We acknowledge the assistance of Research Navigation at Maine Medical Center for assisting with the procurement of clinical tissue, and the Histopathology and Histomorphometry Core (supported by 1P20GM121301, L. Liaw PI) at Maine Medical Center Research Institute for sectioning and staining. This work was supported by NIH grant R01 HL141149 (L. Liaw).

The use of human tissues in this study was evaluated and approved by the Institutional Review Board of Maine Medical Center, and all personnel received appropriate training prior to experimentation.
1. Preparations
<ol>
	<li>Make dissociation buffer by reconstituting 50 mg animal-free collagenase/dispase blend I solution with 1 mL of nanopure H2O. Prepare 1 mg/mL working solution by adding 49 mL of high glucose DMEM containing 1% w/v BSA to the reconstituted collagenase/dispase solution. Store 5 mL working aliquots at -20 &#176;C and warm to 37 &#176;C using a water bath prior to use.</li>
	<li>Make antibiotic solution by adding 1 mL of 100x antibiotic/antimycotic solution (final concentration is 200 units/mL penicillin, 200 units/mL streptomycin and 0.5 &#181;g/mL fungizone) to 49 mL of HBSS, and store on ice.</li>
	<li>Prepare gelatin solution (0.2% w/v) by dissolving 200 mg gelatin from bovine skin in 100 mL of nanopure H2O. Autoclave the solution for 20 min and store at 4 &#176;C.</li>
	<li>Prepare 500 mL of PVAT growth media: high glucose DMEM/F12 with glutamine supplement, sodium pyruvate, and sodium bicarbonate, supplemented with 10% FBS and 100 &#181;g/mL antimicrobial agent (see Table of Materials).</li>
	<li>Prepare 50 mL adipogenic induction media: 40.8 mL high glucose DMEM, supplemented with 7.5 mL PVAT growth media, 500 &#181;L of 100x antibiotic/antimycotic solution (final concentration is 100 units/mL penicillin, 100 units/mL streptomycin and 0.25 &#181;g/mL fungizone), 0.5 mM IBMX (500 mM stock in 0.1 M NaOH), 1 &#181;M dexamethasone (1 mM stock in ethanol), 5 &#181;M rosiglitazone (5 mM stock in DMSO), 33 &#181;M biotin (66 mM stock in DMSO), 100 nM insulin (200 &#181;M stock in 0.1% acetic acid), and 20 &#181;M pantothenic acid (100 mM stock in H2O).</li>
	<li>Prepare 50 mL of control induction media for the adipogenic lineage: 40.8 mL high glucose DMEM supplemented with 7.5 mL PVAT growth media and 500 &#181;L of pen-strep (100x stock).</li>
	<li>Prepare 50 mL adipogenic maintenance media: 41.5 mL high glucose DMEM supplemented with 7.5 mL PVAT growth media from step 1.3, 500 &#181;L pen-strep (100x stock), 1 &#181;M dexamethasone (1 mM stock in EtOH), 33 &#181;M biotin (66 mM stock in DMSO), 100 nM insulin (200 &#181;M stock in 0.1% acetic acid), and 20 &#181;M pantothenic acid (100 mM stock in H2O).</li>
	<li>Prepare 500 mL of growth media for the osteogenic and chondrogenic lineages: high glucose &#945;MEM supplemented with 10% FBS, 1x glutamine supplement (see Table of Materials), and 5 mL of pen-strep (100x stock).</li>
	<li>Prepare 50 mL of induction media for the osteogenic lineage: 50 mL of the bone marrow MSC growth media supplemented with 10 nM dexamethasone and 10 mM &#946;-glycerophosphate.</li>
	<li>Prepare 50 mL of induction media for the chondrogenic lineage: 50 mL of the bone marrow MSC growth media supplemented with 100 nM dexamethasone, 50 &#181;g/mL sodium ascorbate-2-phosphate, and 10 ng/mL TGF&#946;1. For the osteogenic and chondrogenic conditions, the basal bone marrow MSC growth media will serve as the non-induction media.</li>
</ol>
2. Protocol 1: Culture Human PVAT Cells from the Stromal Vascular Fraction
NOTE: PVAT is resected from the site of graft anastomosis on the ascending aorta of anesthetized patients undergoing coronary artery bypass graft procedures. Aortic PVAT is placed in a 15 mL conical containing 10 mL ice cold high glucose DMEM F12 and transferred from the operating room to the laboratory within 2 h of resection. Aortic PVAT is discarded tissue during bypass procedure and has been deemed as non-human subjects research by Maine Medical Center&#8217;s Internal Review Board.
<ol>
	<li>This protocol is for a ~500 mg piece of human PVAT (approximately 3&#160;x 1&#160;x 0.5 cm3). Transfer fresh human PVAT from DMEM to a 50 mL conical tube containing 25 mL antibiotic solution. Incubate with rocking for 20 min at 4 &#176;C. While the PVAT is in antibiotic solution, thaw an aliquot of dissociation buffer at 37 &#176;C.</li>
	<li>Add 50 &#181;L of 100x antibiotic/antimycotic solution to 5 mL dissociation buffer and sterilize using a 0.22 &#181;m syringe filter. Add 1 mL of gelatin solution to 1 well of a 24-well plate. In a laminar flow hood, use sterile forceps and scissors to transfer PVAT from the antibiotic solution to a sterile Petri dish. Add 1 mL of pre-warmed dissociation buffer to the tissue and finely mince the entire tissue into a slurry (no pieces larger than ~2&#160;x 2 mm2) using sterile forceps and dissection scissors.</li>
	<li>Transfer the 1 mL slurry to 4 mL dissociation buffer and incubate the tube on its side in a pre-warmed 37 &#176;C orbital shaker at 200 rpm for 1 h. After 1 h, no visible tissue pieces will be present, and the solution will appear as a cloudy cell suspension.</li>
	<li>Filter the solution through a 70 &#181;m cell strainer set atop a 50 mL conical tube. Rinse the strainer with an additional 10 mL antibiotic solution to capture as many cells as possible. Do not squeeze the strainer.</li>
	<li>Pellet the cells for 12 min at 300 x g in a swinging bucket centrifuge. 
	NOTE: After centrifugation, the tube will be separated into a fatty top layer of adipocytes, an interphase, and a pellet. The pellet is the stromal vascular fraction containing endothelial cells, immune cells, blood cells, and progenitor cells.</li>
	<li>Resuspend the pellet in 10 mL of HBSS and centrifuge for 5 min at 300 x g. Repeat this step for a total of 2 washes in HBSS. After the final wash, do not lyse the red blood cells. Repeated attempts with several commercially-available buffers and incubation times have led to marked reductions in progenitor cell attachment and viability.</li>
	<li>Aspirate gelatin from the 24-well plate. Gently wash the well 1x with HBSS to remove unbound gelatin.</li>
	<li>Resuspend stromal vascular fraction pellet with intact red blood cells in 1 mL of growth media and seed onto the gelatin-coated well. Add human FGF2 (resuspended in PBS supplemented with 0.01% BSA w/v) to a final concentration of 25 ng/mL in culture medium. Incubate for 24 h at 37 &#176;C with 5% CO2.</li>
	<li>On the next day, remove growth media and wash wells 5x with HBSS to remove red blood cells and dead cells. Add in 1 mL of fresh growth media supplemented with 25 ng/mL FGF2.</li>
	<li>Change the media every 48 h, making sure to supplement with 25 ng/mL fresh FGF2 each time.</li>
	<li>Cells typically reach 100% confluence 7-10 days after explant; passage cells then:
	<ol>
		<li>Aspirate growth media and wash monolayer 2x in 1 mL HBSS. Aspirate all HBSS from the wells and add a few drops of cell dissociation solution.</li>
		<li>Tap and swirl the plate several times and incubate at 37 &#176;C with 5% CO2 for 5&#8211;7 min to lift the cells. Add ~1 mL of fresh culture medium to the detached cells and distribute 500 &#181;L to 2 wells of a 24-well plate, each containing 500 &#181;L growth media and 25 ng FGF2.</li>
	</ol>
	</li>
	<li>Continue to expand human PVAT-derived cells as indicated in the previous step. Each passage should be no greater than a 1:2 split. Cells are passaged 5&#8211;7 times before allocating for differentiation assays.</li>
</ol>
3. Protocol 2: Culture Human Bone Marrow MSC Colonies
NOTE: Human bone marrow MSC are isolated as described8 and stored as early passage frozen stocks in freeze media (70% FBS 20% basal DMEM and 10% DMSO) at ~100,000 cell/mL in liquid N2.
<ol>
	<li>Rapidly thaw a vial of bone marrow MSC from the liquid N2 in a 37 &#176;C water bath and plate to one well of a 6-well culture plate containing 3 mL of MSC growth media and incubate overnight at 37&#176;C and 5% CO2.</li>
	<li>On the next day, aspirate MSC culture media and wash the cells 3x in 2 mL of HBSS. At 100% confluence, aspirate growth media and wash the cells 3x in 2 mL of HBSS. Add 500 &#181;L/well cell detachment solution and incubate at 37 &#176;C and 5% CO2 for 5 min. Tap the plate to ensure all cells are dislodged and evenly distribute contents to 2 wells of a 6-well plate containing 2 mL MSC growth media.</li>
	<li>Expand the human bone marrow and PVAT-derived MSC in parallel for approximately 5&#8211;7 passages or until sufficient quantities for testing has been achieved.</li>
</ol>
4. Protocol 3: Plate and Induce Adipogenic, Osteogenic, and Chondrogenic Lineages
<ol>
	<li>Plate appropriate numbers of bone marrow and PVAT-derived cells per well of a 12-well plate and appropriate replicates for each experimental condition. For adipogenic and osteogenic conditions, ~200,000&#8211;225,000 cells/well are needed, for chondrogenic conditions, 150,000&#8211;175,000 cells/well are needed. 
	NOTE: We ran a minimum of N = 3 replicate wells for control and induced conditions for each experimental lineage for a total of 2 independent runs (N = 6 replicates total).</li>
	<li>Disassociate cells from both the human PVAT progenitor cell population and the human bone marrow MSC population using cell detachment solution and incubation at 37 &#176;C and 5% CO2 for 5 min. Pool populations into separate 15 mL conical vials. Spin the vial down at 500 x g for 7 min to pellet the cells. Resuspend in 1 mL of PBS and use a hemocytometer to estimate the cell number.</li>
	<li>Plate the cells in 12-well dishes as indicated in step 4.1. Provide separate dishes for induced and non-induced adipogenic, osteogenic, and conditions so that the non-induced condition can be fixed at an earlier time point without disrupting continuing culture of the induced condition.</li>
	<li>Add 1.5 mL of adipogenic and osteogenic induction media to each well of the induced condition. Add 1.5 mL of adipogenic and osteogenic non-induction media to each well of the non-induced condition. Begin incubation of the adipogenic and osteogenic induced and non-induced cell populations at 37 &#176;C and 5% CO2.</li>
	<li>Spin down the remaining volume of human PVAT progenitor cells and human bone marrow MSCs for 7 min at 500 x g.</li>
	<li>Determine the volume needed to resuspend remaining bone marrow and PVAT-derived cell pellets to achieve a density of 100,000 cells/10 &#181;L (106 cells/mL). Resuspend pellets in the calculated volume of MSC growth media for chondrogenic lineage induction. Gently move the volume of cells up and down using a pipet to ensure a homogenous distribution.</li>
	<li>Pipette a 10 &#181;L droplet of the concentrated cell solution into the center of each well to form a micromass of 100,000 cells. Place 1 mL of sterile H2O in the adjacent well to prevent evaporation. Incubate the micromass cultures for 2 h at 37 &#176;C and 5% CO2 to allow the micromass to aggregate.</li>
	<li>After 2 hours, carefully add chondrogenic differentiation media spiked with 10 ng/mL human TGF&#946;1 to each of the induced-condition wells. Carefully add 1.5 mL of the non-induction media (bone marrow MSC growth media) to the non-induced condition wells. Use separate 12-well plates for the induced and non-induced conditions so that the non-induced condition can be fixed at an earlier time point.</li>
</ol>
5. Protocol 4: Culture Adipogenic, Osteogenic, and Chondrogenic Lineages for 14 Days
<ol>
	<li>Culture the induced and non-induced conditions of all three lineages for 4 days at 37 &#176;C and 5% CO2, refreshing media every 2 days. On day 4, change the induced adipogenic lineage condition from induction media to maintenance media for the remainder of the assay.</li>
	<li>Fix all of the non-induced conditions in 10% formalin for 12 h. Dispose of formalin and wash all fixed wells 2x in PBS to remove all traces of formalin. Store plates in PBS at 4 &#176;C until processing.</li>
	<li>Culture the induced conditions for a total of 14 days, refreshing media every 2 days. Add fresh TGF&#946;1 at 10 ng/mL final concentration with each refresh of the chondrogenic induction media. Continue to culture the adipogenic lineage in the adipogenic maintenance media and osteogenic lineage induction media, refreshing every 2 days. At day 14, fix all induced conditions for 12 h in 10% formalin for staining.</li>
	<li>Scrape or pour the micromass in the induced chondrogenic condition into a cassette for embedding. Dehydrate the micromass in a series of increasingly concentrated alcohol baths for 5 min, beginning at 70%, then 80%, twice in 95%, and twice more in absolute alcohol. Place the cassette in a dealcoholization agent and then finally embed the cassette in paraffin wax for sectioning and staining.</li>
</ol>
6. Protocol 5: Staining Adipogenic Condition with Oil Red O
<ol>
	<li>Prepare Oil Red O stock solution by dissolving 350 mg of Oil Red O in 100 mL of 100% isopropanol. Mix for 2 h with a stir bar and vacuum through a 0.2 &#181;m filter. Stock solution can be stored in the dark at room temperature for 1 year.</li>
	<li>Prepare Oil Red O working solution by mixing 3 parts stock solution to 2 parts diH20 (e.g. 60 mL of stock solution to 40 mL diH20). The final concentration of Oil Red O in the working solution is 2.1 mg/mL.</li>
	<li>Remove all fluid from wells. Wash each well 2x with 60% isopropanol, making sure to completely remove all water from the sides of the wells. Aspirate 60% isopropanol and quickly add Oil Red O working solution without touching the walls of the wells to cover the bottom. It is critical that this step is done quickly so the wells do not dry.</li>
	<li>Once the Oil Red O is added, incubate for 10 min at room temperature with gentle rocking.</li>
	<li>Remove all Oil Red O and immediately add distilled H2O. Wash with distilled H2O for 10m to begin to remove unbound Oil Red O. After 10 min, aspirate Oil Red O and repeat the washing procedure 3x.</li>
	<li>Once washing is complete, add PBS and image the cells. Store stained cells in PBS at 4 &#176;C.</li>
</ol>
7. Protocol 6: Staining Osteogenic Condition with Alizarin Red
<ol>
	<li>Remove all fluid from each well. Add appropriate volume of 2% Alizarin Red stain solution to each well (1.5 mL per well for a 12-well plate) and gently tilt the plate side-to-side until the solution completely covers the bottom of the well.</li>
	<li>Incubate for 15 min at room temperature. Remove Alizarin Red from the wells. Gently rinse each well four times with distilled H2O, taking caution to not dislodge calcium crystals. Allow to dry.</li>
</ol>
8. Protocol 7: Staining Chondrogenic Condition with Masson&#8217;s Trichrome
<ol>
	<li>Bake slides in a 60 &#176;C dry oven for 30 min. Deparaffinize and hydrate sections to distilled water.
	<ol>
		<li>To rehydrate, place slides in three 5 min incubations with dealcoholization agents, followed by 5 min incubations in descending alcohol concentrations as follows: two at 100% (absolute ethanol), two at 95% alcohol, two at 80%, two at 70%, and finally two in distilled H2O. Slides can remain in H2O while Bouin&#8217;s fixative is prepared.</li>
	</ol>
	</li>
	<li>Place 40 mL of Bouin&#8217;s fixative in a plastic microwavable coplin jar (uncovered). Microwave on high to bring temp to 55 &#176;C. Place slide in heated solution for 20 min; let stand on the counter covered. Wash in running tap water for 10 min or until all of the yellow color disappears.</li>
	<li>Stain sections in Weigert&#8217;s hematoxylin for 10 min. Wash in running tap water for 10 min.</li>
	<li>Stain sections in Beibrich&#8217;s scarlet acid fuchsin solution for 10 min. Wash 3x 10 min in tap water. Mordant slides in phosphotungstic/phosphomolybdic acid solution for 10 min. Do not rinse.</li>
	<li>Place slides directly in aniline blue solution for 10 min. Rinse with two brief dips in tap water.</li>
	<li>Place slides in 1% acetic acid solution for 3 to 5 min. Wash in tap water for 1 min. Place in 95% ethanol for 1 min. Dehydrate as indicated in step 5.4 and mount with synthetic resin.</li>
</ol>

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The authors have nothing to disclose.

Adipose progenitor cells from different depots vary widely in phenotype and differentiation potential9. Culturing PVAT-derived progenitors from a single patient donor in simultaneous induction down three different lineages, adipogenic, osteogenic, and chondrogenic, allows for a well-controlled investigation of the pluripotent capacity of this novel population of progenitor cells. The methodology described in this report can be used to test the differentiation capacity of progenitor cells from huma...

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages1. This tissue expands through hypertrophy of mature adipocytes and de novo differentiation of resident MSC to adipocytes. Perivascular adipose tissue (PVAT) surrounds blood vessels and regulates vascular function2,3. Obesity-induced PVAT expansion exacerbates cardiovascular pathologies. While the multipotent potential of MSC from human subcutaneous adipose depots have been well studied4,5, no studies have explanted and evaluated the differentiation capacity of human PVAT-derived progenitor cells, likely due to the invasiveness of procurement. Thus, the goal of this work is to provide a methodology to explant and propagate progenitor cells from human aortic PVAT from patients with cardiovascular disease and to test their propensity to differentiate to osteogenic, chondrogenic, and adipogenic lineages. Our source of PVAT is from the site of anastomosis of the bypass graft on ascending aorta of obese patients undergoing coronary artery bypass graft surgery. Freshly-isolated PVAT is enzymatically-dissociated and the stromal vascular fraction is isolated and propagated in vitro, enabling us to test for the first time the differentiation capacity of human PVAT-derived progenitor cells.
Using primary cultured human PVAT stromal vascular fraction, we tested three assays designed to induce stem/progenitor cells to differentiate toward adipogenic, osteogenic, or chondrogenic lineages. Our prior study identified a population of CD73+, CD105+, and PDGFRa+ (CD140a) cells that can robustly differentiate into adipocytes6, although their multipotency was not tested. PVAT directly regulates vascular tone and inflammation7. The rationale for testing the differentiation potential of this novel cell population is to begin to understand the specialized influence of PVAT on vascular function, and mechanisms of PVAT expansion during obesity. This methodology enhances our understanding of the functions of adipose-tissue derived progenitor cells and enables us to identify and compare similarities and differences of progenitor cells from different tissue sources. We build upon established and validated approaches for isolating and differentiating MSC towards different lineages and optimize procedures to maximize the viability of human PVAT-derived progenitor cells. These techniques have broad applications in the fields of stem and progenitor cell research and adipose tissue development.

<table><tbody><tr><td>Animal-free collagenase/dispase blend I &amp;nbsp;</td><td>Millipore-Sigma</td><td>SCR139</td><td>50mg</td></tr><tr><td>Alcian Blue</td><td>NewComerSupply</td><td>1003A</td><td>1% Aqueous solution pH 2.5</td></tr><tr><td>Alizarin Red</td><td>Amresco</td><td>9436-25G</td><td></td></tr><tr><td>alpha-MEM</td><td>ThermoFisher</td><td>12561056</td><td></td></tr><tr><td>Aniline Blue</td><td>NewComerSupply</td><td>10073C</td><td></td></tr><tr><td>Antibiotic/antimycotic</td><td>ThermoFisher</td><td>15240062</td><td></td></tr><tr><td>Beibrich&#039;s scarlet acid fuchsin</td><td>Millipore-Sigma</td><td>A3908-25G</td><td></td></tr><tr><td>b-glycerophosphate</td><td>Millipore-Sigma</td><td>G9422-10G</td><td></td></tr><tr><td>Biebrich Scarlet</td><td>EKI</td><td>2248-25G</td><td></td></tr><tr><td>Biotin</td><td>Millipore-Sigma</td><td>B4501-100MG</td><td></td></tr><tr><td>Bouin&#039;s fixative</td><td>NewComerSupply</td><td>1020A</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Calbiochem</td><td>12659</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Cell detachment solution</td><td>Accutase</td><td>AT104</td><td></td></tr><tr><td>Bell strainer (70 mm)</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>Dexamethasone</td><td>Millipore-Sigma</td><td>D4902-100MG</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td>4.5 g/L glucose, L-glut and pyruvate</td></tr><tr><td>DMEM/F12 medium</td><td>ThermoFisher</td><td>10565-042</td><td>high glucose, glutamax, sodium bicarbinate</td></tr><tr><td>DMSO</td><td>Millipore-Sigma</td><td>D2650</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals&amp;nbsp;</td><td>S11550</td><td></td></tr><tr><td>FGF2</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>Formalin</td><td>NewComerSupply</td><td>1090</td><td></td></tr><tr><td>Gelatin, bovine skin</td><td>Millipore-Sigma</td><td>G9391-500G</td><td></td></tr><tr><td>Glutamax</td><td>ThermoFisher</td><td>35050061</td><td>glutamine supplement</td></tr><tr><td>HBSS</td><td>Lonza</td><td>10-547F</td><td></td></tr><tr><td>IBMX</td><td>Millipore-Sigma</td><td>I5879-250MG</td><td></td></tr><tr><td>Insulin solution</td><td>Millipore-Sigma</td><td>I9278-5ML</td><td></td></tr><tr><td>Oil red O</td><td>Millipore-Sigma</td><td>O0625-100G</td><td></td></tr><tr><td>Pantothenic acid</td><td>Millipore-Sigma</td><td>P5155-100G</td><td></td></tr><tr><td>Penicillin-streptomycin solution</td><td>ThermoFisher</td><td>15240062</td><td>100ml</td></tr><tr><td>Permount</td><td>Fisher</td><td>SP15-500</td><td></td></tr><tr><td>Phosphotungstic/phosphomoybdic acid solution</td><td>Millipore-Sigma</td><td>P4006-100G/221856-100G</td><td></td></tr><tr><td>Primocin</td><td>Invivogen</td><td>ant-pm-1</td><td>Antimicrobial reagent for culture media.</td></tr><tr><td>Rosiglitazone</td><td>Millipore-Sigma</td><td>R2408-10MG</td><td></td></tr><tr><td>TGFb1</td><td>Peprotech</td><td>100-21</td><td></td></tr><tr><td>Weigert&#039;s hematoxylin</td><td>EKI</td><td>4880-100G</td><td></td></tr></tbody></table>

differentiation capacity of human aortic perivascular adipose progenitor cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

Isolation of stromal vascular fraction from human PVAT
Figure 1A shows a schematic of the anatomical region where the PVAT overlying the ascending aorta was obtained. We previously described the patient populations undergoing coronary artery bypass grafting from which these samples were derived6. Figure 1B shows an example of the...

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Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

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Research

JoVE Journal

Biology

7.9K Views.  Maine Medical Center Research Institute. Perivasculair vetweefsel omringt bloedvaten en reguleert cardiovasculaire fysiologie. Dit protocol wordt gebruikt om belangrijke vragen met betrekking tot de functie van menselijke vetvererwaarden in de vasculaire micro-omgeving te beantwoorden. Vetverervercellen variëren afhankelijk van hun anatomische locatie.We tonen aan dat een populatie van multipotige voorlopers met succes kan worden afgeleid uit perivasculair vetweefsel van patiënten met hart- en vaatziekten. Het aantonen van ...

Video: Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Developmental Biology

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

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

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.

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).

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

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

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) &#62;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&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; 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.

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.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Immunology and Infection

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Culturing Preadipocytes Isolated from Murine Perivascular Adipose Tissue

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Author Spotlight: The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

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