Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Summary

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

Abstract

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.

Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Introduction

Perivascular Adipose Tissue (PVAT), due to its close proximity to blood vessels, is a major paracrine signaling component in vasculature function¹. Expansion of this adipose tissue is dependent on the phenotype of the Adipocyte Progenitor Cells (APC) present^2,3. Isolation of cells from adipose tissues is difficult as primary adipocytes are fragile, buoyant, and range in size. Certain isolation techniques can also alter cell phenotype and morphology by increasing inflammatory protein synthesis and reducing adipogenic gene expression⁴, emphasizing the importance of....

Protocol

All procedures described in this paper follow guidelines established by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University. All buffers and medias should be protected from light.

1. Preparation of Buffers, Media, and Instruments

1. Prepare Krebs Ringer Bicarbonate-Buffered solution (KRBB): 135 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium sulfate, 0.4 mM potassium phosphate dibasic, 5.5 mM glucose, 1% antibiotic/antimycotic (10,000 units/mL penicillin, 10,000 µg/mL streptomycin, 25 µg/mL amphotericin B), and 10 mM HEPES (pH = 7.4). This solution is stable for 3 weeks when ke....

Results

Proliferative capacity of preadipocytes and adipogenic potential of adipocyte precursors are characteristics that are maintained in vitro¹¹. In vitro proliferation of isolated SVF and APC from aPVAT, mPVAT, and GON of male rats was evaluated at 8, 24, 48, and 96 h after plating using a quantitative DNA assay. No site differences in SVF expansion rate were observed at any time point except for the APC from aPVAT, which had less proliferation by 96 .......

Discussion

The central focus of the present experiment is the isolation, expansion, and adipogenic induction of APC from PVAT depots. Here we present a protocol for the isolation of APC based on the identification of cells expressing the surface markers CD34 and PDGFRα. These surface proteins were previously identified on APC with high proliferation rates and the potential to differentiate into white or brown adipocytes in various adipose depots^14,15. By selecting cell.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tissue Dissection
Dissecting Dishes	Handmade with Silicone
Culture Petri Dish	Pyrex		7740 Glass
Silicone Elastomer	Dow Corning	Sylgard 170	Kit
Braided Silk Suture	Harvard Apparatus	51-7615	SP104
Stereomicroscope MZ6	Leica		10447254
Stereomaster Microscope Fiber-Optic Light Source	Fisher Scientific	12562-36
Vannas Scissors	George Tiemann & Co	160-150
Splinter Forceps	George Tiemann & Co	160-55
Tissue Scissors	George Tiemann & Co	105-400
KRBB Solution
Sodium Chloride	Sigma-Aldrich	7647-14-5
Potassium Chloride	Sigma-Aldrich	7447-40-7
Magnesium Sulfate	Sigma-Aldrich	7487-88-9
Potassium Phosphate Dibasic	Sigma-Aldrich	7758-114
Glucose	Sigma-Aldrich	50-99-7
Antibiotic/Antimicotic	Corning	30-004-CI
HEPES	Corning	25-060-CI
Tissue Digestion
Collagenase Type 1	Worthington Biochemical	LS004196
Bovine Serum Albumin (BSA)	Fisher Scientific	9048-46-8
Red Blood Cell Lysis Buffer	BioLegend	420301	1X Working Solution
Water Bath	Thermo-Fisher Scientific	2876	Reciprocal Shaking Bath
Biosafety Cabinet	Thermo-Fisher Scientific	1385
Rotisserie Incubator	Daigger	EF4894C
100 µm Cell Strainer	Thermo-Fisher Scientific	22-363-549	Yellow
40 µm Cell Strainer	Thermo-Fisher Scientific	22-363-547	Blue
Hemocytometer	Cole-Parmer	UX-79001-00
Trypan Blue	Sigma-Aldrich	93595
Cell Isolation
OctoMACS Kit	Miltenyi Biotech	130-042-108
(DMEM):F12 Medium	Corning	90-090	Medium Base
Fetal Bovine Serum (FBS)	Corning	35016CV	USA Origins
Normal Donkey Serum	AbCam	AB7475
Anti-CD34	Santa Cruz	SC-7324	FITC-conjugated
Anti-PDGFRα	Thermo-Fisher Scientific	PA5-17623
Donkey Anti-Rabbit IgG	Jackson ImmunoResearch	712-007-003
Phosphate-Buffered Saline (PBS) 10X	Corning	46-013-CM	1X Working Solution
EDTA	Fisher Scientific	15575020
Cell Culture
CO2 Cell Incubator	Thermo-Fisher Scientific	51030285	Heracell 160i
6-Well Plates	Corning	3516	TC-Treated
48-Well Plates	Corning	3548	TC-Treated
96-Well Plates, Black Wall	Corning	353376	TC-Treated
Sodium Bicarbonate	Sigma-Aldrich	144-55-8	TC-Treated
Fetal Calf Serum (FCS)	Corning	35011CV	USA Origins
Ascorbic Acid	Sigma-Aldrich	50-81-7
Biotin	Sigma-Aldrich	58-85-5
Pantothenate	Sigma-Aldrich	137-08-6
L-Glutamine	Corning	61-030
Bone Morphogenic Protein 4 (BMP4)	Prospec Bio	CYT-081
Epidermal Growth Factor (EGF)	PeproTech	400-25
Leukemia Inhibitory Factor	PeproTech	250-02
Platelet-derived Growth Factor BB	Prospec Bio	CYT-740
Basic Fibroblast Growth Factor (bFGF)	PeproTech	450-33
Insulin	Corning	25-800-CR	ITS Solution
IBMX	Sigma-Aldrich	28822-58-4
Dexamethasone	Sigma-Aldrich	50-02-2
T3 (Triiodothyronine)	Sigma-Aldrich	6893-023
Cell Analysis
CyQUANT Proliferation Assay	Thermo-Fisher Scientific	C7026
AdipoRed Fluorescence Assay Reagent	Lonza	PT-7009
Oil Red O Lipid Dye Reagent	Sigma-Aldrich	O1391	In Solution
M1000 Microplate Reader	Tecan
Eclipse Inverted Microscope	Nikon
Digital Sight DS-Qil Camera	Nikon