Name: differentiation capacity of human aortic perivascular adipose progenitor cells
Uploaded: 2019-03-05T15:00:00
Description: Watch this Scientific Journal Video about Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells at JoVE.com

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 so...

<ol>
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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.

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			<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>
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			<td>1% Aqueous solution pH 2.5</td>
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			<td style="width:139px;">Amresco</td>
			<td style="width:148px;">9436-25G</td>
			<td></td>
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			<td height="21" style="height:21px;width:299px;">alpha-MEM</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:148px;">12561056</td>
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			<td>stored at 4C</td>
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			<td height="21" style="height:21px;width:299px;">cell strainer (70mm)</td>
			<td style="width:139px;">Corning</td>
			<td style="width:148px;">352350</td>
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			<td height="21" style="height:21px;width:299px;">dexamethasone</td>
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			<td style="width:139px;">Corning</td>
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			<td>4.5g/L glucose, L-glut and pyruvate</td>
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		<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>
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			<td height="21" style="height:21px;width:299px;">DMSO</td>
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			<td style="width:148px;">D2650</td>
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			<td style="width:148px;">S11550</td>
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			<td style="width:148px;">1090</td>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:299px;">IBMX</td>
			<td style="width:139px;">Millipore-Sigma</td>
			<td style="width:148px;">I5879-250MG</td>
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			<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;width:299px;">permount</td>
			<td style="width:139px;">Fisher</td>
			<td style="width:148px;">SP15-500</td>
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			<td style="width:148px;">P4006-100G/221856-100G</td>
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			<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>
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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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