The authors are grateful to Lisa Hansen and Kirsten Vestergaard for excellent technical assistance, and P. Scherer, N. Joffin, and C. Crewe for critical reading of the manuscript. The authors thank the UTSW Flow Cytometry Core for excellent guidance and assistance in developing the protocols described here. R.K.G. is supported by NIH NIDDK R01 DK104789, NIDDK RC2 DK118620, and NIDDK R01 DK119163. J.P. is sponsored by a pre-doctoral award from Innovation Fund Denmark.

All animal protocols and procedures have been approved by the University of Texas Southwestern Medical Center Institutional Animal Use and Care Committee.
1. Isolation of stromal vascular fraction (SVF) from gonadal white adipose tissue
<ol>
	<li>Dissect the gonadal white adipose tissue from 6-8-week-old mice and place fat pads in 1x PBS solution.</li>
	<li>Combine up to 4 fat depots (2-4 depots from 1-2 mice recommended) and mince the tissue in a 10 mL beaker containing 200 &#181;L of digestion buffer (1x HBSS, 1.5% BSA and 1 mg/mL collagenase D).</li>
	<li>Transfer the minced tissue to a 50 mL centrifuge tube containing 10 mL digestion buffer.</li>
	<li>Incubate mixture at 37 &#176;C in a water bath for 1 h while shaking.</li>
	<li>Filter the digested tissue through a 100 &#181;m cell strainer to remove undigested tissue.</li>
	<li>Dilute filtered sample to 30 mL with PBS containing 2% FBS, and centrifuge at 600 x g for 5 min at 4 &#176;C. Aspirate the supernatant, which contains mature adipocytes.</li>
	<li>Proceed immediately to preferred cell isolation method.</li>
</ol>
2. Isolation of APCs and FIPs using FACS
<ol>
	<li>Dissolve the pellet in 1 mL of 1x RBC lysis buffer by shaking the tube gently.</li>
	<li>Incubate at RT for 1-2 min.</li>
	<li>Add 10 mL of 2% FBS/PBS and pass through a 40 &#181;m cell strainer into a clean 50 mL centrifuge tube.</li>
	<li>Centrifuge at 600 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate media and resuspend the pellet in 400-800 &#181;L of 2% FBS/PBS containing Fc block (1:200).</li>
	<li>Incubate at 4 &#176;C for 10 min.</li>
	<li>Transfer 400 &#181;L-800 &#181;L of cell suspensions containing &#8804;106 cells per mL to 1.5 mL tubes and add antibodies. Antibody concentrations are listed in the Table of Materials section.
	<ol>
		<li>Prepare separate control tubes for 1) unstained cells, 2) single color controls, and 3) FMO (fluorescence minus one) controls.</li>
		<li>Stain the samples with PDGFR&#946;, CD45, CD31, CD9, LY6C antibodies.</li>
	</ol>
	</li>
	<li>Incubate at 4 &#176;C for 15 min protected from light.</li>
	<li>Centrifuge at 600 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate media and resuspend cells in 400 &#181;L of 2% FBS/PBS.</li>
	<li>Centrifuge at 600 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate media and resuspend cells in 400-800 &#181;L of 2% FBS/PBS. Then pass through 40 &#181;m filter caps into 5 mL polystyrene round-bottom tubes for FACS.</li>
	<li>Follow the following steps for gating.
	<ol>
		<li>Use unstained and single-color controls for compensation.</li>
		<li>Use FMO controls to set experimental gates.</li>
		<li>Use the gating strategy shown in Figure 1 to obtain APCs and FIPs. Gate APCs as CD45- CD31- PDGFR&#946;+ LY6C- CD9- and FIPs as CD45- CD31- PDGFR&#946;+ LY6C+ cells.</li>
	</ol>
	</li>
	<li>Collect sorted cells in tubes containing 500 &#181;L of 100% serum and maintain the cells on ice.</li>
	<li>Centrifuge cells at 600 x g for 5 min at 4 &#176;C. Add 2% FBS/PBS to wash the cells by centrifuging again at 600 x g for 5 min at 4 &#176;C. Discard the supernatant use the pelleted cells.</li>
	<li>Resuspend the pelleted cells in the appropriate volume of gonadal APC culture media (for cell culture) or appropriate buffer for subsequent analysis.</li>
</ol>
3. Immunomagnetic separation of adipogenic and non-adipogenic fractions
<ol>
	<li>Isolate SVF from gonadal white adipose tissue following steps 1.1-1.7.</li>
	<li>Aspirate the supernatant and resuspend the SVF pellet in 10 mL of 1x commercially available MS Buffer.</li>
	<li>Filter the cell suspension through a 40 &#181;m cell strainer.</li>
	<li>Centrifuge at 600 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend pelleted cells in 100 &#181;L of 1x MS Buffer.</li>
	<li>Perform CD31+ and CD45+ cell depletion as discussed below (Figure 2A, Step 1). This is done to remove endothelial and hematopoietic lineage cells.
	<ol>
		<li>Count cells using a cell counter and adjust the concentration to &#8804; 1 x 108 cells/mL.</li>
		<li>Add the biotin conjugated CD31 and CD45 antibodies to the cell suspension, each at a concentration of &#8804; 0.25 &#181;g per 106 cells and incubate on ice for 15 min.</li>
		<li>Wash cells by adding 4 mL of 1x MS Buffer.</li>
		<li>Centrifuge at 600 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant and resuspend the pellet with 100 &#181;L of 1x MS buffer.</li>
		<li>Add 10 &#181;L of streptavidin nanobeads and incubate cells on ice for 15 min.</li>
		<li>Wash cells by adding 4 mL of 1x MS buffer.</li>
		<li>Centrifuge at 600 x g for 5 min at 4 &#176;C, then aspirate the supernatant.</li>
		<li>Resuspend cells in 2.5 mL of 1x MS buffer and transfer to a 5 mL polypropylene tube.</li>
		<li>Place the tube in the magnet rack for 5 min.</li>
		<li>Collect unlabeled cells by pouring the cell suspension into a clean 15 mL centrifuge tube. 
		NOTE: Avoid shaking or blotting off hanging droplets as this leads to contamination from unwanted cell fraction.</li>
		<li>Remove the tube from magnet and repeat steps 3.6.9. to 3.6.11. two more times for a total of 3 washes.</li>
	</ol>
	</li>
	<li>Separation of adipogenic and non-adipogenic fractions (Figure 2A, Step 2)
	<ol>
		<li>Continue working with unlabeled fraction (i.e., CD45- CD31- fraction).</li>
		<li>Centrifuge the 15 mL tube containing unlabeled cells at 600 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 100 &#181;L of 1x MS buffer.</li>
		<li>Add biotin conjugated LY6C and CD9 antibodies, respectively, at a concentration of &#8804; 0.25 &#181;g per 106 cells and incubate on ice for 15 min.</li>
		<li>Follow steps 3.6.3. to 3.6.12. to complete the second separation.</li>
		<li>Collect unbound fractions. Unlabeled cells (LY6C- CD9-) represents adipogenic fraction containing APCs (Figure 2A, Step 3).</li>
		<li>Remove tube from the magnet, resuspend and elute bound cells in 1x MS buffer. Eluates or labeled fraction (LY6C+ CD9+), represents non-adipogenic fraction containing FIPs (Figure 2A, Step 3).</li>
		<li>Centrifuge labeled and unlabeled fractions at 600 x g for 5 min to pellet cells at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Proceed immediately to cell culture (step 6) or use undifferentiated precursors for preferred analyses (step 4 and/or step 5).</li>
</ol>
4. Assess purity of adipogenic and non-adipogenic fractions by flow cytometry
<ol>
	<li>Resuspend bead-isolated cell fractions in 200-400 &#181;L of 2% FBS/PBS containing Fc block (1:200).</li>
	<li>Incubate at 4 &#176;C for 10 min.</li>
	<li>Transfer the cell suspension to 1.5 mL tubes and add antibodies. Antibody concentrations are listed in the Table of Materials.
	<ol>
		<li>Prepare separate control tubes for 1) unstained cells, 2) single color controls, and 3) FMO controls.</li>
		<li>To the samples, add CD45, CD31, CD9, and LY6C antibodies.</li>
	</ol>
	</li>
	<li>Incubate at 4 &#176;C for 15 min protected from light.</li>
	<li>Centrifuge at 600 x g for 5 minutes at 4 &#176;C.</li>
	<li>Aspirate supernatant and resuspend cells in 400 &#181;L of 2% FBS/PBS.</li>
	<li>Centrifuge the suspension at 600 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend cells in 400-800 &#181;L of 2% FBS/PBS. Filter through 40 &#181;m filter caps into 5 mL polystyrene round-bottom tubes for analysis by flow cytometry.</li>
	<li>Flow cytometry analysis to assess purity
	<ol>
		<li>Use unstained and single-color controls for compensation.</li>
		<li>Use FMO controls to set experimental gates.</li>
		<li>Use the gating strategy for live and single cells as shown in Figure 2B.</li>
		<li>Perform the gating as shown in Figure 2B for CD45, CD31, LY6C, and CD9.</li>
		<li>Use flow cytometry software to evaluate the frequencies of CD45- CD31- LY6C- CD9- cells (APCs) and CD45- CD31- LY6C+ cells (FIPs) within isolated fractions.</li>
	</ol>
	</li>
</ol>
5. Gene expression analysis using quantitative PCR to assess purity of FIPs and APCs
<ol>
	<li>Extract mRNA from magnetically or FACS isolated cells using commercially available extraction kit (see Table of Materials) following the manufacturer&#8217;s instructions.</li>
	<li>Use 1 &#181;g of RNA to synthetize cDNA using a cDNA Reverse Transcriptase kit (see Table of Materials) by following the manufacturer&#8217;s instructions.</li>
	<li>Determine relative mRNA levels of APC and FIPs-selective genes (Table 1) by quantitative PCR using SYBR green PCR master mix.
	<ol>
		<li>Set up the sample reaction for PCR as follows: 5 &#181;L of 2x SYBR green dye, 1 &#181;L of cDNA (~50 ng/ &#181;L), 0.5 &#181;L of each forward and reverse primer (10 &#181;M) and 3 &#181;L of water.</li>
		<li>Use the following standard PCR conditions for the quantitative PCR run: hold stage: 50 &#176;C for 2 min, 95&#176;C for 10 min; PCR stage (40 cycles): 95 &#176;C for 15 s, 60 &#176;C for 1 min.</li>
	</ol>
	</li>
	<li>Normalize values to house-keeping gene (Rps18) levels by calculating &#916;&#916;-Ct.</li>
	<li>To evaluate statistical significance, perform unpaired Student&#8217;s t-test.</li>
</ol>
6. Cell culture and differentiation
<ol>
	<li>Centrifuge magnetically- or FACS- isolated cells at 600 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the pellet in 500 &#181;L of gonadal APC culture media and plate 40K cells/well from each fraction in a 48 well culture plate.</li>
	<li>Replace media every 1-2 days. APCs will begin to undergo differentiation into adipocytes as they approach confluence. FIPs maintained in the same media are resistant to undergoing adipogenesis.</li>
</ol>

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T.A.P. is an employee and shareholder in Novo Nordisk A/S

The C57BL/6 strain of mice is the most used mouse strain in studies of diet-induced obesity. C57BL/6 mice rapidly gain weight when placed on a high-fat diet (HFD) and develop some of the prominent features of metabolic syndrome associated with obesity (e.g., insulin resistance and hyperlipidemia). Notably, WAT expansion occurring in association with high-fat diet (HFD) feeding occurs in a depot-specific manner19,20,21,</sup...

White adipose tissue (WAT) represents the principal site for energy storage in mammals. Within this tissue, adipocytes, or &#8220;fat cells,&#8221; store excess calories in the form of triglyceride, packaged into large unilocular lipid droplets. Moreover, adipocytes secrete a multitude of factors that regulate various aspects of energy homeostasis1,2,3. Adipocytes constitute the bulk of WAT volume; however, adipocytes only represent less than 50% of total cells found in WAT4,5. The non-adipocyte compartment of WAT, or stromal-vascular fraction (SVF), is quite heterogeneous and contains vascular endothelial cells, tissue-resident immune cells, fibroblasts, and adipocyte precursor cell (APC) populations.
WAT is exceptional in its remarkable capacity to expand in size as the demand for energy storage increases. Maintaining this tissue plasticity is essential as adequate storage of lipids in WAT protects against deleterious ectopic lipid deposition into non-adipose tissues6. The manner by which individual WAT depots undergo this expansion in response to caloric excess is a critical determinant of insulin sensitivity in the setting of obesity7. Pathologic WAT expansion, observed in obese individuals with metabolic syndrome, is characterized by preferential expansion of visceral WAT depots at the expense of metabolically favorable subcutaneous fat tissue. Moreover, insulin resistance in obesity is associated with pathologic remodeling of WAT. This is characterized by hypertrophic growth of existing adipocytes (increase in size), inadequate angiogenesis, chronic metabolic-inflammation, accumulation of extracellular matrix components (fibrosis), and tissue hypoxia8,9. These WAT phenotypes of obesity are associated with hepatic steatosis and insulin resistance, similar to what is observed in the condition of lipodystrophy (absence of functional WAT). In contrast, healthy WAT expansion is observed in the metabolically healthy obese population and is characterized by preferential expansion of protective subcutaneous WAT and depot expansion through adipocyte hyperplasia10. The recruitment of new adipocytes is mediated by de novo adipocyte differentiation from adipocyte precursor cells (APCs) (termed, &#8220;adipogenesis&#8221;). Adipocyte hyperplasia coincides with relatively lower degrees of WAT fibrosis and metabolic inflammation6,11. A multitude of cell types within the WAT microenvironment directly influence the health and expandability of WAT in obesity12. As such, defining the function of the various cell types present in WAT remains a high priority for the field.
Over the past decade, several strategies have been employed to define and isolate native APCs from human and mouse WAT SVF13. Such strategies isolate APCs based on the cell surface expression of common mesenchymal stem/progenitor cell markers using antibody-based cell separation techniques. These approaches include fluorescence-activated cell sorting (FACS), using fluorophore-labelled antibodies, or immunomagnetic bead separation (i.e., chemically modified antibodies). Cell surface proteins targeted for the isolation of APCs include PDGFR&#945;, PDGFR&#946;, CD34, and SCA-1. These approaches have helped enrich for APCs; however, cell populations isolated based on these markers are quite heterogeneous. Very recent single-cell RNA-sequencing (scRNA-seq) studies have highlighted the molecular and functional heterogeneity of stromal cells within the isolated stromal-vascular fraction (SVF) of murine WAT14,15,16,17. From our own scRNA-seq and functional analyses, we have identified and characterized functionally distinct immune-modulating and adipogenic PDGFR&#946;+ perivascular cell subpopulations in the stromal compartment of intra-abdominal WAT in adult mice15. Fibro-inflammatory precursors, or FIPs, represent a prominent subpopulation of PDGFR&#946;+ cells and can be isolated based on LY6C expression (LY6C+ PDGFR&#946;+ cells)15. FIPs lack adipogenic capacity, exert a strong pro-inflammatory response to various stimuli, produce collagen, and secrete anti-adipogenic factors15. The pro-inflammatory and fibrogenic activity of these cells increases in association with obesity in mice, implicating these cells as regulators of WAT remodeling. The LY6C- CD9- PDGFR&#946;+ subpopulation represents adipocyte precursor cells (APCs). These APCs are enriched in the expression of Pparg and other pro-adipogenic genes, and readily differentiate into mature adipocytes in vitro and in vivo15. Here, we provide a detailed protocol for the isolation of these distinct cell populations from intra-abdominal WAT depots of adult mice using FACS, and immunomagnetic bead separation with biotinylated antibodies. This protocol can be used to isolate functionally distinct adipose progenitor subpopulations from multiple intra-abdominal WAT depots of adult male and female mice15. Studying these functionally distinct cell populations in isolation may contribute greatly to our current understanding of the molecular mechanisms that regulate adipogenesis and intra-abdominal adipose tissue remodeling in health and disease.
The protocol below details the isolation of adipose progenitors from murine epididymal WAT; however, the same procedure can be used to isolate corresponding cells from the mesenteric and retroperitoneal WAT depots of both male and female mice15. A detailed protocol on how to identify and isolate these depots in mice can be found in Bagchi et al.18. This protocol has been optimized for the use of mice 6-8 weeks of age. The frequency and differentiation capacity of APCs may decline in association with ageing.

<table><tbody><tr><td>&lt;strong&gt;Mechanical Tissue Preparation and SVF Isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>40 and 100 &amp;micro;m cell strainers</td><td>Fisher Scientific</td><td>352340/352360</td><td></td></tr><tr><td>1X Phosphate buffered saline (PBS)</td><td>Fisher Scientific</td><td>21040CV</td><td></td></tr><tr><td>5ml polypropylene tubes</td><td>Fisher Scientific</td><td>352053</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Digestion Buffer (for 10mL)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 ml HBSS</td><td>Sigma</td><td>H8264</td><td></td></tr><tr><td>10 mg Collagenase D (1 mg/ml final cc.)</td><td>Roche</td><td>11088882001</td><td></td></tr><tr><td>0.15 g BSA (1.5 % final cc.)</td><td>Fisher Scientific</td><td>BP1605-100</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Immunomagnetic separation of APCs and non-APCs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5X MojoSort Buffer (MS buffer)</td><td>BioLegend</td><td>480017</td><td></td></tr><tr><td>5 ml MojoSort Magnet (MS magnet)</td><td>BioLegend</td><td>480019</td><td></td></tr><tr><td>100 &amp;micro;L MojoSort Streptavidin Nanobeads</td><td>BioLegend</td><td>480015</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Purity Check and FACS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10X Red Blood Cell Lysis Buffer</td><td>eBioscience</td><td>00-4300-54</td><td></td></tr><tr><td>Fc block (Mouse CD16/CD32)</td><td>eBioscience</td><td>553141</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biotin CD45</td><td>BioLegend</td><td>103103</td><td>Concentration: &amp;le; 0.25 &amp;micro;g per 10^6 cells&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: 30-F11</td></tr><tr><td>Biotin CD31</td><td>BioLegend</td><td>102503</td><td>Concentration: &amp;le; 0.25 &amp;micro;g per 10^6 cells&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: MEC13.3</td></tr><tr><td>Biotin CD9</td><td>BioLegend</td><td>124803</td><td>Concentration: &amp;le; 0.25 &amp;micro;g per 10^6 cells&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: MZ3</td></tr><tr><td>Biotin LY6C</td><td>BioLegend</td><td>128003</td><td>Concentration: &amp;le; 0.25 &amp;micro;g per 10^6 cells&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: HK1.4</td></tr><tr><td>CD31-PerCP/Cy5.5</td><td>BioLegend</td><td>102419</td><td>Concentration: Dilution 1:400&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: 390</td></tr><tr><td>CD45-PerCP/Cy5.5</td><td>BioLegend</td><td>103131</td><td>Concentration: Dilution 1:400&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: 30-F11</td></tr><tr><td>CD140b PDGFR&amp;beta;-PE</td><td>BioLegend</td><td>136006</td><td>Concentration: Dilution 1:50&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: APB5</td></tr><tr><td>LY6C-APC</td><td>BioLegend</td><td>128016</td><td>Concentration: Dilution 1:400&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: HK1.4</td></tr><tr><td>CD9-FITC</td><td>BioLegend</td><td>124808</td><td>Concentration: Dilution 1:400&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: MZ3</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cell Culture and Differentiation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Gonadal APC Culture media (for 500mL)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>288 mL DMEM with 1 g/L glucose</td><td>Corning</td><td>10-014-CV</td><td></td></tr><tr><td>192 mL MCDB201</td><td>Sigma</td><td>M6770</td><td></td></tr><tr><td>10 mL Fetal bovine serum (FBS)** lot#14E024</td><td>Sigma</td><td>12303C</td><td></td></tr><tr><td>5 mL 100% ITS premix</td><td>BD Bioscience</td><td>354352</td><td></td></tr><tr><td>5 mL 10 mM L-ascorbic acid-2-2phosphate</td><td>Sigma</td><td>A8960-5G</td><td></td></tr><tr><td>50 &amp;micro;L 100 g/ml FGF-basic</td><td>R&amp;amp;D systems</td><td>3139-FB-025/CF</td><td></td></tr><tr><td>5 mL Pen/Strep</td><td>Corning</td><td>30-001-CI</td><td></td></tr><tr><td>500 &amp;micro;L Gentamycin</td><td>Gibco</td><td>15750-060</td><td></td></tr><tr><td>**NOTE: The adipogenic capacity of primary APCs can vary from lot to lot of commercial FBS. Multiple lots/sources of FBS should be tested.</td><td></td><td></td><td></td></tr></tbody></table>

isolation of adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal adipose depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes two strategies that allow for the isolation of distinct stromal cell populations from intra-abdominal WAT depots of adult mice. APCs and FIPs can be isolated by FACS (Figure 1) or immunomagnetic bead separation with biotinylated antibodies (Figure 2). Both approaches utilize reagents and antibodies that are all commercially available. Immunomagnetic bead separation leads to the separation of adipogenic from non-adipogenic cells from the g...

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Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

isolation-adipogenic-fibro-inflammatory-stromal-cell-subpopulations

Research

JoVE Journal

Immunology and Infection

2.5K Views.  Novo Nordisk A/S. Dit protocol beschrijft de technische benadering om adipogene en fibro-inflammatoire stromale celsubpopulaties te isoleren uit intra-abdominale witte vetweefseldepots (WAT) van muizen door middel van fluorescentie-geactiveerde celsortering of immunomagnetische parelscheiding.

Video: Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

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. ...

Developmental Biology

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

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 ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Medicine

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 ...

Biology

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

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.

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown ...

Culturing Murine Adipocytes and Explants for Functional and Imaging Studies

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and mobilization, thermoregulation, and the secretion of endocrine factors. The expansion of white adipose tissue due to caloric imbalance results in both the enlargement of existing adipocytes and the generation of additional adipocytes from adipocyte progenitor cells. Obesity-driven changes to white adipose tissue, including those affecting adipocytes, are associated with numerous comorbidities, such as type 2 diabetes and 13 types of cancer. A significant barrier to studying how adipocytes contribute to disease is the inability to readily isolate and culture mature adipocytes. This article describes a protocol to isolate murine lean and obese adipocytes from the subcutaneous and visceral fat depots of male and female C57BL/6 mice. The protocol details how isolated primary adipocytes can be cultured in a membrane adipocyte aggregate system for up to 2 weeks, facilitating their functional analysis in co-culture experiments, lipolysis assays, or through the collection of conditioned media containing adipocyte-secreted factors. Additionally, the protocol outlines methods for culturing adipose tissue explants in basement membrane matrix domes and imaging primary isolated adipocytes. Importantly, this approach can be integrated with existing protocols for the isolation of adipose tissue-resident adipocyte&#160;progenitor cells using fluorescence-activated cell sorting (FACS). Together, these protocols provide researchers with tools to functionally study adipocytes, adipocyte progenitor cells, and whole adipose tissue from lean and obese, male and female mice.

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

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