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

脂肪是储存脂质以产生能量的重要器官，也是调节食物摄入和胰岛素敏感性的激素的来源。由于环境和/或遗传因素，脂肪组织功能障碍会导致脂肪储存在错误的地方，增加2型糖尿病（T2D）和心血管疾病的风险。人类全基因组关联（GWA）已经确定了bbbb403 T2D和bbbb500 BMI和腰臀比肥胖风险位点。一个主要的挑战是如何研究脂肪分布的遗传决定因素以及控制脂肪储存和代谢信号的机制。整个动物模型，如小鼠，功能强大，但吞吐量低。简单的细胞二维培养系统是一种替代品，但体内组织明显的表型差异限制了它们的实用性。为了弥合二维和体内组织之间的差距，Cox实验室和SME OxSyBio合作开发和优化了三维（3D）脂肪培养系统（Graham等人，IOP bifabrication (2019) https://doi.org/10.1088/1758-5090/ab56fe）。该实验使用了来自组织的3T3-L1脂肪细胞和原代脂肪细胞。这种新的3D生物制造方法可以帮助连接现有的肥胖和糖尿病研究模型。该项目的主要目的是改进我们的3D模型，以获得并充分表征一个“类器官”型系统，在这个系统中，我们可以操纵基因、环境和应用实验治疗。首先，通过添加其他对白色脂肪组织功能完整至关重要的细胞成分，包括内皮血管细胞和巨噬细胞。我们将评估这些系统对体内已知的脂肪生成、脂肪分解和药物反应信号的反应。我们将通过引入基因突变进一步提高我们的3D系统的效用。例如，来自Cox实验室的数据表明，小鼠Wars2基因的一个分形点突变会导致体内脂肪组织的变化。利用已建立的体外脂肪细胞CRISPR/Cas9基因修饰方案，我们将在脂肪前细胞中进行相同的突变，并评估其对在2D和3D中生长的细胞的影响，与体内组织进行比较。这项工作将为基因操作在3D脂肪中产生功能效应提供原理证明。此外，为了促进对WARS2/TBX15腰臀比GWA位点和其他感兴趣的位点的机制研究。最终目标是利用诱导多能干细胞或培养原代细胞使该系统人性化，以期开发和验证高通量治疗筛选系统。本项目的学术新颖之处在于完善白色脂肪模型，用于初步研究目的，以了解脂肪沉积的基本机制。因此，帮助利用代谢GWA的数据来了解疾病的易感性，满足MRC的战略目标#1分子数据集和疾病。改进的脂肪组织类器官可用于生物活性化合物和蛋白质的高通量筛选。这将为英国研发界和制药行业提供一种采用和利用的机制，从而满足MRCs战略目标#2：确保医学研究的影响。

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.