CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease

Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as “brown” and “brite/beige” adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.

Hypertrophied human adipocyte spheroids as in vitro model of weight gain and adipose tissue dysfunction

The rise in obesity prevalence has created an urgent need for new and improved methods to study human adipocytes and the pathogenic effects of weight gain in vitro. Despite numerous studies showing the advantages of culturing adipocyte progenitors as 3D structures, the majority continue using traditional 2D cultures which result in small, multilocular adipocytes with poor representability. We hypothesized that providing differentiating pre-adipocytes with a vascular growth niche would mimic in vivo adipogenesis and improve the differentiation process. Here we present a simple, easily applicable culture protocol that allows for the differentiation and culturing of human adipocytes with a more unilocular morphology and larger lipid droplets than previous protocols. We moreover offer a protocol for inducing adipocyte enlargement in vitro, resulting in larger lipid droplets and the development of several key features of adipocyte dysfunction, including altered adipokine secretions and impaired lipolysis. Taken together, our hypertrophied human adipocyte spheroids offer an improved culture system for studying the cellular and molecular mechanisms causing metabolic dysfunction and inflammation during weight gain.

Isolation and Characterization of Human Adipocyte-derived Extracellular Vesicles Using Filtration and Ultracentrifugation

ABSTRACTExtracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte- derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances including, obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis including, transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing, microarray and utilized in functional in vitro/in vivo studies.SUMMARYWe describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).