Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Abstract

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease. This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) >35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2-3 g of fat. These cells can be expanded in culture over 10-14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5-6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPARγ agonist-rosiglitazone. This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The AAV-PCSK9 murine model of atherosclerosis and metabolic dysfunction

“…Epididymal adipose tissues (AT) were digested as previously described, 6 and single-cell suspension was stained for CD45 and a Ly6C marker for pro-inflammatory Ly6C high/mid monocytes (Biolegend, #103130, #128006), analyzed via FACSCalibur (BD Biosciences/Cytek Inc.), and FlowJo analysis software (Tree Star Inc.). B6-Chow AT data represent two mice pooled together.…”

Effects of age-dependent changes in cell size on endothelial cell proliferation and senescence through YAP1

“…The ECs were isolated as described before [77]. Briefly, after removing the fibrotic and cauterized regions, about 5 g of subcutaneous adipose tissue was minced using small scissors and transferred into 15 ml tubes containing 5 ml of 1 mg/ml Collagenase A (Roche, Basel Switzerland).…”

White-to-Beige and Back: Adipocyte Conversion and Transcriptional Reprogramming

“…This approach enables an opportunity to counteract obesity and related diseases such as type 2 diabetes mellitus, hyperlipidemia, dyslipidemia, cardiovascular disease, etc. With the use of small molecules, not only adipocytes but also fibroblasts [ 138 ], endothelial cells [ 139 ], and myoblasts [ 140 ] can be transformed into beige adipocytes.…”