Optimized nucleus isolation protocol from frozen mouse tissues for single nucleus RNA sequencing application

The single cell RNA sequencing technique has been particularly used during the last years, allowing major discoveries. However, the widespread application of this analysis has showed limitations. Indeed, the direct study of fresh tissues is not always feasible, notably in the case of genetically engineered mouse embryo or sensitive tissues whose integrity is affected by classical digestion methods. To overcome these limitations, single nucleus RNA sequencing offers the possibility to work with frozen samples. Thus, single nucleus RNA sequencing can be performed after genotyping-based selection on samples stocked in tissue bank and is applicable to retrospective studies. Therefore, this technique opens the field to a wide range of applications requiring adapted protocols for nucleus isolation according to the tissue considered. Here we developed a protocol of nucleus isolation from frozen murine placenta and pancreas. These two complex tissues were submitted to a combination of enzymatic and manual dissociation before undergoing different steps of washing and centrifugation. The entire protocol was performed with products usually present in a research lab. Before starting the sequencing process, nuclei were sorted by flow cytometry. The results obtained validate the efficiency of this protocol which is easy to set up and does not require the use of commercial kits. This specificity makes it adaptable to different organs and species. The association of this protocol with single nucleus RNA sequencing allows the study of complex samples that resist classical lysis methods due to the presence of fibrotic or fatty tissue, such as fibrotic kidney, tumors, embryonic tissues or fatty pancreas

Optimized nucleus isolation protocol from frozen mouse tissues for single nucleus RNA sequencing application.

The single cell RNA sequencing technique has been particularly used during the last years, allowing major discoveries. However, the widespread application of this analysis has showed limitations. Indeed, the direct study of fresh tissues is not always feasible, notably in the case of genetically engineered mouse embryo or sensitive tissues whose integrity is affected by classical digestion methods. To overcome these limitations, single nucleus RNA sequencing offers the possibility to work with frozen samples. Thus, single nucleus RNA sequencing can be performed after genotyping-based selection on samples stocked in tissue bank and is applicable to retrospective studies. Therefore, this technique opens the field to a wide range of applications requiring adapted protocols for nucleus isolation according to the tissue considered. Here we developed a protocol of nucleus isolation from frozen murine placenta and pancreas. These two complex tissues were submitted to a combination of enzymatic and manual dissociation before undergoing different steps of washing and centrifugation. The entire protocol was performed with products usually present in a research lab. Before starting the sequencing process, nuclei were sorted by flow cytometry. The results obtained validate the efficiency of this protocol which is easy to set up and does not require the use of commercial kits. This specificity makes it adaptable to different organs and species. The association of this protocol with single nucleus RNA sequencing allows the study of complex samples that resist classical lysis methods due to the presence of fibrotic or fatty tissue, such as fibrotic kidney, tumors, embryonic tissues or fatty pancreas

Table1_Optimized nucleus isolation protocol from frozen mouse tissues for single nucleus RNA sequencing application.pdf

The single cell RNA sequencing technique has been particularly used during the last years, allowing major discoveries. However, the widespread application of this analysis has showed limitations. Indeed, the direct study of fresh tissues is not always feasible, notably in the case of genetically engineered mouse embryo or sensitive tissues whose integrity is affected by classical digestion methods. To overcome these limitations, single nucleus RNA sequencing offers the possibility to work with frozen samples. Thus, single nucleus RNA sequencing can be performed after genotyping-based selection on samples stocked in tissue bank and is applicable to retrospective studies. Therefore, this technique opens the field to a wide range of applications requiring adapted protocols for nucleus isolation according to the tissue considered. Here we developed a protocol of nucleus isolation from frozen murine placenta and pancreas. These two complex tissues were submitted to a combination of enzymatic and manual dissociation before undergoing different steps of washing and centrifugation. The entire protocol was performed with products usually present in a research lab. Before starting the sequencing process, nuclei were sorted by flow cytometry. The results obtained validate the efficiency of this protocol which is easy to set up and does not require the use of commercial kits. This specificity makes it adaptable to different organs and species. The association of this protocol with single nucleus RNA sequencing allows the study of complex samples that resist classical lysis methods due to the presence of fibrotic or fatty tissue, such as fibrotic kidney, tumors, embryonic tissues or fatty pancreas.</p

Découverte et caractérisation d’une nouvelle forme de pancréatite chronique

Les causes à l’origine de la pancréatite chronique chez l’enfant sont en partie différentes de celles trouvées chez l’adulte et restent mal connues. Ainsi, la présence de mutations génétiques prédisposant à la pancréatite est suspectée être une cause plus fréquente chez l’enfant. Dans ce contexte, parmi des patients pédiatriques présentant des signes précurseurs de pancréatite, nous avons identifié une catégorie de patients souffrant de symptômes typiquement associés aux ciliopathies, un groupe de désordres génétiques liés à un dysfonctionnement du cil primaire. Dans ce travail, nous avions donc pour objectif d’identifier des mutations de gènes ciliaires chez ces patients, et d’établir un lien entre ces mutations et le développement d’une pancréatite. Via le Whole Exome Sequencing de l’ADN génomique de 43 patients pédiatriques présentant une pancréatite chronique idiopathique, nous avons tout d’abord identifié chez trois d’entre eux une mutation dans les gènes ciliaires/ciliogéniques PKHD1, HNF1β et NPHP3. Nous nous sommes ensuite focalisés sur le gène NPHP3 et avons généré par la technique de CRISPR/Cas9 deux nouveaux modèles murins transgéniques ; l’un reproduit les mutations du gène NPHP3 présentes chez le patient correspondant (modèle NPHP3mut1/mut2), tandis que l’autre est une inactivation conditionnelle de NPHP3 (modèle NPHP3f/f). L’analyse phénotypique de ces deux modèles nous a permis de conclure qu’une perte de fonction de NPHP3 dans les cellules canalaires pancréatiques conduisait à une inflammation et une fibrose légère associées à une atrophie acinaire importante et une lipomatose sévère. Ce tableau fait penser à la présence, dans ces modèles de souris, d’une nouvelle forme de pancréatite caractérisée par une origine ciliopathique. Afin de confirmer la présence de lipomatose pancréatique chez des patients présentant des mutations de HNF1β et NPHP3, des premières analyses par IRM ont été réalisées et ont permis de révéler un pourcentage important de tissu adipeux au sein du pancréas de ces patients. Une analyse plus approfondie des modèles murins, notamment pour caractériser les mécanismes moléculaires impliqués dans les phénotypes observés, et le recrutement d’un plus grand nombre de patients et de sujets contrôles doit nous permettre de confirmer l’existence de cette nouvelle forme de pancréatite d’origine ciliopathique

IL-21 promotes the development of a CD73-positive Vγ9Vδ2 T cell regulatory population

International audienceVγ9Vδ2 T cells contribute to the immune response against many tumor types through their direct cytotoxic activity and capacity to regulate the biological functions of other immune cells, such as dendritic cells and IFN-γ-producing CD8+ T cells. However, their presence in the tumor microenvironment has also been associated with poor prognosis in breast, colon and pancreatic cancers. Additionally, recent studies demonstrated that cytokines can confer some plasticity to Vγ9Vδ2 T cells and promote their differentiation into cells with regulatory functions. Here, we demonstrated that activation of Vγ9Vδ2 T cells isolated from healthy donors and cultured in the presence of IL-21 favors the emergence of a subpopulation of Vγ9Vδ2 T cells that express the ectonucleotidase CD73 and inhibits T cell proliferation in a CD73/adenosine-dependent manner. This subpopulation produces IL-10 and IL-8 and displays lower effector functions and cytotoxic activity than CD73-negative Vγ9Vδ2 T cells. We also showed, in a syngeneic mouse tumor model, the existence of a tumor-infiltrating γδ T cell subpopulation that produces IL-10 and strongly expresses CD73. Moreover, maturation, IL-12 production and induction of antigen-specific T cell proliferation are impaired in DC co-cultured with IL-21-amplified Vγ9Vδ2 T cells. Altogether, these data indicate that IL-21 promotes Vγ9Vδ2 T cell regulatory functions by favoring the development of an immunosuppressive CD73+ subpopulation. Thus, when present in the tumor microenvironment, IL-21 might negatively impact γδ T cell anti-tumor functions