Differentiation and Induction of Lipid Droplet Accumulation in Human Adipocytes as a Model to Study Obesity In Vitro

Summary

The presented protocol aims to develop a cellular model that mimics human obesity. To achieve this, human adipose-derived stem cells are differentiated into adipocytes, which are next fattened using a mixture of selected fatty acids.

Abstract

Obesity-related changes in adipose tissue alter local secretory profiles and influence interactions with other surrounding cells, including cancer cells. The continued delivery of nutrients shifts the metabolism of fat cells, making them act as a lipid reservoir. Typically, research on obesity mechanisms relies on animal models, which, however, do not allow analysis of metabolic processes at the cellular level. Hence, there is a need to develop a reliable in vitro model of human obesity that can help explore changes in the function of obese adipocytes compared to their lean counterparts. This work presents a protocol for the differentiation of human adipocytes from their precursors (adipose-derived stem cells) using a mixture of insulin, isobutyl-1-methylxanthine, dexamethasone, and indomethacin. Subsequently, differentiated adipocytes were incubated with a set of fatty acids, including linoleate, palmitate, and oleate, to promote lipid accumulation. As a result, a valuable model mimicking the hypertrophic cells under obesity conditions was achieved, which can be used for further studies.

Introduction

Obesity is thought to be a major risk factor for a wide range of diseases, such as musculoskeletal disorders, cardiovascular diseases, insulin resistance, metabolic syndrome, diabetes, and selected types of cancer¹. Obesity is one of the most perceptible and under-estimated public health problems, with causes that are multi-elemental. Briefly, obesity is triggered by an imbalance between calories consumed and expended, resulting in enhanced fat storage². The caloric excess leads to pathological expansion and dysfunction of adipose tissue (AT), the key element in the body's control of energy homeostasis.

Protocol

Use sterile techniques to perform all the steps of the protocol in a laminar flow cell culture hood. See Table of Materials for details about all reagents and equipment. The differentiation protocol described below is suitable for human adipose-derived stem cells and may differ among other cell types and their origin. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of ADSCs for differentiation

1. Prepare ADSCs growth medium (ADSCs GM): Supplement Dulbecco's Modified Eagle Medium (DMEM) containing 1 g/L glucose with 10% fetal bovine serum (FBS), 2 mM glut....

Results

The method described herein allows differentiation of human ADSCs into mature adipocytes, followed by enhanced lipid accumulation, mimicking the hypertrophic cells observed under obesity conditions. The time course of the differentiation of human ADSCs is shown in Figure 1. At day 0, when cells reach 100% confluency (Figure 1A), ADSCs exhibit elongated, spindle-like morphology. At this time point, the differentiation protocol can be applied using DM containing a.......

Discussion

The present protocol is a replicable two-step procedure for the differentiation of ADSCs, followed by incubation with FAs to achieve a cellular model mimicking obesity. The critical step determining its effectiveness is the number of passages of ADSCs after its isolation. Multiple passages lead to a decrease in the differentiation potential of these cells¹⁰.

During adipocyte differentiation, ADSCs are shifted from dividing cells to growth-arrested adipocytes. Growth arr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-Isobutyl-1-methylxanthine (IBMX)	Merck	I5879
0.25% trypsin/0.05% EDTA pH 7.2	IITD PAN, Wrocaw, Poland	20-500
96-well plate	Thermo	130188
antibiotics-antimycotic solution	Sigma-Aldrich	A5955
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A3294
Cell counter	N/A	N/A
Centrifuge	N/A	N/A
Dexamethasone	Sigma-Aldrich	D4902
Dulbecco's Modified Eagle Medium (DMEM)	IITD PAN, Wrocaw, Poland	10-500	containing 1 g/L glucose
Dulbecco's Modified Eagle Medium:Nutrient Mixture F12	Gibco	11320-033
Ethanol	Chempur	113964200#1L
Fetal bovine serum (FBS)	Gibco	A5256701
hADSC Human Adipose-Derived Stem Cells	Lonza	PT-5006
Incubator	N/A	N/A
Indomethacin	Sigma-Aldrich	I8280-5G
Insulin	Sigma-Aldrich	I9278
Inverted microscope	N/A	N/A
L-glutamine	Sigma-Aldrich	25030-024
Linoleic acid sodium salt	Sigma-Aldrich	L8134
LipidSpot fluorescent	Biotium	70065
Phosphate-buffered saline (PBS)	Thermo	10010023
Soduim oleate	Sigma-Aldrich	O7501
Soduim palmitate	Sigma-Aldrich	P9767
T75 cell culture flask	Thermo	130190
Trypan blue	Sigma-Aldrich	95896HJ