Development of in vitro 3D white adipose cell culture models for investigating white adipose cell biology and genetics of fat distribution

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Fat is an important organ that stores lipid to produce energy but is also a source of hormones that regulate food intake and insulin sensitivity. Dysfunction, due to environmental and/or genetic factors, of fat tissue can lead to fat being stored in the wrong place and increased risk of type 2 diabetes (T2D) and cardiovascular disease. Human genome-wide-association (GWA) has identified >403 T2D and >500 BMI and waist-hip-ratio adiposity risk loci.A major challenge is how to investigate genetic determinants of fat distribution and the mechanisms that control fat storage and metabolic signalling. Whole animal models, such as the mouse, are powerful but low throughput. Simple cellular 2D culture systems are a substitute but clear phenotypic differences to in vivo tissue limits their usefulness. To bridge the gap between 2D and in vivo tissue, the Cox Lab and an SME OxSyBio have collaborated to develop and optimise a 3-dimensional (3D) adipose culture system (Graham et al., IOP Biofabrication (2019) https://doi.org/10.1088/1758-5090/ab56fe). This has used 3T3-L1 adipocyte and primary fat cells from tissue. This new 3D bio-fabrication approach could help bridge existing models for obesity and diabetes research.The primary aim of this project is to improve our 3D model to obtain and fully characterize an 'organoid' type system in which we can manipulate genes, environment and apply treatments experimentally. Firstly, by adding other cellular components critical for fully functional white adipose tissue, including endothelial vascular cells and macrophages. We will evaluate these systems in response to known in vivo signals for lipogenesis, lipolysis and drug response.We will further increase the utility of our 3D system by introducing genetic mutations. For example, data from the Cox Laboratory has shown that a hypomorphic point mutation in the mouse Wars2 gene results in changes in adipose tissue in vivo. Using established in vitro protocols for CRISPR/Cas9 genetic modification in adipose cells, we will make the same mutation in preadipocytes and evaluate its effects on cells grown in both 2D and 3D, compared to in vivo tissue. This work will provide proof of principle for genetic manipulation giving rise to functional effects in 3D adipose. Further, to facilitate mechanistic studies on the WARS2/TBX15 waist-hip-ratio GWA locus and other loci of interest. A final goal is to humanise the system using induced pluripotent stem cells or cultured primary cells with a view to developing and validating a high throughput therapeutic screening system.The academic novelty of this project lies in the refinement of white adipose models for primary research purposes, to understand the basic mechanisms of fat deposition. Thus, helping exploit data from metabolic GWA to understand predispositions to disease, meeting MRC strategic aim #1 Molecular datasets and disease. An improved adipose tissue organoid can be used in high-throughput screens of biologically active compounds and proteins. This will provide a mechanism for adoption and exploitation by the UK research and development community and pharmaceutical industry, thus meeting the MRCs strategic Aim #2: Securing impact from medical research.