Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Summary

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Abstract

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco's modified Eagle medium (HG-DMEM), β-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific β-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Introduction

Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are among the most widely used cell types for regenerative medicine^1,2. They are classified as adult stem cells and characterized by multilineage differentiation potential and self-renewal capacity³. MSCs can be isolated and obtained from various sources, including adipose tissue, bone marrow, peripheral blood, umbilical cord tissue and blood, hair follicles, and teeth^4,5.

The isolation of stem cells from adipose tissue is seen as....

Protocol

All experiments were carried out according to the approved guidelines, and all procedures were approved by the Ethical Committee of the Faculty of Pharmacy, The British University in Egypt (BUE), Cairo, Egypt. The Ad-MSC isolation protocol was adopted from Lopez and Spencer, with modifications¹⁵.

1. Isolation of Ad-MSCs from rat epididymal fat pads

1. Use male Sprague-Dawley rats weighing 250-300 g that are not more than 1 month of age (two per isolation). Anesthetize the animals, then euthanize them by cervical dislocation. Spray the euthanized animal with 70% ethanol. Excise the skin and t....

Results

Isolation and characterization of Ad-MSCs
As shown in Figure 2, the isolated cells from adipose tissue showed a heterogeneous population of rounded and fibroblast-like cells starting from the next day of isolation (Figure 2A). 4 days post isolation, the fibroblast cells started to increase in number and size and grow as a homogenous population by passage 1 (Figure 2B,C). These cells continued .......

Discussion

In this protocol, we managed to present a detailed protocol for the isolation of Ad-MSCs from rat epididymal fat and the differentiation of these Ad-MSCs into IPCs. In fact, rat epidydimal fat is an easily attainable source of adipose tissue for obtaining Ad-MSCs and does not require any special equipment, neither for collection nor for processing^15,26,27. The isolated Ad-MSCs showed excellent culture expansion and exhibited all.......

Materials

Name	Company	Catalog Number	Comments
Albumin, bovine serum Fraction V	MP Biomedicals
Alcian Blue 8GX	Sigma-Aldrich, USA	A3157
Alizarin Red S	Sigma-Aldrich, USA	A5533
Ammonium hydroxide	Fisher Scientific, Germany
Antibody for Rat CD90, FITC	Stem Cell Technologies	60024FI
Bovine serum albumin	Sigma Aldrich	A3912
Calcium Chloride	Fisher Scientific, Germany
CD105 Monoclonal Antibody, FITC	Thermo Fisher Scientific, Invitrogen, USA	MA1-19594
CD34 Polyclonal Antibody	Thermo Fisher Scientific, Invitrogen, USA	PA5-85917
Chloroform	Fisher Scientific, USA
Collagenase type I, powder	Gibco, Thermo Fisher, USA	17018029
D-Glucose anhydrous, extra pure	Fisher Scientific, Germany	G/0450/53
Dimethyl sulfoxide (DMSO)	Fisher Scientific, Germany	BP231-100
Dithizone staining	Sigma-Aldrich, USA	D5130
DMEM - High Glucose 4.5 g/L	Lonza, Switzerland	12-604F
DMEM - Low Glucose 1 g/L	Lonza, Switzerland	12-707F
DMEM/F12 medium	Lonza, Switzerland	BE12-719F
DNAse/RNAse free water	Gibco Thermo Fisher, USA	10977-035
Ethanol absolute, Molecular biology grade	Sigma-Aldrich, Germany	24103
Exendin-4	Sigma-Aldrich, Germany	E7144
Fetal Bovine Serum (FBS)	Gibco Thermo Fisher, Brazil	10270-106
Formaldehyde 37%	Fisher Scientific
Hydrochloric acid (HCl)	Fisher Scientific, Germany
Isopropanol, Molecular biology grade	Fisher Scientific, USA	BP2618500
L-Glutamine	Gibco Thermo Fisher, USA	25030-024
Magnesium Chloride (Anhydrous)	Fisher Scientific, Germany
Mesenchymal Stem Cell Functional identification kit	R&D systems Inc., MN, USA	SC006
Nicotinamide	Sigma-Aldrich, Germany	N0636
Oil Red Stain	Sigma-Aldrich, USA	O0625
Penicillin-Streptomycin-Amphotericin	Gibco Thermo Fisher, USA	15240062
Phosphate buffered saline, 1X, without Ca/Mg	Lonza, Switzerland	BE17-516F
Potassium Chloride	Fisher Scientific, Germany
Rat Insulin ELISA Kit	Cloud-Clone Corp., USA	CEA682Ra
Sodium Bicarbonate	Fisher Scientific, Germany
Sodium Chloride	Fisher Scientific, Germany
Sodium Phosphate Dibasic (Anhydrous)	Fisher Scientific, Germany
Sodium Phosphate Monobasic (Anhydrous)	Fisher Scientific, Germany
SYBR Green Maxima	Thermo Scientific, USA	K0221
Syringe filter, 0.2 micron	Corning, USA	431224
TRIzol	Thermo Scientific, USA	15596026
Trypan blue	Gibco Thermo Fisher, USA	15250061
Trypsin-Versene-EDTA, 1X	Lonza, Switzerland	CC-5012
Verso cDNA synthesis kit	Thermo Scientific, USA	AB-1453/A
β-mercaptoethanol	Sigma-Aldrich, Germany	M3148