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

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 adipocytes^1,2,3. The phenotype of such cells is a subject of controversy, with evidence supporting endothelial cells, smooth muscle/pericyte, or a spectrum of intermediate phenotypes^4,5,6,7. The scope of developing this methodology was to test the adipogenic potential of CD34+CD31+ endothelial cells isolated from different fat depots from obese humans. Other studies in the literature are focusing on the adipogenic potential of the total stromal vascular fraction or of the known adipocyte progenitors^2,8,9. Since currently existing technologies can target specifically adipose tissue endothelial cells for drug delivery¹⁰, understanding the potential of such cells to undergo adipogenic induction towards white or beige adipocytes is important for future targeted therapies.

Different groups reported the combination of CD31 and CD34 markers as surrogates to isolate endothelial cells from human adipose tissue^11,12,13. Typically, the isolation is performed using two sequential steps and a positive selection using magnetic beads. In this report, immunoseparation using CD34+ magnetic beads combined with CD31 plastic beads was utilized. We found this technique superior to the sequential magnetic immunoseparation with respect to the preservation of typical cobblestone endothelial morphology. Also, we were able to generate enough cells required for the expansion and adipogenic induction starting from as little as 1–2 g of fat. A small sample biopsy of subcutaneous fat is enough to produce the required quantity of cells for downstream applications. This aspect is potentially important, particularly if this method will be utilized for screening for a responsiveness to adipogenic induction in human subjects.

Unlike other systems reported in the literature, this method utilizes only two ingredients for the adipogenic induction of the CD34+CD31+ cells: a PPARγ agonist—rosiglitazone—and human insulin. Importantly, the amount of insulin used falls within the normal/high range of circulating post-absorptive insulin in humans¹⁴. The degree of responsiveness to insulin of the cells in vitro, measured by Akt phosphorylation, does not correlate with their ability to respond to the induction cocktail. Interestingly, using this induction cocktail and experimental conditions, a mix of white and beige/brown cells were obtained as determined by the size and numbers of intracellular lipid droplets and the expression of molecular markers. This straightforward and cost-effective induction protocol along with the quantitative evaluation of the phenotype of the responder cells (white vs. beige) allows for a screening of agents that can potentially alter the balance of differentiated beige:white adipocytes.

This method also provides a translational approach for understanding the underlying mechanisms of adipogenesis of vascular endothelial progenitors in human adipose tissue. Using this specific isolation/differentiation technique, investigators can interrogate various pathways responsible for adipogenesis in a subset of vascular endothelial cells from various fat depots in lean and obese humans.

Protocol

The Institutional Review Board Committee at Eastern Virginia Medical School approved the research and collection of human adipose tissue samples used in the study. Informed written consent was collected from the patients.

1. Preparation of Buffers, Media, and Instruments

1. Prepare a Krebs Ringer Bicarbonate-Buffered Solution (KRBBS): 135 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium sulfate, 0.4 mM potassium phosphate dibasic, 5.5 mM glucose, 1 mM adenosine, 0.01% antibiotic/antimycotic mix (50 µg/mL of penicillin, 50 µg/mL of streptomycin, 30 µg/mL of gentamicin, 15 ng/mL of amphotericin) and 10 mM HEPES (pH = 7.4). This solution is prepared fresh each time for the tissue collection and digestion.
2. Prepare a fresh collagenase solution: 1 mg/mL of collagenase, type 1, in KRBSS; make 3 mL/g of fat. Pre-warm the collagenase solution at 37 °C in a water bath prior to the tissue digestion.
3. Prepare a CD34 Cell Isolation Buffer: 2% fetal bovine serum (FBS) and 1 mM EDTA in sterile phosphate-buffered saline (PBS).
4. Prepare Adipogenesis Media: DMEM/F12, 5% FBS, 50 µg/mL of penicillin/streptomycin, 1.25 µL/mL of human insulin (5 µg/mL or 144 mU/mL), and 0.36 µL/mL of 2.8 mM rosiglitazone (1 µM).
5. Prepare a 0.3% Oil Red O (ORO) stock solution (weight/volume) by dissolving 0.3 g of ORO into 100 mL of isopropanol.

2. Adipose Stromal Vascular Fraction Isolation

NOTE: The study included a cross-sectional cohort of morbidly obese type 2 diabetic (T2D) and non-diabetic subjects, aged 18–65 years, undergoing bariatric surgery at the Sentara Metabolic and Weight Loss Surgery Center (Sentara Medical Group, Norfolk, VA). Exclusion criteria included an autoimmune disease including type 1 diabetes mellitus, conditions requiring chronic immunosuppressive therapy, anti-inflammatory medications, thiazolinendiones, active tobacco use, chronic or acute infections, or a history of malignancy treated within the last 12 months. T2D was defined as a fasting plasma glucose of 126 mg/dL or greater, a glucose of 200 mg/dL or greater after a 2 h glucose tolerance test, or the use of antidiabetic medications.

1. Collect human omental (OM) and subcutaneous (SC) adipose tissue (AT) from human subjects undergoing bariatric surgery.
2. Keep the OM and SC AT in separate vials containing Hank’s Buffered Salt Solution with 50 µg/mL of penicillin/streptomycin at room temperature immediately after tissue extraction.
3. Carefully clean and remove fibrotic and cauterized sections of the tissue by using 70% ethanol swabbed scissors and tweezers when the sample arrives at the lab after the tissue extraction.
4. Weigh the adipose tissue and partition it into 5 g (or less) aliquots for the collagenase digestion.
5. Finely mince 5 g of the AT in 5 mL of a collagenase solution in a scintillation vial at room temperature, using two pairs of scissors.
6. Add an additional 10 mL of collagenase solution to the minced tissue for 15 mL total.
7. Digest the samples for 1 h, at 37 °C, in a water bath with continuous shaking.
8. Cut the tip off a 20 mL syringe to make a blunt end.
9. Cut a 9 cm x 9 cm square from a 250 µm mesh and push it halfway into a 50 mL conical tube using the blunt end syringe.
10. Pour the samples into the 20 mL blunt end syringe and filter through the 250 µm nylon mesh into the 50 mL conical tube to separate adipocytes and stromal vascular cells from undigested tissue.
    NOTE: Do not use more than 5 g of AT per 50 mL conical tube, as the mesh will get clogged due to excess fibrotic undigested material.
11. Wash out the scintillation vial with 10 mL of KRBBS and pour it through the filter to collect residual cells.
12. Incubate the filtered samples for 5 min at room temperature.
    Note: This step is important to allow for the adipocytes to float at the top of the tube and some of the stromal vascular cells to settle on the bottom of the tube.
13. Utilize a 20 mL syringe and a 20 G x 6 in pipetting needle to remove the stromal vascular fraction layer and transfer it to a clean 50 mL conical tube.
14. Add 10 mL of KRBBS and allow the samples to sit on the bench for 5 min, at room temperature.
15. Repeat steps 2.12–2.14 twice.
    Note: These steps will ensure that there is no contamination of the stromal vascular cells with the floating adipocytes.
16. Keep the stromal vascular fractions from both depots on ice for further processing, as described in the steps below.
    Note: Do not leave the cells on ice for more than 1 h, as this may have an impact on the cell viability. The adipocytes can be flash-frozen in liquid nitrogen for long-term storage or used fresh for experiments.

3. Isolation of Adipose Tissue Endothelial Cells

1. Spin the stromal vascular fractions (SVF) at 500 x g for 5 min, at 4 °C.
2. Remove the samples from the centrifuge and carefully pour off the supernatant; keep the pellet containing the SVF cells.
3. Gently resuspend the cell pellets in 5 mL of PBS.
   NOTE: If more than 5 g of an AT depot was processed into multiple aliquots (see step 2.4), pool the cell pellets together.
4. Spin the samples at 500 x g for 5 min, at 4 °C.
5. Remove the supernatant, resuspend the cells in 1 mL of PBS, and count the cells.
   NOTE: Here, an automatic cell counter was used for cell counting. The cell viability was obtained by mixing 12 µL of cell suspension with 12  µL of an acridine orange/propidium iodide (AO/PI) solution (see Table of Materials). The average number of cells per gram of AT for each depot is: (a) OM Depot: 1.69 x 10⁵ ± 4.51 x 10⁴; (b) SC Depot: 1.24 x 10⁵ ± 6.45 x 10⁴. The viability of the cells from both depots was >90%.
6. Pellet the samples at 500 x g for 5 min, at 4 °C.
   NOTE: A CD34 immunomagnetic selection protocol (see Table of Materials) was adapted for steps 3.7–3.15.
7. Resuspend the pellet in 100 µL of CD34 isolation buffer.
   NOTE: The total cell counts require the following re-suspension volumes: (a) <2 x 10⁷ cells: resuspend the pellet in 100 µL; (b) 2 x 10⁸–5 x 10⁸ cells: resuspend the pellet in 1 mL. In steps 3.9–3.16, the volumes of the reagents used were scaled down for <2 x 10⁷ cells.
8. Add 10 µL of CD34 cocktail and incubate the sample for 15 min, at room temperature.
9. Add 5 µL of magnetic beads and incubate the sample for 10 min, at room temperature.
10. Add 2.5 mL of CD34 isolation buffer to each sample and place the samples into the magnet, for 5 min, at room temperature.
11. Invert the magnet for 5 s to pour off the isolation buffer.
12. Remove the samples from the magnet and repeat steps 3.10 and 3.11 4x.
13. Remove the tube from the magnet and add 2 mL of CD34 isolation buffer.
14. Pellet the cells at 500 x g for 5 min, at 4 °C.
15. Resuspend the cell pellets in 1 mL of CD34 isolation buffer and count the cells.
    NOTE: Here, the average number of cells per gram of AT is: (a) OM Depot: 4.75 x 10⁴ ± 3.36 x 10⁴; (b) SC Depot: 1.06 x 10⁴ ± 9.53 x 10³. A CD31 plastic immunobead protocol was adapted for steps 3.16–3.34 (see Table of Materials).
16. Pellet cells at 500 x g for 5 min and resuspend the pellet in 250 µL of Buffer B and 250 µL of wash buffer.
17. Thoroughly resuspend the CD31 beads by vortexing the tube.
18. Add 20 µL of CD31 beads.
    NOTE: Due to total cell counts >1 x 10⁶, the volume was scaled down according to the manufacturer’s input.
19. Incubate the sample with rocking for 30 min, at room temperature.
20. Attach a strainer to a sterile 50 mL conical tube.
    NOTE: The larger opening of the strainer must be on top.
21. Prior to the cell separation, add 1 mL of wash buffer to equilibrate the strainer. Apply the mixture generated under step 3.16 to the strainer.
22. Wash the strainer with 5 mL of wash buffer in a circular motion for a total volume of 20 mL. Attach the connector to a sterile 50 mL conical tube and close the luer-lock.
23. Attach the strainer to the connector. Add 1 mL of wash buffer along the wall of the strainer. Add 1 mL of activated buffer D along the wall of the strainer. Swirl the sample gently and incubate it for 10 min, at room temperature.
24. Add 1 mL of wash buffer. Separate the cells from the beads by pipetting up and down 10x.
    NOTE: Avoid generating air bubbles.
25. Open the luer-lock and allow the detached cells to flow into the 50 mL conical tube. Wash the strainer 10x with 1 mL of wash buffer each time.
26. Discard the connector and strainer and centrifuge the samples at 300 x g for 10 min, at 4 °C. Resuspend the cell pellet in 2 mL of complete EGM-2 Media for culture.
    Note: The average number of cells per gram of AT at this point is: (a) OM Depot: 5.92 x 10³ ± 4.27 x 10³; (b) SC Depot: 4.12 x 10³ ± 2.1 x 10³. The viability of cells from both depots was over 90%.

4. Induction of Adipogenesis in Isolated Endothelial Cells

1. Grow the cells in 6-well tissue culture-treated plates with complete EGM-2 media in a 37 °C, 5% CO₂ incubator. Replace the media every 3–4 days until the cells reach 80–90% confluence.
   NOTE: The population doubling is between 2–3 days.
2. Expand the cells by plating them on 100 mm Petri dishes and split the cells 1:3 once. At this stage, the cells are on passage 2.
3. Count the cells using an automatic cell counter (see Table of Materials).
4. Seed approximately 100–200,000 cells into 6-well plates ensuring duplicate wells for each depot and culture them in complete EGM-2 media for 2 days.
5. Aspirate off any growth media and replace it with 2 mL of DMEM/F12 with 5% FBS and 50 µg/mL of penicillin/streptomycin for the control cells and replace it with 2 mL of Adipogenesis media (see step 1.4) for the treatment cells.
6. Incubate the cells at 37 °C in a humidified 5% CO₂ incubator for 13 days, replacing the respective media every 3 to 4 days.
   NOTE: Cells will begin to accumulate lipids after 4 days of treatment.
7. Conduct ORO and Nile red staining according to published methods^15-17.
8. To isolate cells for an RNA extraction, remove the media from the 6-well induction plates and wash it with 1 mL of sterile PBS.
9. Add 350 µL of a guanidium thiocyanate-phenol-chloroform solution (see Table of Materials) to each well and incubate it at room temperature for 5–10 min.
10. Scrape the cells off the plate using a cell scraper and transfer the cells to a 1.5 mL microcentrifuge tube.
    Note: The samples can be stored in the -80 °C freezer for RNA extraction and further processing at a later date.
11. Extract mRNA from the samples using an adapted RNA extraction protocol (see Table of Materials).
12. Perform a real-time polymerase chain reaction (RT-PCR) to assess the gene expression using the following probes: adiponectin, UCP-1, and CIDEA (see Table of Materials).
    NOTE: Here, RT-PCR procedures were carried out using the following standard protocol: denaturation (95 °C; 15 s), annealing, and extension (60 °C; 1 min) for 40 cycles. The samples that expressed genes with C_T values >35 were considered undetectable.

Results

Our protocol aims to provide an in vitro approach to determine the adipogenic potential of CD34+CD31+ vascular cells from different depots of human adipose tissue. A simplified flowchart diagram is shown in Figure 1A. The first step using a positive selection of CD34 expressing cells results in > 95% CD34+ cells in the population of the freshly isolated cells (Figure 1A). Importantly, this marker is lost after the ce...

Discussion

The focus of this paper is to provide a methodology for the isolation, expansion and adipogenic induction of CD34+CD31+ endothelial cells from visceral and subcutaneous depots of human adipose tissue.

Methodologies have been reported for the isolation of endothelial cells from various vascular beds of rodents or humans that involve primarily techniques using CD31 antibodies either fluorescently labeled or coupled to magnetic beads^18,19...

Materials

Name	Company	Catalog Number	Comments
Large Equipment
Biosafety Cabinet	Nuaire	nu-425-400
Cell Culture Incubator	Thermo-Fisher Scientific	800 DH
Water Bath	Forma Scientific	2568	Reciprocal Shaker
RT-PCR Machine	BIO-RAD	CFX96-C1000
Electrophoresis Box	BIO-RAD	Mini PROTEAN 3 Cell
Transblot Box	BIO-RAD	Mini Trans-Blot Cell
Electrophoresis Power Supply	BIO-RAD	PowerPac Basic
ELISA Reader	Molecular Devices	SpectraMax M5
Blot Reader	LI-COR	Odyssey	Near Infrared
Refrigerated Centrifuge	Eppendorf	5810 R
Tabletop Centrifuge	Eppendorf	MiniSpin Plus
Fluorescent Microscope	Olympus	BX50
Inverted Microscope	Nikon	TMS
KRBSS Buffer
HEPES	Research Products International	H75030
Sodium bicarbonate	Sigma-Aldrich	792519
Calcium chloride dihydrate	Sigma-Aldrich	C7902
Potassium phosphate monobasic	Sigma-Aldrich	P5655
Magnesium sulfate	Sigma-Aldrich	M2643
Sodium chloride	Sigma-Aldrich	746398
Sodium phosphate monobasic monohydrate	Sigma-Aldrich	S9638
Potassium chloride	Sigma-Aldrich	P9333
Glucose	Acros Organics	410950010
Adenosine	Acros Organics	164040250
Bovine Serum Albumin	GE Healthcare Bio-Sciences	SH30574.02
Penicillin/Streptomycin	Thermo-Fisher Scientific	15070063
Tissue Digestion
20 mL Syringe	Global Medical	67-2020
Nylon Mesh, 250 µm	Sefar	03-250/50
Pipetting Needles	Popper	7934
Fine Scissors	Fine Science Tools	14058-11
Tissue Forceps	George Tiemann & Co	160-20
Collagenase, Type I	Worthington Biochemical	LS004196
Petri Dishes, 100 mm	USA Scientific	5666-4160	TC Treated
Eppendorf Tubes, 1.5 mL	USA Scientific	1615-5500
Conical Tubes, 15 mL	Nest Scientific	601052
Conical Tubes, 50 mL	Nalgene	3119-0050
Scintillation Vials	Kimble	74505-20	Tissue Dicing
Cell Isolation
Cellometer	Nexcelom	Auto 2000
Cellometer Slides	Nexcelom	CHT4-SD100-002
Cellometer Viability Stain	Nexcelom	CS2-0106-5mL	Acridine Orange/Propidium Iodine
Anti-CD34 Magnetic Beads	StemCell Technologies	18056	Kit
EasySep Magnet	StemCell Technologies	18000
Anti-CD31 Plastic Beads	pluriSelect USA	19-03100-10
pluriSelect 10x Wash Buffer	pluriSelect USA	60-00080-10
pluriSelect Connector Ring	pluriSelect USA	41-50000-03
pluriSelect Detachment Buffer	pluriSelect USA	60-00046-12
pluriSelect Incubation Buffer	pluriSelect USA	60-00060-12
pluriSelect S Cell Strainer	pluriSelect USA	43-50030-03
Cell Culture
6-well Plates	USA Scientific	CC7682-7506	TC Treated
4-well chambered slides	Corning Life Sciences	354559	Fibronectin coated
4-well chambered slides	Thermo-Fisher Scientific	154526PK	Uncoated glass
Human Adipose Microvascular Endothelial Cells (HAMVEC)	Sciencell Research Laboratories	7200	Primary cell line
Endothelial Cell Media (ECM)	ScienCell Research Laboratories	1001	Complete Kit
DMEM/F12 Basal Media	Thermo-Fisher Scientific	11320082
Fetal Bovine Serum (FBS)	Rocky Mountain Biologicals	FBS-BBT
Insulin	Lilly	U-100	Humalog
Rosiglitazone	Sigma-Aldrich	R2408
Cell Analysis
Oil Red O Dye	Sigma-Aldrich	O0625	Prepared in isopropanol
96 well plates	USA Scientific	1837-9600
96 well PCR plates	Genesee Scientific	24-300
RNA Extraction	Zymo Research	R2072	Kit
cDNA Synthesis	BIO-RAD	1708841	Supermix
JumpStart PCR Polymerase	Sigma-Aldrich	D9307-250UN	Hot start, with PCR Buffer N
Magnesium Chloride Solution	Sigma-Aldrich	M8787-5ML	3 mM final in PCR reaction
dNTPs	Promega	U1515
TaqMan AdipoQ	Thermo-Fisher Scientific	Hs00605917_m1
TaqMan CIDEA	Thermo-Fisher Scientific	Hs00154455_m1
TaqMan RPL27	Thermo-Fisher Scientific	Hs03044961_g1
TaqMan UCP1	Thermo-Fisher Scientific	Hs00222453_m1
BCA Assay	Sigma-Aldrich	QPBCA-1KT	Kit
Bis-acrylamide	BIO-RAD	1610146	40% stock solution
Ammonium Persulfate	BIO-RAD	1610700
TEMED	BIO-RAD	1610800
Tris	Sigma-Aldrich	T1503
Glycine	BIO-RAD	1610718
Sodium Dodecal Sulfate	Sigma-Aldrich	L3771
EDTA	Fisher Scientific	S311-100
Bromophenol Blue	Sigma-Aldrich	B8026
Blot Membrane	EMD Millipore	IPFL00010
Methanol	Fisher Scientific	A452-SK4
Odyssey Blocking Buffer, Tris	LI-COR	927-50000
Anti-AKT antibody	Cell Signaling Technology	2920S	Mouse monoclonal
Anti-pAKT antibody	Cell Signaling Technology	9271S	Rabbit polyclonal
Anti-UCP1 antibody	Abcam	ab10983	Rabbit polyclonal
Anti-Mouse IgG antibody	LI-COR	926-68070	Goat Polyclonal, IRDye 680RD
Anti-Rabbit IgG antibody	LI-COR	926-32211	Goat Polyclonal, IRDye 800CW
Anti-Rabbit IgG antibody	Jackson ImmunoResearch	111-025-003	Goat Polyclonal, TRITC
Phosphate Buffer Saline	Thermo-Fisher Scientific	10010049
37% Formaldehyde Solution	Electron Microscopy Sciences	15686	4% solution for cell fixation
Normal Goat Serum	Vector Laboratories	S-1000	10% blocking solution
Triton X-100	Sigma-Aldrich	X-100	0.1% permeabilization solution
DAPI	Thermo-Fisher Scientific	D1306
Calcein AM	Thermo-Fisher Scientific	65-0853-39	Cell fluorescent visualization
Matrigel Basement Membrane Matrix	Corning Life Sciences	356231	Growth factor reduced
DiI labeled Acetylated LDL	Thermo-Fisher Scientific	L3484