functional-human-liver-preservation-recovery-means-subnormothermic

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Massachusetts General Hospital, Harvard Medical School

The non-invasive high-resolution spatial mapping of cell metabolism within tissues could provide substantial advancements in assessing the efficacy of stem cell therapy and understanding tissue development. Here, using two-photon excited fluorescence microscopy, we elucidate the relationships among endogenous cell fluorescence, cell redox state, and the differentiation of human mesenchymal stem cells into adipogenic and osteoblastic lineages. Using liquid chromatography/mass spectrometry and quantitative PCR, we evaluate the sensitivity of an optical redox ratio of FAD/(NADH + FAD) to metabolic changes associated with stem cell differentiation. Furthermore, we probe the underlying physiological mechanisms, which relate a decrease in the redox ratio to the onset of differentiation. Because traditional assessments of stem cells and engineered tissues are destructive, time consuming, and logistically intensive, the development and validation of a non-invasive, label-free approach to defining the spatiotemporal patterns of cell differentiation can offer a powerful tool for rapid, high-content characterization of cell and tissue cultures.

Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation.

Mechanical property elaboration of engineered tissues is often assumed on the basis of gene and protein characterizations, rather than mechanical testing. However, we recently demonstrated that mechanical properties are not consistently correlated with matrix content and organization during embryonic tissue development. Based on this, mechanical properties should be assessed independently during natural or engineered tissue formation. Unfortunately, mechanical testing is destructive, and thus alternative means of assessing these properties are desirable. In this study, we examined lysyl oxidase (LOX)-mediated crosslinks as markers for mechanical properties during embryonic tendon formation and the potential to detect them non-destructively. We used tandem mass spectrometry (LC-MS/MS) to quantify changes in hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) crosslink density in embryonic chick tendon as a function of developmental stage. In addition, we assessed a multiphoton imaging approach that exploits the natural fluorescence of HP and LP. With both techniques, we quantified crosslink density in normal and LOX-inhibited tendons, and correlated measurements with mechanical properties. HP and LP crosslink density varied as a function of developmental stage, with HP-to-dry mass ratio correlating highly to elastic modulus, even when enzymatic crosslink formation was inhibited. Multiphoton optical imaging corroborated LC-MS/MS data, identifying significant reductions in crosslink density from LOX inhibition. Taken together, crosslink density may be useful as a marker of tissue mechanical properties that could be assessed with imaging non-destructively and perhaps non-invasively. These outcomes could have significant scientific and clinical implications, enabling continuous and long-term monitoring of mechanical properties of collagen-crosslinked tissues or engineered constructs.

Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation.

Lipid accumulation in adipocytes reflects a balance between enzymatic pathways leading to the formation and breakdown of esterified lipids, primarily triglycerides. This balance is extremely important, as both high and low lipid levels in adipocytes can have deleterious consequences. The enzymes responsible for lipid synthesis and breakdown (lipogenesis and lipolysis, respectively) are regulated through the coordinated actions of several transcription factors (TFs). In this study, we examined the dynamics of several key transcription factors (TFs) - PPARγ, C/EBPβ, CREB, NFAT, FoxO1, and SREBP-1c - during adipogenic differentiation (week 1) and ensuing lipid accumulation. The activation profiles of these TFs at different times following induction of adipogenic differentiation were quantified using 3T3-L1 reporter cell lines constructed to secrete the Gaussia luciferase enzyme upon binding of a TF to its DNA binding element. The dynamics of the TFs was also modeled using a combination of logical gates and ordinary differential equations, where the logical gates were used to explore different combinations of activating inputs for PPARγ, C/EBPβ, and SREBP-1c. Comparisons of the experimental profiles and model simulations suggest that SREBP-1c could be independently activated by either insulin or PPARγ, whereas PPARγ activation required both C/EBPβ as well as a putative ligand. Parameter estimation and sensitivity analysis indicate that feedback activation of SREBP-1c by PPARγ is negligible in comparison to activation of SREBP-1c by insulin. On the other hand, the production of an activating ligand could quantitatively contribute to a sustained elevation in PPARγ activity.

Analysis of transcription factor network underlying 3T3-L1 adipocyte differentiation.

Metabolites produced by the intestinal microbiota are potentially important physiological modulators. Here we present a metabolomics strategy that models microbiota metabolism as a reaction network and utilizes pathway analysis to facilitate identification and characterization of microbiota metabolites. Of the 2,409 reactions in the model, ~53% do not occur in the host, and thus represent functions dependent on the microbiota. The largest group of such reactions involves amino-acid metabolism. Focusing on aromatic amino acids, we predict metabolic products that can be derived from these sources, while discriminating between microbiota- and host-dependent derivatives. We confirm the presence of 26 out of 49 predicted metabolites, and quantify their levels in the caecum of control and germ-free mice using two independent mass spectrometry methods. We further investigate the bioactivity of the confirmed metabolites, and identify two microbiota-generated metabolites (5-hydroxy-L-tryptophan and salicylate) as activators of the aryl hydrocarbon receptor.

Article	Year
Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation. Scientific reports\| PubMed ID: 24305550	2013
Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation. Acta biomaterialia\| PubMed ID: 24316363	2014
Analysis of transcription factor network underlying 3T3-L1 adipocyte differentiation. PloS one\| PubMed ID: 25075860	2014
Prediction and quantification of bioactive microbiota metabolites in the mouse gut. Nature communications\| PubMed ID: 25411059	2014

Gautham V. Sridharan

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