TGFbeta Family Members Are Key Mediators in the Induction of Myofibroblast Phenotype of Human Adipose Tissue Progenitor Cells by Macrophages

International audienceOBJECTIVE: The present study was undertaken to characterize the remodeling phenotype of human adipose tissue (AT) macrophages (ATM) and to analyze their paracrine effects on AT progenitor cells. RESEARCH DESIGN AND METHODS: The phenotype of ATM, immunoselected from subcutaneous (Sc) AT originating from subjects with wide range of body mass index and from paired biopsies of Sc and omental (Om) AT from obese subjects, was studied by gene expression analysis in the native and activated states. The paracrine effects of ScATM on the phenotype of human ScAT progenitor cells (CD34(+)CD31(-)) were investigated. RESULTS: Two main ATM phenotypes were distinguished based on gene expression profiles. For ScAT-derived ATM, obesity and adipocyte-derived factors favored a pro-fibrotic/remodeling phenotype whereas the OmAT location and hypoxic culture conditions favored a pro-angiogenic phenotype. Treatment of native human ScAT progenitor cells with ScATM-conditioned media induced the appearance of myofibroblast-like cells as shown by expression of both α-SMA and the transcription factor SNAIL, an effect mimicked by TGFβ1 and activinA. Immunohistochemical analyses showed the presence of double positive α-SMA and CD34 cells in the stroma of human ScAT. Moreover, the mRNA levels of SNAIL and SLUG in ScAT progenitor cells were higher in obese compared with lean subjects. CONCLUSIONS: Human ATM exhibit distinct pro-angiogenic and matrix remodeling/fibrotic phenotypes according to the adiposity and the location of AT, that may be related to AT microenvironment including hypoxia and adipokines. Moreover, human ScAT progenitor cells have been identified as target cells for ScATM-derived TGFβ and as a potential source of fibrosis through their induction of myofibroblast-like cells

Native human adipose stromal cells: localization, morphology and phenotype

International audienceObjectives:Beside having roles in energy homeostasis and endocrine modulation, adipose tissue (AT) is now considered a promising source of mesenchymal stromal cells (adipose-derived stromal cells or ASCs) for regenerative medicine. Despite numerous studies on cultured ASCs, native human ASCs are rarely investigated. Indeed, the phenotype of ASCs in their native state, their localization within AT and comparison with bone marrow-derived mesenchymal stromal cells (BM-MSCs) has been poorly investigated.Design:To address these issues, the stroma vascular fraction (SVF) of human AT was extracted and native cell subtypes were isolated by immunoselection to study their clonogenic potential in culture. Immunohistology on samples of human AT in combination with reconstruction of confocal sections were performed in order to localize ASCs.Results:Compared with BM-MNCs, all native ASCs were found in the CD34(+) cell fraction of the AT-SVF. Native ASCs expressed classical mesenchymal markers described for BM-MSCs. Interestingly, CD34 expression decreased during ASC cell culture and was negatively correlated with cell proliferation rate. Immunohistological analysis revealed that native ASCs exhibited specific morphological features with protrusions. They were found scattered in AT stroma and did not express in vivo pericytic markers such as NG2, CD140b or alpha-smooth muscle actin, which appeared during the culture process. Finally, ASCs spontaneous commitment to adipocytic lineage was enhanced in AT from obese humans.Conclusions:The use of complementary methodological approaches to study native human ASCs revealed their immunophenotype, their specific morphology, their location within AT and their stemness. Furthermore, our data strongly suggest that human ASCs participate in adipogenesis during AT development.International Journal of Obesity advance online publication, 25 January 2011; doi:10.1038/ijo.2010.269

D’un genre à l’autre.

Harold Garfinkel a mis en évidence le processus par lequel un individu apprend à « passer » en s’employant à maintenir le sens de la réalité de ce qu’il se dit être. Pour maintenir le sens de ce qu’ils sont pour eux-mêmes et pour autrui, les transsexuels et les transgenres ne font pas qu’intervenir sur l’image de leur apparence physique, ils élaborent aussi a posteriori un récit de vie subjectif dont la narration permet l’enracinement de la « nouvelle » identité de genre.Les degrés d’intervention sur l’apparence en public ou en privé et les spécificités de la narration de l’histoire personnelle sont en partie contingents, mais ils sont aussi fonction d’un ordre social inscrit dans les institutions – institutions qui façonnent les individus – auxquelles les transsexuels et les transgenres sont confrontés. Les tensions entre les attentes individuelles et les exigences des institutions médicales et juridiques ont contribué à la formation de l’ébauche d’une identité collective transgenre.From one gender to the otherHarold Garfinkel has shed light on the process whereby individuals learn to “pass” so as to maintain the meaning of the reality of what they say they are. To maintain the meaning of what they are for themselves and for others, transsexuals and transgenders do not just work on their physical appearances, they also work out a posteriori a life story that they can narrate so as to give roots to their new gender identity. Their interventions on their public or private appearances and the narration of their own life-stories are partly contingent, but they also depend on the social order established in institutions. These institutions shape individuals; and transsexuals and transgenders have to cope with them. The tensions between individual expectations and the requirements of medical and legal institutions have helped shape a collective transgender identity

Développement de méthodes de séquençage de seconde génération pour l'analyse des profils de méthylation de l'ADN

The analysis of DNA methylation patterns has become of great interest as methylome alterations have been found in many diseases. MeDIP (Methylated DNA ImmunoPrecipitation) immunoprecipitates genome-wide methylated sequences many of which are located in the repetitive sequences. Such sequences are difficult to align unambiguously after sequencing (MeDIP-Seq) leading to a large number of sequences that are currently not used for further analysis. We present an innovative method called MeDIP-dep-Seq which depletes a significant part of several classes of these highly repetitive sequences (up to 300-fold decrease), while unique sequences of interest are not affected. After sequencing on a second generation sequencer (GAIIx, Illumina) the alignment rate substantially enhanced increasing thus the amount of usable sequences. We have further developed a pipeline for the analysi of MeDIP-Seq datasets. Potential candidate regions identified in this genome-wide assay can then be validated by the use of selector probes that specifically capture genomic regions of interest. We introduced a bisulfite treatment in the selection protocol and developed a novel multiplex assay. 98 gene loci were enriched in 6 samples and were then sequenced in parallel on a bench sequencer (GS Junior, Roche). The combination of these technologies will permit the establishment of methylome maps and the identification of novel epigenetic biomarkers for cancer and complex diseases diagnostics and prognosis.L'analyse des profils de méthylation présente un grand intérêt car des altérations du méthylome sont impliquées dans de nombreuses pathologies. Le MeDIP (Methylated DNA ImmunoPrecipitation) immunoprécipite les séquences méthylées sur le génome entier, la plupart étant localisées dans les séquences répétées. De telles séquences sont difficiles à aligner après séquençage (MeDIP-Seq) et bon nombre d'entre elles ne peuvent donc être utilisées pour la suite des analyses. Nous présentons une méthode innovante appelée MeDIP-dep-Seq permettant de supprimer une quantité significative de plusieurs familles de ces éléments répétés (diminution d'un facteur de 300 au maximum) tandis que les séquences uniques d'intérêt ne sont pas affectées. Après séquençage sur un séquenceur de seconde génération (GAIIx, Illumina), le taux d'alignement est amélioré de façon conséquente permettant ainsi d'augmenter la quantité de séquences analysables. Nous avons également développé une plateforme d'analyse des données issues du MeDIP-Seq. De potentielles régions candidates identifiées par cette technique sur le génome entier peuvent ensuite être validées en utilisant des sélectors, sondes permettant la capture de régions génomiques d'intérêt. Nous avons introduit un traitement au bisulphite dans le protocole de sélection afin de développer un nouvel outil pour une analyse multiplexe. 98 loci ont été enrichis dans 6 échantillons puis séquencés en parallèle sur un séquenceur de paillasse (GS Junior, Roche). La combinaison de ces technologies permettra d'établir des cartes du méthylome et d'identifier des nouveaux biomarqueurs épigénétiques pour diagnostiquer et pronostiquer les cancers et maladies complexes

Et si l’origine des progéniteurs fibro-adipeux contribuait à leur hétérogénéité dans le muscle ?

Les progéniteurs fibro-adipogéniques (FAPs), cellules stromales mésenchymateuses (CSMs) résidentes du muscle squelettique, jouent un rôle crucial dans l’homéostasie et la régénération musculaire via leur activité paracrine. Les avancées technologiques récentes dans le domaine du séquençage de l’ARN en cellule unique ont permis la description de l’hétérogénéité de cette population cellulaire. Dans cet article, nous présenterons les différentes sous-populations de FAPs en condition basale, lésionnelle ou de dégénérescence, ainsi que leurs fonctions associées chez la souris et l’homme. Nous discuterons ensuite de l’origine extra-musculaire possible d’une population de FAPs post-lésionnelle. En effet, nos travaux récents démontrent que des CSMs provenant du tissu adipeux et infiltrées dans le muscle pourraient participer à l’hétérogénéité des FAPs

The role of endothelial cells in inflamed adipose tissue.

International audienceIn recent years, the general concept has emerged that chronic low-grade inflammation can be the condition linking excessive development of adipose tissue (AT) and obesity-associated pathologies such as type II diabetes and atherosclerosis. Moreover, the evidence that the growth of the fat mass was associated with an accumulation of adipose tissue macrophages (ATM) has raised the hypothesis that the development of an inflammatory process within the growing fat mass is a primary event involved in the genesis of systemic metabolic and vascular alterations. As ATM originate from the bone marrow/blood compartment, enhanced macrophage recruitment to growing AT is suspected. However, the mechanisms responsible for attracting the blood cells and their entry into the fat mass remain to be clearly defined. The present review highlights the key role of endothelial cells in the control of the inflammatory process and describes the potential involvement of AT-endothelial cells as well as the factors involved in the regulation of their phenotype in the 'inflamed fat tissue'

Chemotaxis and differentiation of human adipose tissue CD34+/CD31- progenitor cells: role of stromal derived factor-1 released by adipose tissue capillary endothelial cells.

International audienceThe native CD34+/CD31- cell population present in the stroma-vascular fraction of human adipose tissue (hAT) displays progenitor cell properties since they exhibit adipocyte- and endothelial cell-like phenotypes under appropriate stimuli. To analyze the signals within hAT regulating their phenotypes, the influence of hAT-derived capillary endothelial cells (CECs) was studied on the chemotaxis and differentiation of the hAT-CD34+/CD31- cells. Conditioned medium from hAT-CECs led to a strong chemotaxis of the hAT-CD34+/CD31- cells that was inhibited with pretreatments with pertussis toxin, CXCR-4 antagonist, or neutralizing antibodies. Furthermore, hAT-CECs produced and secreted the CXCR-4 ligand, that is, the stromal derived factor-1 (SDF-1). Finally, hAT-CECs induced the differentiation of hAT-CD34+/CD31- cells toward an endothelial cell (EC) phenotype. Indeed, hAT-CECs and -CD34+/CD31- cell coculture stimulated in a two-dimensional system the expression of the EC CD31 marker by the hAT-progenitor cells and, in a three-dimensional approach, the formation of capillary-like structures via a SDF-1/CXCR-4 dependent pathway. Thus, the migration and differentiation of hAT progenitor cells are modulated by hAT-CEC-derived factors. SDF-1, which is secreted by hAT-derived CECs, and its receptor CXCR-4, expressed by hAT-derived progenitor cells, may promote chemotaxis and differentiation of hAT-derived progenitor cells and thus contribute to the formation of the vascular network during the development of hAT