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HUMAN Report Summary

Project ID: 602757
Funded under: FP7-HEALTH
Country: Sweden

Periodic Report Summary 3 - HUMAN (Health and the Understanding of Metabolism, Aging and Nutrition)

Project Context and Objectives:
The prevalence of metabolic diseases has dramatically risen owing to the great increase of life expectancy and cultural transitions in lifestyle and nutrition, with a predicted massive economic burden for the European health systems. In the last decade, great research efforts have been devoted to identify the genetic basis of age related and metabolic diseases, mostly by means of large genome-wide association studies (GWAS). GWAS studies have reported associations of around 4,000 single nucleotide polymorphisms (SNPs) for more than 200 traits/diseases. Despite the highly significant association with the trait of interest, the functional role of all these genetic variants is still not yet completely elucidated. This reduces their potential clinical and pharmaceutical application for i.e. the treatment and prevention of metabolic diseases. To overcome these limitations one of the main goals of the HUMAN consortium is the generation of mouse models highly repopulated with human hepatocytes or carrying pancreatic beta-cells from either primary cells (hepatocytes) or induced pluripotent stem cells (iPSCs). This innovative approach offers the unique possibility of studying the function of genetic risk variants associated to metabolic diseases in an integrated living system (i.e the mouse body), but in human-derived organs. A specific strength of the project is that the iPSCs used to generate hepatocytes and beta-cells derive from patients affected by severe metabolic diseases such as type 2 diabetes (T2D) with or without complications, or subjects selected for nearly the complete opposite phenotype, for their nearly complete lack of disease and exceptional longevity. We have selected the most relevant genes amongst those that emerged from GWAS studies for their association with T2D and metabolic syndrome, and cross-referenced and “filtered” them against genes associated with longevity. This allows us to arrive at the allelic differences for the genes most likely to be associated with a risk for, or protection from, disease.
Mice with humanised liver and/or pancreatic beta-cells are generated, and the phenotype that results from the risk or protective alleles will be characterised. The model will also allow testing of the effect of different nutritional regimes (e.g. high fat diet, caloric restriction), to disentangle the complex circuitry across organs (e.g. the hypothalamus-liver axis) and the impact of endocrine regulations (e.g. thyroid hormones) through nuclear receptors (e.g. LXR, THR). Since complex diseases such as T2D and metabolic syndrome are polygenetic and partially dependent on the genes selected here, the creation of the risk and protective cell lines and mouse models will provide a platform technology for discovery of genes and metabolic pathways that cooperate with these known risk alleles to manifest the full spectrum of the disease. The main goal of the project is to offer European research and industries exclusive tools to tackle the challenge of functional validation of metabolic disease-associated genetic variants by offering innovative and robust humanised animal and cellular models and a portfolio of new and validated therapeutic targets for better understanding of metabolic diseases and healthy aging.

Project Results:
During the 3rd year we have selected hepatocytes from donors with protective alleles for all selected SNPs, from donors with risk alleles for TCF7L2, APOE (e4/e4), combination FTO and APOE (e4/e4), and from a donor homozygous for both FTO and TCF7L2 risk alleles.
Fibroblasts from LLS offspring with low glucose and LLS controls with high glucose, and fibroblast from healthy semi-supercentenarians and from patients with T2D have been selected for iPSC generation. Robust differentiation of iPSC to hepatocyte-like cells has also been obtained. A method was established for comparison of gene expression in stem cell-derived hepatocyte-like cells versus authentic human liver. CRISPR technology has been applied in one donor and a corrected clone for the OTC mutation was obtained. We have designed and cloned several gRNAs targeting selected SNPs in TCF7L2, FTO and MC4R and are applying CRISPR targeting TCF7L2 in iPSCs derived from a subject from the T2D cohort. Experiments have shown that mice with >80% repopulation of human hepatocytes can be generated in primary transplants in some recipients without the need for serial transplants. However, the timeframe for high repopulation level differs between donors of high age (>30 years) and younger donors. Thus, serial transplantation will be a strategy to adopt when cell showing a low repopulation-rate have to be used.
As proof of concept OTCD hepatocytes have been successfully transplanted into mice and we have isolated the hepatocytes from mice highly repopulated with these donor cells. We have also successfully generated liver humanised mice with hepatocytes bearing some of the selected SNPs, to produce mice with risk/protection for specific diseases, and the phenotypic characterization has started. Mice engrafted with hepatocyte bearing risk allele for FTO and APOE seems to have altered glucose tolerance. Further studies on carbohydrate metabolism have shown that liver humanised mice have resistance to murine growth hormone (GH), giving us a possibility to show how GH signalling is fundamental for regulation of glucose homeostasis. The humanisation of lipoprotein profiles that was previously shown to be independent of CETP, seem instead dependent on the lack of editing of APOB mRNA inside human hepatocytes. An almost complete humanisation of faecal bile acid composition has been achieved in our mice with a resulting impact on intestinal microbiota. Thus, we were able to demonstrate the importance of primary bile acid composition in the determination of gut microbiota.
Robust methods and SOPs have been developed for metabolic profiling of different areas of the brain and various cells (fibroblasts, iPSCs). Comparisons have been made between metabolic profiles of five different areas of brain and plasma from liver-humanised mice and un-transplanted controls in order to investigate the effects of humanisation of the liver on the metabolic profiles in these samples. Metabolic profiling has been performed on cell media and cells from cultured fibroblasts and corresponding iPSCs in order to understand how reprogramming of fibroblasts to iPSCs changes the metabolites secreted.
We have assessed the specificity of the Illumina Human Methylation 450K Assay, evaluated the discriminative capability and characterised the age-associated methylation signatures in liver. ChIP assays for the histone marks H3K4me3 and H3K27ac from humanised livers for subsequent deep sequencing has been established and performed on humanised versus murinized FRGKO/NOD mice. We have also discovered 10-20 fold elevation in mRNA levels of mouse FGF21 in the humanised livers of FRGKO/NOD mice upon high-fat, high-sucrose diet compared to murinized mice.
In order to work with metadata, we finalised the sample naming convention, defined metadata formats and integration algorithms. The quality control and first analysis of proteomics data from seven organs from two mouse strains under different diet conditions has been done.

Potential Impact:
During the third year the results from the experimental work done by HUMAN Consortium has expanded the exceptional insights in the regulation of metabolism and ageing that will have great impact in the understanding of human physiology and in particular on the cross talk between liver, brain, and gut. Furthermore, the insights on human lipoprotein metabolism and on species-related differences between humans and mice have been expanded and the factor causing the humanization of lipoprotein profiles has likely been identified. We could also demonstrated how humanization of fecal bile acid affects macrobiota. We were able to achieve a high grade of hepatocyte differentiation of iPS cells reaching the level of expression of some specific hepatocyte genes similar to the one of fetal and neonatal livers. The metabolic profiling of different areas of brain tissue of five different areas of brain (striatum, cerebellum, cortex, hypothalamus and hippocampus) and plasma from humanised liver mice and un-transplanted controls have been already performed. The metabolic profiling of cell media and cells from cultured fibroblasts and corresponding iPSCs has been done in order to understand how reprogramming of fibroblasts to iPSCs changes the metabolome and identify biomarker for successful differentiation. We the progressed have been almost according to plan and have had only minor deviations from the initial strategy and few postponements of deliverables that have been approved during the mid-term review. Changes in priority (boost of transplantation with primary hepatocytes have been done while waiting for the survival of mice transplanted with iPSC-derived hepatocytes) mainly been done to optimize the efficiency, outcome, and the number of animals that will be studied in the future experimental work. We strongly believe that our data will be of great value for European researchers and industries. Some companies have indeed already approached us being interested of our results.

According to the WHO European Region non communicable diseases (NCDs) are the leading cause of death, disease and disability in Europe. Among them metabolic disease, T2D, and its complications like cardiovascular disease, account for the majority of the disease burden and of premature mortality in the European Region. This puts increasing pressure on health systems, economic development and the well-being of large parts of the European population, in particular people aged 50 years and older. Lately, there is great concern that metabolic disease and T2D risk factors increasingly affect younger age groups, with considerable consequences for public health trends in Europe in the future. As individuals age, metabolic diseases and T2D become one of the principal causes of morbidity, disability and mortality. As consequence, an enormous amount of health care costs are and will be increasingly concentrated in the latter decades of European citizen’s lives. Thus, the understanding of healthy ageing and how age and disease interrelate is of strategic importance. To reach this aim the HUMAN consortium has made a substantial effort to construct a careful selection of the most promising target genes associated to known human metabolic diseases and healthy aging. Furthermore, these genes are linked to metabolic phenotypes of healthy aging that we could verify in our unique human cohorts of longevity – including healthy “super-centenarians” 105+ years old. Using unique humanised animal and cellular models and large-scale metabolic phenotyping on a multi-center basis, HUMAN will provide the European research community and industry with standardised operating protocols of validated therapeutic targets. This synergy will increase the competitiveness and boost the innovative capacity of European health-related industries and businesses while improving human health.

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Life Sciences
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