Characterization of Adipocyte-Derived Extracellular Vesicle Secretion Using a CD63-GFP Reporter Mouse Model In Vivo and In Vitro

We study adipocyte-derived extracellular vesicle, EVs, to define their roles in adipose tissue, particularly how they regulate the crosstalk between mature adipocytes and their progenitor cells. There are currently no in vivo systems to study adipocyte EV secretions, and the existing purification protocols are not yet fully optimized. To begin, use sterile technique to excise inguinal adipose tissue from euthanized mice.

Place one gram of tissue into a two milliliter tube containing 0.5 milliliters of digestion buffer. Using sterile scissors, mince the tissue into pieces no larger than one millimeter. Suspend the minced tissue in 10 milliliters of digestion buffer containing 0.5 milligrams per milliliter Liberase and 50 units per milliliter DNase I.Incubate the sample at 37 degrees Celsius on an orbital shaker.

During incubation, gently invert the tube several times. When the solution appears cloudy and no visible tissue fragments remain, the digestion is complete. To isolate stromal vascular fraction or SVF, dilute the digested suspension with two volumes of digestion buffer.

Gently invert the tube three to four times to mix well. Pass the suspension through a sterile 100-micrometer filter to remove undigested tissue, then centrifuge the filtrate at 300g for five minutes at four degrees Celsius. Label the pellet as SVF.

Incubate the stromal vascular fraction in freshly prepared red blood cell lysis buffer on ice for five minutes, protected from light. Add two volumes of wash buffer and centrifuge at 400g for five minutes at four degrees Celsius. Resuspend the stromal vascular fraction in 80 microliters of buffer per gram of tissue and transfer the contents to a new tube.

Add 20 microliters of non-adipocyte progenitor depletion cocktail per gram of tissue. Incubate the sample for 15 minutes at two to eight degrees Celsius in the dark. Pass the sample through an LS column and collect the flow through containing the lineage negative cell fraction.

Centrifuge the flow through at 300g for five minutes at four degrees Celsius to obtain the adipocyte progenitor cells. Incubate 250 microliters of isolated mouse adipocyte progenitor cells mice onto collagen-coated eight-well chamber slides for six hours. Fix the cells with 4%paraformaldehyde for 10 minutes to preserve cellular and extracellular vesicle structures for imaging.

Next, wash the slides three times with PBS, then mount with anti-fade medium. Next, after counting the adipocyte progenitor cells, suspend them and dilute viability dye. Incubate on ice for 20 minutes, protected from light.

Wash the cells once with wash buffer and centrifuge. Suspend the cells in brilliant staining buffer so that the final concentration is 1 million cells per 100 microliters. Block nonspecific binding by adding Fc receptor blocking reagent at a one to 100 ratio and incubate.

Free-spin the SCA-1 antibody at 6000g for 10 minutes at four degrees Celsius. Add the SCA-1 antibody to the APC tube at a one to 100 dilution, then place the tube on ice, protected from light. Wash the cells twice with one milliliter of wash buffer, then centrifuge at 300g for five minutes at four degrees Celsius.

Suspend the cells in wash buffer for flow cytometry sorting, then acquire the data for SCA-1 positive and GFP positive cells on a spectral flow cytometer equipped with a violet and blue laser. To begin differentiation, coat dishes with 0.2%gelatin, for 30 minutes at room temperature. Seed freshly isolated APCs in supplemented DMEM/F12 medium and incubate until confluency.

Replace the medium from day one to day eight with MesenCult adipogenic differentiation medium. On day nine, wash the adipocytes twice with PBS, then replace the medium with mature adipocyte culture medium containing DMEM 1%BSA and 0.2%Primocin. To isolate adipocyte-secreted extracellular vesicles, first pass 15 milliliters of culture medium through a 40-micrometer filter, followed by a 30-micrometer filter.

Centrifuge the filtrate at 500g for five minutes then centrifuge the supernatant at 2000g for 10 minutes to remove any residual cells and debris. After passing the resulting supernatant through a 0.8-micrometer filter and transferring the filtrate into a 15-milliliter ultrafiltration unit, centrifuge at 4000g for 15 minutes to concentrate the medium. Load the concentrated sample onto a size exclusion chromatography column connected to an automatic fraction collector and collect fractions F1 through F8.To perform Western blot analysis, concentrate each fraction from 400 microliters to 25 microliters.

Pipette 50 microliters of radio immunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors to the concentrate. Add 75 microliters of two 2X SDS loading buffer to denature the sample and resolve proteins on a 4%to 12%Tris-Glycine gel. For nanoparticle tracking analysis, dilute extracellular vesicle samples with 0.1 micrometer filtered sterile PBS and mix gently.

Load the diluted sample into the NTA instrument equipped with a high sensitivity scientific complementary metal oxide semiconductor camera and a 532-nanometer green laser. Set detection for particles between 10 nanometers and 1, 000 nanometers, and within a concentration of 10 to the power of six to 10 to the power of nine particles per milliliter. Adipose progenitor cells from stop-flock-flock/CD63GFP mice showed no detectable green fluorescent protein puncta under confocal microscopy.

While cells from AdipCD63 mice displayed numerous distinct green fluorescent protein puncta throughout the cytoplasm. Flow cytometry analysis showed that 1.2%of skull 1 positive cells in the reporter mice expressed green fluorescent protein compared to 12.6%in AdipCD63-GFP mice. Western blotting of size exclusion chromatography fractions showed that extracellular vesicle markers CD63 and HSP70 were enriched in fractions F1 and F2.While markers of endoplasmic reticulum and mitochondria were absent, confirming vesical purity.

Nanoparticle tracking analysis revealed that adipocyte-derived extracellular vesicles ranged in size from 50 nanometers to 500 nanometers, and transmission electron microscopy confirmed the vesicles exhibited intact double membrane structures. Western blot confirmed knockout of STX4 in adipocytes. Confocal microscopy showed that CD63-GFP positive vesicles were dispersed throughout the cytosol in wild type adipocytes, but accumulated near the plasma membrane in STX4 knockout adipocytes.

The concentration of secreted adipocyte-derived extracellular vesicles was significantly reduced in STX4 knockout adipocytes compared to wild type. Western blot of equal protein amounts of intracellular vesicles showed reduced expression of CD63 and CD81 in STX4 knockout adipocytes compared to wild type. The protocol provides a robust and reproducible system to study adipocyte EV secretion in vivo and in vitro, as well as an optimized adipocyte EV purification method.

The protocol for the first time describes and validates the use of serial low speed centrifugations combined with size exclusion chromatography to purify EVs derived from mature adipocytes. Our findings allow us to ask new questions about how adipocytes use extracellular vesicles to communicate with other cell types under physiological and pathological conditions.