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

Podsumowanie

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).

Streszczenie

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.

Wprowadzenie

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 a protocol that maintains the integrity of the cells.

Culture of primary cells and specific preadipocyte subpopulations gives a reductionist approach to in vivo growth and maintains equivalent cellular genetic makeup⁵, although working time with these cells is limited due to deterioration with aging, or senescence⁶. Preadipocytes from various adipose depots, including subcutaneous and omental depots, also demonstrate differences in proliferation⁷, which emphasizes the importance of collecting cells from specific anatomical sites. Precursor cells from non-PVAT white adipose depots have been characterized in previous studies^7,8,9, but less is known about PVAT APC phenotypes.

The techniques described here allow for the analysis of specific and defined APC populations with minimal impact on their morphology, viability, and potential to proliferate and differentiate. Magnetic-activated Cell Sorting (MCS) is amenable to downstream applications, such as culture, as the beads dissolve without altering the cell. MCS is also economical, and once the antibody concentrations have been standardized, the need for flow cytometry assays is minimal. In vitro studies with PVAT precursors can also give a glimpse of the potential that these primary cells may have.

Protokół

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 kept sterile and at 4 °C.
2. Prepare Collagenase Type 1 Solution: 1 mg/mL in KRBB with 4% Bovine Serum Albumin (BSA). This solution should be kept at 37 °C and is stable for 4 h.
3. Prepare Erythrocyte Lysis Buffer Solution: 154 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM EDTA. Keep at 4 °C for up to one month.
4. Prepare MCS Blocking Buffer: DMEM/F12 Media base, 10% Fetal Bovine Serum (FBS), 5% normal donkey serum, 40 µL/mL F(ab) Fragment Donkey Anti-Rat IgG. Keep at 4 °C for up to one month.
5. Prepare MCS Buffer Solution: Phosphate-Buffered Saline (PBS, pH = 7.5), 0.5% BSA, and 2 mM EDTA. Keep at 4 °C for up to one month. De-gas solution by heating to 37 °C in a glass container and then applying a vacuum for15 s. Leave unused buffered sealed.
6. Prepare Stromal Vascular Fraction (SVF) Basal Media: Dulbecco's-Modified Eagles Medium (DMEM):F12, 15% Fetal Calf Serum (FCS), 1% antibiotic/antimycotic (10,000 units/mL penicillin, 10,000 µg/mL streptomycin, 25 µg/mL amphotericin B), 44.05 mM sodium bicarbonate, 100 µM ascorbic acid, 33 µM biotin, 17 µM pantothenate, 2 mM L-glutamine, and 20 mM HEPES. Keep sterile and at 4 °C for up to 2 weeks.
7. Prepare APC Media: Basal Media with additional growth factors including epidermal growth factor 10 ng/mL), leukemia inhibitory factor (10 ng/mL), platelet-derived growth factor BB (10 ng/mL), and basic fibroblast growth factor (5 ng/mL). Keep sterile and at 4 °C for up to 2 weeks.
8. Prepare APC Induction Media: APC Media with 10% FBS, 2.5 µg/mL insulin, 0.5 mM 2-isobutyl-1-methylaxanthine (IBMX), 1 µM dexamethasone, and 200 pM T3 (triiodothyronine thyroid hormone). Keep sterile and at 4 °C for up to 2 weeks.

2. Adipocyte Progenitors Isolation

1. Anesthetize rat according to institutional guidelines. Place the rat in dorsal recumbency. Confirm depth of anesthesia via a toe-pinch and the loss of reflex response to this painful stimulus.
   NOTE: This protocol uses 10-week-old Sprague Dawley rats and 70 mg/kg of pentobarbital delivered via an intraperitoneal injection.
2. Make a vertical midline incision with scissors along the sternum in the thoracic area and to the perineal area. Access the abdominal cavity and expose the superior mesenteric artery, the small mesenteric resistance vessels (mPVAT) and the thoracic aorta (aPVAT).
   1. Sever all connections to the mesentery and aorta and remove vessels from animal. Isolate PVAT by using a dissecting microscope and Petri dish filled with KRBB to view vessels and isolate PVAT.
   2. In this experiment, collect gonadal (GON) adipose to represent a non-PVAT adipose depot. Place isolated fat pads on ice in KRBB with 10 mM HEPES (pH = 7.4).
   3. In a biosafety hood, transfer about 50 mg of tissue to a 1.7 mL tube with 1 mL of collagenase type I solution and mince with tissue scissors (1 - 3 mm pieces).
3. Digest samples by incubating at 37 °C in a rotisserie incubator (or incubator with an orbital shaker) for 1 h. In a biosafety hood, sequentially filter digested material through 100 and 40 µm cell strainers into a 50-mL tube. Centrifuge resulting filtrate at 4 °C for 10 min at 300 × g.
   NOTE: All steps in the protocol from here forward are to be performed in a biosafety hood to keep cells sterile.
4. Pour off supernatant and resuspend pellets containing the SVF cells in 1 mL of 1X Erythrocyte Lysis Buffer Solution and transfer to a 1.7 mL microfuge tube. Incubate cells for 5 min at RT protected from light and centrifuge at 4 °C for 5 min at 300 x g.
5. Pour off supernatant and resuspend remaining cell pellet in SVF Basal Media. Collect a 20 µL sub-sample to count live cells with Trypan Blue Solution.
   NOTE: The number of SVF cells that can be isolated will vary by site. Average numbers of SVF harvested per mg of tissue are: aPVAT = 5.0 ±2.0x10³, mPVAT = 1.04 ±0.62x10⁴, GON = 2.4 ±1.2x10⁵.

3. Magnetic-activated Cell Sorting

NOTE: Isolate APC from SVF based on CD34 and PDGFRα cell surface markers by performing all steps at 4 °C.

1. Spin cells for 5 min at 300 x g. Pour off supernatant and resuspend the cell pellet in MCS Blocking Buffer at 1 x 10⁶ cells/mL and incubate for 20 min.
   NOTE: Cell suspensions of 1 x 10⁶ - 2 x 10⁸ cells/mL can be separated effectively.
2. Incubate cells with 5 µL of FITC-conjugated Mouse anti-CD34 (1 µg/1 x 10⁶ cells) for 30 minutes at 4 °C. Spin cells for 5 min at 300 x g and 4 °C.
   1. Incubate with 4 µL of anti-FITC microbeads and 96 µL of MCS buffer (total volume of 100 µL) for 5 min at 4 °C in the dark to separate CD34⁺ and CD34^- cells.
3. Attach magnetic separator to the stand and place the MultiSort (MS) Column, with the column wings to the front, into the separator. Place a 5 mL collection tube under the MS Column in the upper tube holder.
4. Prepare MS Column by rinsing with MCS Buffer Solution. Apply 500 µL of MCS Buffer on top of the column and let the buffer run through. Discard effluent and change collection tube.
5. Load antibody labeled cell suspension onto the prepared MS Column. Collect flow-through containing unlabeled cells.
6. Wash MS Column with 500 µL of degassed MCS Buffer 3x. Collect unlabeled cells that pass through and combine with the flow through from previous step.
7. Remove MS Column from the magnetic separator and place it on a new collection tube. Pipette 1 mL of MCS Buffer onto the MS Column. Immediately flush out fraction with the magnetically labeled cells by firmly, but slowly, applying the plunger supplied with the column as to not allow excess gas into the column.
   NOTE: Isolated cell numbers will vary by site. Average numbers of CD34⁺ APC isolated from the SVF population per mg of tissue are: aPVAT = 2.6 ±0.43x10², mPVAT = 9.6 ±1.4x10², GON = 1.3 ±0.22x10³.
8. Spin cells for 5 min at 300 × g and 4 °C. Incubate the CD34⁺ fraction collected in 10 µL of a 1:200 solution/1 x 10⁶ cells Rabbit anti-PDGFRα for 30 min at 4 °C.
   1. Centrifuge cells again for 5 min at 300 x g and 4 °C. Incubate labeled cells with 4 µL of anti-rabbit IgG microbeads and 96 µL of MCS buffer by repeating steps 3.3 through 3.7 to isolate.
      NOTE: Isolated cell numbers will vary by site. Average numbers of PDGFRα⁺ APC isolated from the CD34⁺ population per mg of tissue are: aPVAT = 2.4 ±0.64, mPVAT = 8.4 ±2.4, GON = 10.4 ±1.9, which is 0.5 - 10% of the previously isolated population.

4. Cell Culture and Adipogenesis Induction

1. Culture the SVF and APC in 6-Well tissue culture plates in basal media with replacement every 2 days. After 3 serial passages, plate in black 96-Well tissue culture plates at 1x10² cells/well for proliferation assays, which are evaluated at 8, 24, 48, and 96 h, and at 50,000 cells/well in 24-well plates or 10,000 cells/well in 48-Well tissue culture plates for adipogenesis assays, which are qualitative and quantitative.
2. Supplement APC basal media for 48 h post-confluency and prior to induction with bone morphogenic protein 4 (3.3 nmol/L) as indicated¹⁰ for differentiation. Induce cells after 48 h of 100% confluency (day 0) using the APC Induction Media to incubate the cells.
3. After 48 h, change media to maintain cells in APC Induction media without IBMX and dexamethasone, for 14 days with media changes every 48 h.
4. MCS isolation validated by FACS.
   1. Take a 50 µL (50,000 cell) sub sample of magnetically separated cells and wash with FACS solution.
   2. Centrifuge cells and resuspend in 100 µL of a 1:1,000 solution of donkey anti-rabbit IgG Dylight 405 to label the PDGFRα⁺ cells. Incubate for 30 min protected from light and at 4 °C.
   3. Wash cells and resuspend in 200 µL of 2% formaldehyde solution until time for analysis using 488 nm (FITC) and 405 nm (Dylight 405) filters on a flow cytometer.
      NOTE: Lipid accumulation by cultured cells is assessed quantitatively using a lipophilic adipogenesis fluorescence assay in a microplate reader measuring fluorescence and using preadipocytes as calibrators for lipid accumulation. Lipid accumulation is also measured qualitatively by lipid dye staining and imaging performed on an inverted microscope equipped with a camera, making the percentage of total cells that do or do not contain lipid observable. Any plate reader that is able to measure fluorescence with excitation at 485 nm and emission at 572 nm is suitable for analysis as well as any microscope with a camera capable of capturing digital images.

Wyniki

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 ...

Dyskusje

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...

Materiały

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