory functions characteristic of airway epithelial tissue. These results open new perspectives for cell therapy of injured epithelium in airway diseases, such as bronchopulmonary dysplasia, cystic fibrosis, or bronchiolitis obliterans.

International audience; In plants, the shoot apical meristem (SAM) serves as a reservoir of pluripotent stem cells from which all above ground organs originate. To sustain proper growth, the SAM must maintain homeostasis between the self-renewal of pluripotent stem cells and cell recruitment for lateral organ formation. At the core of the network that regulates this homeostasis in Arabidopsis are the WUSCHEL (WUS) transcription factor specifying stem cell fate and the CLAVATA (CLV) ligand-receptor system limiting WUS expression. In this study, we identified the ERECTA (ER) pathway as a second receptor kinase signaling pathway that regulates WUS expression, and therefore shoot apical and floral meristem size, independently of the CLV pathway. We demonstrate that reduction in class III HD-ZIP and ER function together leads to a significant increase in WUS expression, resulting in extremely enlarged shoot meristems and a switch from spiral to whorled vegetative phyllotaxy. We further show that strong upregulation of WUS in the inflorescence meristem leads to ectopic expression of the AGAMOUS homeotic gene to a level that switches cell fate from floral meristem founder cell to carpel founder cell, suggesting an indirect role for ER in regulating floral meristem identity. This work illustrates the delicate balance between stem cell specification and differentiation in the meristem and shows that a shift in this balance leads to abnormal phyllotaxy and to altered reproductive cell fate.

International audience; The study of human cortical development has major implications for brain evolution and diseases but has remained elusive due to paucity of experimental models. Here we found that human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), cultured without added morphogens, recapitulate corticogenesis leading to the sequential generation of functional pyramidal neurons of all six layer identities. After transplantation into mouse neonatal brain, human ESC-derived cortical neurons integrated robustly and established specific axonal projections and dendritic patterns corresponding to native cortical neurons. The differentiation and connectivity of the transplanted human cortical neurons complexified progressively over several months in vivo, culminating in the establishment of functional synapses with the host circuitry. Our data demonstrate that human cortical neurons generated in vitro from ESC/iPSC can develop complex hodological properties characteristic of the cerebral cortex in vivo, thereby offering unprecedented opportunities for the modeling of human cortex diseases and brain repair.

Self-renewal and differentiation of stem cell depend on a dynamic interplay of cell-extrinsic and -intrinsic regulators. However, how stem cells perceive the right amount of signal and at the right time to undergo a precise developmental program remains poorly understood. The cell surface proteins Glypicans act as gatekeepers of environmental signals to modulate their perception by target cells. Here, we show that one of these, Glypican4 (Gpc4), is specifically required to maintain the self-renewal potential of mouse embryonic stem cells (ESCs) and to fine tune cell lineage commitment. Notably, Gpc4-mutant ESCs contribute to all embryonic cell lineages when injected in blastocyts but lose their intrinsic tumorigenic properties after implantation into nude mice. Therefore, our molecular and functional studies reveal that Gpc4 maintains distinct stemness features. Moreover, we provide evidence that self-renewal and lineage commitment of different stem cell types is fine tuned by Gpc4 activity by showing that Gpc4 is required for the maintenance of adult neural stem cell fate in vivo. Mechanistically, Gpc4 regulates self-renewal of ESCs by modulating Wnt/β-catenin signaling activities. Thus, our findings establish that Gpc4 acts at the interface of extrinsic and intrinsic signal regulation to fine tune stem cell fate. Moreover, the ability to uncouple pluripotent stem cell differentiation from tumorigenic potential makes Gpc4 as a promising target for cell-based regenerative therapies.

International audience; Gene expression-based scores used to predict risk in cancer frequently include genes coding for DNA replication, repair or recombination. Using two independent cohorts of 206 and 345 previously-untreated patients with Multiple Myeloma (MM), we identified 50 cell cycle-unrelated genes overexpressed in multiple myeloma cells (MMCs) compared to normal human proliferating plasmablasts and non-proliferating bone marrow plasma cells and which have prognostic value for overall survival. Thirty-seven of these 50 myeloma genes (74%) were enriched in genes overexpressed in one of 3 normal human stem cell populations--pluripotent (18), hematopoietic (10) or mesenchymal stem cells (9)--and only three genes were enriched in one of 5 populations of differentiated cells (memory B lymphocytes, T lymphocytes, polymorphonuclear cells, monocytes, osteoclasts). These 37 genes shared by MMCs and adult or pluripotent stem cells were used to build a stem cell score ((SC)score), which proved to be strongly prognostic in the 2 independent cohorts of patients compared to other gene expression-based risk scores or usual clinical scores using multivariate Cox analysis. This finding highlights cell cycle-unrelated prognostic genes shared by myeloma cells and normal stem cells, whose products might be important for normal and malignant stem cell biology.

International audience; Vertebrate development requires progressive commitment of embryonic cells into specific lineages through a continuum of signals that play off differentiation versus multipotency. In mammals, Nanog is a key transcription factor that maintains cellular pluripotency by controlling competence to respond to differentiation cues. Nanog orthologs are known in most vertebrates examined to date, but absent from the Anuran amphibian Xenopus. Interestingly, in silico analyses and literature scanning reveal that basal vertebrate ventral homeobox (ventxs) and mammalian Nanog factors share extensive structural, evolutionary and functional properties. Here, we reassess the role of ventx activity in Xenopus laevis embryos and demonstrate that they play an unanticipated role as guardians of high developmental potential during early development. Joint over-expression of Xenopus ventx1.2 and ventx2.1-b (ventx1/2) counteracts lineage commitment towards both dorsal and ventral fates and prevents msx1-induced ventralization. Furthermore, ventx1/2 inactivation leads to down-regulation of the multipotency marker oct91 and to premature differentiation of blastula cells. Finally, supporting the key role of ventx1/2 in the control of developmental potential during development, mouse Nanog (mNanog) expression specifically rescues embryonic axis formation in ventx1/2 deficient embryos. We conclude that during Xenopus development ventx1/2 activity, reminiscent of that of Nanog in mammalian embryos, controls the switch of early embryonic cells from uncommitted to committed states.

International audience; BACKGROUND: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. CONCLUSION/SIGNIFICANCE: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.

International audience; Toti- or pluripotent cells proliferation and/or differentiation have been shown to be strongly related to nuclear chromatin organization and structure over the last past years. We have recently identified ZFPIP/Zfp462 as a zinc finger nuclear factor necessary for correct cell division during early embryonic developmental steps of vertebrates. We thus questioned whether this factor was playing a general role during cell division or if it was somehow involved in embryonic cell fate or differentiation. To achieve this goal, we performed a knock-down experiment in the pluripotent P19 and differentiated 3T3 cell lines, both expressing endogenous ZFPIP/Zfp462. Using specific shRNA directed against ZFPIP/Zfp462 transcripts, we demonstrated that depletion of this protein induced cell death in P19 but had no effect in 3T3 cells. In addition, in the absence of the protein, the P19 cells exhibited a complete destructuration of pericentromeric domains associated with a redistribution of the HP1alpha proteins and an increase in DNA satellites transcribed RNAs level. These data suggested an instrumental role of ZFPIP/Zfp462 in maintaining the chromatin structure of pluripotent cells.

International audience; Human embryonic stem cells (hESC) are obtained from the inner cell mass from the early embryo at blastocyste stage. Derived in cell lines for the first time in 1998, they can be maintained in culture in an undifferentiated state indefinitely under certain conditions. Two essential properties characterize hESC: pluripotency and self-renewal. Pluripotency is convey by the expression of specific transcription factors such as OCT4 and NANOG, and is under the control of growth factors such as IGF2 and FGFb. Markers used to characterize these cells include surface antigens, notably SSEA-3 and SSEA-4, and nuclear markers such as OCT4. HESC can differentiate into different cell types in vitro. They represent a unique and essential model for early human development research and for regenerative medicine. By their self-renewal capacity and their potential to differentiate into several cell types, hESC are an unlimited source of cells enabling to replace or restore lost or damaged cells in numerous diseases. Even if it is not conceivable today to use them in clinical practice for ethic and scientific reasons, it seems essential to explore the numerous potentialities of these cells. This knowledge might be relevant to handle adult stem cells in vitro and will be mandatory for a therapeutic use of hESC in the future.

Although the properties of embryonic stem (ES) cells make these cells very attractive in the field of replacement therapy, the molecular mechanisms involved in the maintenance of their pluripotency are not fully characterized. Starting from the observation that most pluripotent markers are also expressed by spermatogonia stem cells, we identified Tex19 as a new potential pluripotency marker. We show that Tex19 is a mammalian-specific protein duplicated in mouse and rat, renamed Tex19.1 and Tex19.2, whereas only one form is found in human. In mouse, both forms are localized on chromosome 11 and transcribed in opposite directions. Tex19 proteins are well conserved, showing two highly conserved domains that do not present any similarity with any other known domains. We show that Tex19.2 is specifically detected in the male somatic gonad lineage, whereas Tex19.1 expression is very similar to that of Oct4. Transcripts are maternally inherited, and expression starts as soon as the early embryo and later is limited to the germ line. Tex19.1 transcripts were also detected in mouse pluripotent stem cells, and expression of Tex19.1, like that of Oct4, decreases after murine embryonic stem and germ cell differentiation. Human TEX19 was more closely related to murine Tex19.1 and was also detected in adult testis and in undifferentiated ES cells. By immunofluorescence, we found that Tex19.1 protein localizes to the nucleus of mouse ES and inner cell mass cells. All these results suggest that Tex19.1, as well as human TEX19, could be a new factor involved in the maintenance of self-renewal or pluripotency of stem cells. Disclosure of potential conflicts of interest is found at the end of this article.

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice., Genetic manipulations reveal a novel role of Notch signaling in promoting and directing embryonic stem cells toward neural fates and suppressing differentiation into other lineages.

International audience; Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.

International audience; Adipocytes and osteoblasts are derived from a common precursor cell. It has been proposed that the bone loss commonly seen during aging or in the pathology of osteoporosis might be partly caused by a deregulation of the normal balance between osteoblast and adipocyte differentiation. In vitro differentiation of mouse embryonic stem cells toward the adipogenic and the osteogenic lineages provides a powerful system for testing effects of compounds on the developmental switch between adipogenesis and osteogenesis and identification of pharmacological targets. In this chapter, an improved protocol to commit mouse embryonic stem cells into both lineages at a correct rate is detailed.

Les cellules souches embryonnaires pluripotentes (ES) ont des propriétés de prolifération et de différenciation qui font d’elles un outil prometteur pour la thérapie cellulaire. Malgré la légitime controverse sur les nombreuses questions éthiques que cette technologie suscite, et en dépit de ses potentiels dans des domaines aussi variables que la thérapie cardiaque, pulmonaire ou hépatique, ou la régénération osseuse et articulaire, c’est dans le traitement des maladies cérébrales que la technique ouvre les perspectives les plus intéressantes. De très nombreux travaux réalisés sur les cellules ES rapportent une régénération du cerveau et de la moelle épinière chez des rongeurs et la réparation des problèmes cognitifs causés lors de dommages apportés aux tissus lésés. Cet article offre une synthèse des avancées récentes dans le domaine de la manipulation des cellules souches (murines) en vue de sélectionner des populations de neurones, astrocytes et oligodendrocytes. En parallèle, nous mettons l’accent sur les similitudes frappantes qui existent dans les programmes génétiques mis en oeuvre au cours du développement embryonnaire in utero et au cours de la différenciation in vitro des cellules., Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.

Recent studies demonstrated that stromal cells isolated from adult bone marrow have the competence of differentiating into neuronal cells in vitro and in vivo . However, the capacity of marrow stromal cells or mesenchymal stem cells (MSCs) to differentiate into diverse neuronal cell populations and the identity of molecular factors that confer marrow stromal cells with the competence of a neuronal subtype have yet to be elucidated. Here, we show that Sonic hedgehog (Shh) and retinoic acid (RA), signaling molecules secreted from tissues in the vicinity of peripheral sensory ganglia during embryogenesis, exert synergistic effects on neural-competent MSCs to express a comprehensive set of glutamatergic sensory neuron markers. Application of Shh or RA alone had little or no effect on the expression of these neuronal subtype markers. In addition, incubation of MSCs with embryonic hindbrain/somite/otocyst conditioned medium or prenatal cochlea explants promoted up-regulation of additional sensory neuron markers and process outgrowth. These results identify Shh and RA as sensory competence factors for adult pluripotent cells and establish the importance of interactions between adult pluripotent cells and the host microenvironment in neuronal subtype specification.

International audience; Protocadherin 12 protein (PCDH12, VE-cadherin 2) is a cell adhesion molecule that has been isolated from endothelial cells. Here, we have used Northern and Western blots, immunohistology, and flow cytometry to examine the distribution of PCDH12 in mouse tissues. It is an N-glycosylated protein of 150-kDa mass. In the endothelium, PCDH12 immunoreactivity was variable and dependent upon the vascular bed. In both the embryo and embryonic stem cell differentiation system, signals were localized in vasculogenic rather than angiogenic endothelium. In addition, the protein was strongly expressed in a subset of invasive cells of the placenta, which were identified as glycogen-rich trophoblasts. In adult mice, strong PCDH12 signals were observed in mesangial cells of kidney glomeruli whereas expression was not detected in other types of perivascular cells. As opposed to most protocadherins, PCDH12 is not expressed in early embryonic (day 12.5) and adult brains. As a first approach to obtain insight into PCDH12 function, we produced transgenic mice deficient in PCDH12, which were viable and fertile. They did not display any obvious histomorphological defects. We conclude that PCDH12 has a unique expression pattern and that its deficiency does not lead to conspicuous abnormalities. Moreover, PCDH12 is the first specific marker for both glycogen-rich trophoblasts and mesangial cells.

The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. Introduction: Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. Materials and Methods: C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10−7 M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. Results and Conclusions: Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.

During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells., Synopsis Chromatin is the support of our genetic information. It is composed of numerous repeated units called nucleosomes, in which DNA wraps around a core of histone proteins. Modifications in the composition and biochemical properties of nucleosomes play major roles in the regulation of genome function. Such modifications are termed “epigenetic” when they are inherited across cell divisions and confer new information to chromatin, in addition to the genetic information provided by DNA. It is usually believed that during genome replication, the basic chromatin assembly machinery builds up “naïve” nucleosomes, and, in a subsequent step, nucleosomes are selectively modified by a series of enzymes to acquire epigenetic information. Here, the authors studied the role of a basic chromatin assembly factor (CAF-1) in mouse embryonic stem cells and early embryos. Surprisingly, they show that CAF-1 confers epigenetic information to specific genomic regions. In addition, this study revealed that CAF-1 is required for the proper spatial organization of chromosomes in the nucleus. This new knowledge may contribute to better understanding the role of chromatin in the maintenance of embryonic stem cell identity and plasticity.