Innovative techniques for gene editing have enabled accurate animal models of human diseases to be established. In order for these methods to be successfully adopted in the scientific community, the optimization of procedures used for breeding genetically altered mice is required. Among these, the in vitro fertilization (IVF) procedure is still suboptimal and the culture methods do not guarantee the development of competent embryos. Critical aspects in traditional in vitro embryo culture protocols include the use of mineral oil and the stress induced by repetitive handling of the embryos.A new microfluidic system was designed to allow for efficient in vitro culture of mouse embryos. Harmful fluidic stress and plastic toxicity were excluded by completing the industry gold standard Mouse Embryo Assay. The potential competence of the embryos developed in the device was quantified in terms of blastocyst rate, outgrowth assay, energy substrate metabolism and expression of genes related to implantation potential.Mass spectrometry analyses identified plastic-related compounds released in medium, and confirmed leaching of low molecular weight species into the culture medium that might be associated to un-crosslinked PDMS.Finally, these data show the potential for the system to study preimplantation embryo development and to improve human IVF techniques.
Inflammatory cytokines are key players in the pathogenesis of autoimmune disorders where their signaling leads to inappropriate balance between effector CD4 T-cells (Teff), Th1 and Th17, and the immunosuppressive regulatory T-cells (Treg). Each subset utilizes a distinctive metabolic program but how cytokines induce differential metabolic rewiring and what aspects of it are critical for the functioning and lineage stability of Teff subsets and Treg is not understood. To delineate pathogenicity-associated metabolic programs, we performed global untargeted metabolomics mass spectrometry (MS) in in vitro cytokine-differentiated mouse Th1, Th17 and Treg cells with varying degrees of pathogenicity over time. Integrating our data-driven metabolic network with global proteomics profiling and cytokine secretion allowed us to examine metabolic pathways at metabolite-enzyme level. Metabolites at the interface of amino acid metabolism, particularly glutamine and serine, glycolysis, the TCA cycle and nucleotide synthesis were the most differentially changed. Serine limitation or pharmacological inhibition indicated differential coupling of serine/one-carbon metabolism to T-cell proliferation, lineage choices and the production of cytokines IL-17 or IFNγ. Furthermore, targeting this pathway promoted Treg lineage development in the presence of pathogenic cytokines and prevented loss of Treg transcription factor FoxP3 upon re-stimulation. In vivo, spinal cord infiltrating T-cells in murine experimental autoimmune encephalomyelitis had altered serine metabolic pathway. Our findings identify key areas of T-cell metabolism that may offer a new category of therapeutic targets for autoimmune and inflammatory disease.
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