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 border="0" cellpadding="0" cellspacing="0" style="width:884px;" width="884">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">animal-free collagenase/dispase blend I &#160;</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">SCR139</td>
			<td style="width:299px;">50mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Alcian Blue</td>
			<td style="width:139px;">NewComerSupply</td>
			<td style="width:148px;">1003A</td>
			<td>1% Aqueous solution pH 2.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Alizarin Red</td>
			<td style="width:139px;">Amresco</td>
			<td style="width:148px;">9436-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">alpha-MEM</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:148px;">12561056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Aniline Blue</td>
			<td style="width:139px;">NewComerSupply</td>
			<td style="width:148px;">10073C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">antibiotic/antimycotic</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:148px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Beibrich&#39;s scarlet acid fuchsin</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">A3908-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">b-glycerophosphate</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">G9422-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Biebrich Scarlet</td>
			<td style="width:139px;">EKI</td>
			<td style="width:148px;">2248-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">biotin</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">B4501-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Bouin&#39;s fixative</td>
			<td style="width:139px;">NewComerSupply</td>
			<td style="width:148px;">1020A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">bovine serum albumin</td>
			<td style="width:139px;">Calbiochem</td>
			<td style="width:148px;">12659</td>
			<td>stored at 4C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Cell detachment solution</td>
			<td style="width:139px;">Accutase</td>
			<td style="width:148px;">AT104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">cell strainer (70mm)</td>
			<td style="width:139px;">Corning</td>
			<td style="width:148px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">dexamethasone</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">D4902-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DMEM</td>
			<td style="width:139px;">Corning</td>
			<td style="width:148px;">10-013-CV</td>
			<td>4.5g/L glucose, L-glut and pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DMEM/F12 medium</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:148px;">10565-042</td>
			<td>high glucose, glutamax, sodium bicarbinate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DMSO</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">fetal bovine serum</td>
			<td style="width:139px;">Atlanta Biologicals&#160;</td>
			<td style="width:148px;">S11550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">FGF2</td>
			<td style="width:139px;">Peprotech</td>
			<td style="width:148px;">100-18B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">formalin</td>
			<td style="width:139px;">NewComerSupply</td>
			<td style="width:148px;">1090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">gelatin, bovine skin</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">G9391-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">glutamax</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:148px;">35050061</td>
			<td>glutamine supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">HBSS</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:148px;">10-547F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">IBMX</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">I5879-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">insulin solution</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">I9278-5ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Oil red O</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">O0625-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">pantothenic acid</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">P5155-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">penicillin-streptomycin solution</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:148px;">15240062</td>
			<td>100ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">permount</td>
			<td style="width:139px;">Fisher</td>
			<td style="width:148px;">SP15-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">phosphotungstic/phosphomoybdic acid solution</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">P4006-100G/221856-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">primocin</td>
			<td style="width:139px;">Invivogen</td>
			<td style="width:148px;">ant-pm-1</td>
			<td>Antimicrobial reagent for culture media.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">rosiglitazone</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">R2408-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">TGFb1</td>
			<td style="width:139px;">Peprotech</td>
			<td style="width:148px;">100-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Weigert&#39;s hematoxylin</td>
			<td style="width:139px;">EKI</td>
			<td style="width:148px;">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...

Watch this Scientific Journal Video about Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells at JoVE.com

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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JoVE Journal

Biology

7.9K Views.  Maine Medical Center Research Institute. 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.

Video: Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex&#160;vivo culture will represent the in&#160;vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

Isolated pancreatic acini retain their in&#160;vivo morphology and activity and offer powerful ways for monitoring and manipulating secretion. This work demonstrates how acini can be isolated from the mouse pancreas, and how their secretory capacities can be assessed.

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...