An orchestrated intron retention program in meiosis controls timely usage of transcripts during germ cell differentiation

Global transcriptome reprogramming during sper-matogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Sper-matocytes and spermatids require particularly exten-sive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete func-tion. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differ-ently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis

Tax ubiquitylation and SUMOylation control the dynamic shuttling of Tax and NEMO between Ubc9 nuclear bodies and the centrosome

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Global changes in transcriptome regulation in postmeiotic spermatids

The male germ cell undergoes a proliferative step of amplification of the cell number, a meiotic step that reduces the genome to haploid, and a morphological differentiation stage to form spermatozoa. Although each phase requires a dynamic repertoire of functional factors, transcription is not always active in spermatogenesis, being characterized by a mitotic phase in spermatogonia, a meiotic phase in pachytene spermatocytes and a post-meiotic phase in round spermatids. We have performed deep-sequencing analysis of the transcriptome of purified populations of pachytene spermatocytes and round spermatids. We generated strend-specific RNAseq data for polyadenylated RNAs, resulting in >100 million 100bp mapped reads for each sample. Comparison of the spermatid versus spermatocytes transcriptome revealed 5508 up regulated and 7218 down-regulated genes in post-meiotic germ cells. Cell cycle and splicing were the most significantly up regulated pathways in post-meiotic versus meiotic cells. As for the splicing, 3278 exons in 2185 genes were differentially regulated in spermatids and spermatocytes. The most represented classes were alternative first exons, intron retention, alternative last exons and exon cassettes. These changes were accompanied by changes in the expression of key splicing factors, like the overall down regulation of the serine-arginine rich (SR) family, involved in positive regulation of constitutive and alternative splicing events. Our results indicate that male germ cells extensively modify the pattern of gene expression during the meiotic divisions and identify key molecular players involved in this transition. The global reduction in the SR protein family, known positive regulators of splicing events, may drive the extensive changes in alternative splicing observed between meiotic and post-meiotic germ cells

EMMPRIN/CD147 is a novel coreceptor of VEGFR-2 mediating its activation by VEGF

EMMPRIN/CD147 is mainly known for its protease inducing function but a role in promoting tumor angiogenesis has also been demonstrated. This study provides evidence that EMMPRIN is a new coreceptor for the VEGFR-2 tyrosine kinase receptor in both endothelial and tumor cells, as it directly interacts with it and regulates its activation by its VEGF ligand, signalling and functional consequences both in vitro and in vivo. Computational docking analyses and mutagenesis studies identified a molecular binding site in the extracellular domain of EMMPRIN located close to the cell membrane and containing the amino acids 195/199. EMMPRIN is overexpressed in cancer and hence is able to further potentiate VEGFR-2 activation, suggesting that a combinatory therapy of an antiangiogenic drug together with an inhibitor of EMMPRIN/VEGFR-2 interaction may have a greater impact on inhibiting angiogenesis and malignancy.This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), La Ligue Nationale contre le Cancer (LNCC), La Société Française de Dermatologie and Université Paris Diderot. F.K was supported by a PhD fellowship from Cancéropôle-Ile de France and from Fondation ARC pour la Recherche sur le Cancer. L.P.C was supported by a FPU fellowship from Spanish Ministry of Science. This work was supported by grant BIO2010–22324 from Plan NacionalI+D+iMICINN. We thank the core facility of the Institut Universitaire d’Hématologie for confocal microscopy analyses. The core facility is supported by grants from the Association Saint-Louis, Conseil Regional d’Ile-de-France, and the Ministère de la Recherche.Peer ReviewedPostprint (published version

BCL-2 Inhibitor ABT-737 effectively targets leukemia-initiating cells with differential regulation of relevant genes leading to extended survival in a NRAS/BCL-2 mouse model of high risk-myelodysplastic syndrome

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment

Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation

International audienceIn recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells

Toward the creation of 2D or 3D clusters of cells in acoustic levitation

International audienceIntroduction Today, three-dimensional (3D) cell cultures tend to replace 2D conventional method because of their more relevant tissue-mimicking characteristics. Indeed, the 3D cell architecture (spheroïd, organoïd, etc) and the microenvironment is closer to In Vivo physiological behaviour [1, 2]. The main difficulties remain in creating a scaffold compatible with the targeted cells and tissues. Bioprinting is one the great objective for tissue engineering. For instance, stereolithography is a 3D printing technology where the freestanding object is built layer by layer with a photosensitive polymer resin through the projection of a UV image in the top plane. In recent promising work, stereolithography has been applied to create 3D hydrogel structures to guide cells like hepatocytes [3]. Nevertheless, ideally, scaffold-free methods are needed. We propose a new method combining microfluidic channels and the acoustic radiation force (ARF) to structure and to control the shape of stem-cells aggregates

Self-organization of human mesenchymal stem cells into spheroids trapped in acoustic levitation

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Osteogenic-differentiated Mesenchymal Stem Cell-secreted Extracellular Matrix as a Bone Morphogenetic Protein-2 delivery system for ectopic bone formation

International audienceWhile human bone morphogenetic protein-2 (BMP-2) is a promising growth factor for bone regeneration, a major challenge in biomedical applications is finding an optimal carrier for its delivery at the site of injury. Because of their natural affinities for growth factors (including BMP-2) as well as their role in instructing cell function, cultured cell-derived extracellular matrices (ECM) are of special interest. We hereby hypothesized that a “bony matrix” containing mineralized, osteogenic ECM is a potential efficacious carrier of BMP-2 for promoting bone formation and, therefore, compared the efficacy of the decellularized ECM derived from osteogenic-differentiated human mesenchymal stem cells (hMSCs) to the one obtained from ECM from undifferentiated hMSCs. Our results provided evidence that both ECMs can bind BMP-2 and promote bone formation when implanted ectopically in mice. The osteoinductive potential of BMP-2, however, was greater when loaded within an osteogenic MSC-derived ECM; this outcome was correlated with higher sequestration capacity of BMP-2 over time in vivo. Interestingly, although the BMP-2 mainly bound onto the mineral crystals contained within the osteogenic MSC derived-ECM, these mineral components were not involved in the observed higher osteoinductivity, suggesting that the organic components were the critical components for the matrix efficacy as BMP-2 carrier