Immunogenicity of cardiac progenitors derived from embryonic stem cells

Le sujet de ce travail de thèse a concerné l'analyse de l’immunogénicité de progéniteurs cardiaques issus de cellules souches embryonnaires humaines. Le but a été double. D’une part, comprendre les mécanismes cellulaires et moléculaires qui sous-tendent cette immunogénicité et, d’autre part, mettre en place des stratégies d’immuno-intervention permettant de la surmonter.Le travail a comporté deux volets, l’un in vitro et l’autre in vivo utilisant des modèles expérimentaux murins. Les analyses in vitro, ont utilisé une méthode de culture lymphocytaire mixte où des progéniteurs cardiaques humains ont été mis en culture avec des lymphocytes allogéniques. Les résultats ont montré que les progéniteurs cardiaques sont effectivement immunogènes et que la réponse immunitaire qu’ils suscitent peut-être modulée efficacement par des cellules mésenchymateuses dérivées du tissu adipeux. De plus, nous avons confirmé l’expression des molécules d’histocompatibilité de classe I à la surface de progéniteurs cardiaques, une expression qui semble modulée au cours de la culture.Les modèles in vivo que nous avons utilisés ont consisté en l’implantation de corps embryoïdes et des progéniteurs cardiaques de souris dans un contexte allogénique. Divers sites d’implantation ont été utilisés (myocarde, capsule rénale, muscle gastrocnemius) chez des souris immunocompétentes. Les résultats ont montré qu’à la fois les corps embryoïdes et les progéniteurs cardiaques sont rejetés chez les receveurs immunocompétents non traités, avec une cinétique différente en fonction du site d’implantation. Par ailleurs, l’utilisation d’un traitement par anticorps anti-CD3, appliqué à différents temps suivant l’implantation nous a permis de prolonger la survie des cellules implantées en induisant, en fonction de la fenêtre thérapeutique, soit une immunosuppression soit une tolérance immunitaire.The present work concerned the analysis of the immunogenicity of cardiac progenitors derived from human embryonic stem cells. Our aim was to understand the cellular and molecular mechanisms which underlie this immunogenicity and to surmount it by setting up strategies of immune-intervention. The study consisted in two major components, one in vitro and the other in vivo using experimental mice models. The in vitro analyses were assessed by the mixed leukocyte reaction method, where human cardiac progenitors were cultured with allogeneic lymphocytes. The results showed that the cardiac progenitors are indeed immunogenic and that the immune response that they induce could be modulated by mesenchymal stromal cells derived from adipose tissue. Moreover, we confirmed the expression of class I histocompatibility molecules on the surface of cardiac progenitors, an expression which seems modulated during the culture. The in vivo models that we used consisted of the grafting of embryoïdes bodies and cardiac progenitors derived from mouse embryonic stem cells in an allogeneic context. Cells were grafted in different sites of immunocompetent mice (myocardium, renal capsule, muscle gastrocnemius). The results showed highlighted that at the same time both embryoïdes bodies and cardiac progenitors are rejected among untreated immunocompetents hosts, whereas their survival is extended by anti-CD3 treatments, In addition, anti-CD3 treatment prolongs the survival of grafted cells, either by immunosuppression or by inducing immune tolerance according to the timing when it is applied

Long-Term Functional Benefits of Epicardial Patches as Cell Carriers

International audienceBoth enzymatic dissociation of cells prior to needle-based injections and poor vascularization of myocardial infarct areas are two important contributors to cell death and impede the efficacy of cardiac cell therapy. Because these limitations could be overcome by scaffolds ensuring cell cohesiveness and codelivery of angiogenic cells, we used a chronic rat model of myocardial infarction to assess the long-term (6 months) effects of the epicardial delivery of a composite collagen-based patch harboring both cardiomyogenesis-targeted human embryonic SSEA-1(+) (stem cell-derived stage-specific embryonic antigen-1 positive) cardiovascular progenitors and autologous (rat) adipose tissue-derived angiogenesis-targeted stromal cells (n=27). Cell-free patches served as controls (n=28). Serial follow-up echocardiographic measurements of left ventricular ejection fraction (LVEF) showed that the composite patch group yielded a significantly better preservation of left ventricular function that was sustained over time as compared with controls, and this pattern persisted when the assessment was restricted to the subgroup of rats with initial LVEFs below 50%. The composite patch group was also associated with significantly less fibrosis and more vessels in the infarct area. However, although human progenitors expressing cardiac markers were present in the patches before implantation, none of them could be subsequently identified in the grafted tissue. These data confirm the efficacy of epicardial scaffolds as cell carriers for ensuring long-term functional benefits and suggest that these effects are likely related to paracrine effects and call for optimizing cross-talks between codelivered cell populations to achieve the ultimate goal of myocardial regeneration

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

International audiencePrimary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function