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

Bronson A. Haynes; Ryan W. Huyck; Ashley J. James; Meghan E. Carter; Omnia U. Gaafar; Marjorie Day; Avennette Pinto; Anca D. Dobrian

doi:10.3791/57804

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Method Article

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

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DOI:

10.3791/57804

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July 17th, 2018

Bronson A. Haynes¹ , Ryan W. Huyck¹ , Ashley J. James¹ , Meghan E. Carter¹ , Omnia U. Gaafar¹ , Marjorie Day¹ , Avennette Pinto¹ , Anca D. Dobrian¹

¹Department of Physiological Sciences, Eastern Virginia Medical School

Summary

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

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.

Introduction

Recent evidence shows that both in mice and in humans, a subset of cells residing in the adipose tissue vasculature can be differentiated into either white or beige/brown adipocytes¹^,²^,³. The phenotype of such cells is a subject of controversy, with evidence supporting endothelial cells, smooth muscle/pericyte, or a spectrum of intermediate phenotypes⁴^,⁵^,⁶^,⁷. The scope of developing this methodology was to test the adipogenic potential of CD34+CD31+....