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

Podsumowanie

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.

Streszczenie

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 PDGFRβ+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, “FIPs”) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFRβ+ 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.

Wprowadzenie

White adipose tissue (WAT) represents the principal site for energy storage in mammals. Within this tissue, adipocytes, or “fat cells,” 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 homeostasis^1,2,3. Adipocytes constitute the bulk of WAT volume; however, adipocytes only represent less than 50% of total cells found in WAT^4,5. 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 hypoxia^8,9. 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, “adipogenesis”). Adipocyte hyperplasia coincides with relatively lower degrees of WAT fibrosis and metabolic inflammation^6,11. 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 PDGFRα, PDGFRβ, 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 WAT^14,15,16,17. From our own scRNA-seq and functional analyses, we have identified and characterized functionally distinct immune-modulating and adipogenic PDGFRβ+ perivascular cell subpopulations in the stromal compartment of intra-abdominal WAT in adult mice¹⁵. Fibro-inflammatory precursors, or FIPs, represent a prominent subpopulation of PDGFRβ+ cells and can be isolated based on LY6C expression (LY6C+ PDGFRβ+ 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- PDGFRβ+ 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.

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Protokół

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

1. Dissect the gonadal white adipose tissue from 6-8-week-old mice and place fat pads in 1x PBS solution.
2. 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 µL of digestion buffer (1x HBSS, 1.5% BSA and 1 mg/mL collagenase D).
3. Transfer the minced tissue to a 50 mL centrifuge tube containing 10 mL digestion buffer.
4. Incubate mixture at 37 °C in a water bath for 1 h while shaking.
5. Filter the digested tissue through a 100 µm cell strainer to remove undigested tissue.
6. Dilute filtered sample to 30 mL with PBS containing 2% FBS, and centrifuge at 600 x g for 5 min at 4 °C. Aspirate the supernatant, which contains mature adipocytes.
7. Proceed immediately to preferred cell isolation method.

2. Isolation of APCs and FIPs using FACS

1. Dissolve the pellet in 1 mL of 1x RBC lysis buffer by shaking the tube gently.
2. Incubate at RT for 1-2 min.
3. Add 10 mL of 2% FBS/PBS and pass through a 40 µm cell strainer into a clean 50 mL centrifuge tube.
4. Centrifuge at 600 x g for 5 min at 4 °C.
5. Aspirate media and resuspend the pellet in 400-800 µL of 2% FBS/PBS containing Fc block (1:200).
6. Incubate at 4 °C for 10 min.
7. Transfer 400 µL-800 µL of cell suspensions containing ≤10⁶ cells per mL to 1.5 mL tubes and add antibodies. Antibody concentrations are listed in the Table of Materials section.
   1. Prepare separate control tubes for 1) unstained cells, 2) single color controls, and 3) FMO (fluorescence minus one) controls.
   2. Stain the samples with PDGFRβ, CD45, CD31, CD9, LY6C antibodies.
8. Incubate at 4 °C for 15 min protected from light.
9. Centrifuge at 600 x g for 5 min at 4 °C.
10. Aspirate media and resuspend cells in 400 µL of 2% FBS/PBS.
11. Centrifuge at 600 x g for 5 min at 4 °C.
12. Aspirate media and resuspend cells in 400-800 µL of 2% FBS/PBS. Then pass through 40 µm filter caps into 5 mL polystyrene round-bottom tubes for FACS.
13. Follow the following steps for gating.
    1. Use unstained and single-color controls for compensation.
    2. Use FMO controls to set experimental gates.
    3. Use the gating strategy shown in Figure 1 to obtain APCs and FIPs. Gate APCs as CD45^- CD31^- PDGFRβ⁺ LY6C^- CD9^- and FIPs as CD45^- CD31^- PDGFRβ⁺ LY6C⁺ cells.
14. Collect sorted cells in tubes containing 500 µL of 100% serum and maintain the cells on ice.
15. Centrifuge cells at 600 x g for 5 min at 4 °C. Add 2% FBS/PBS to wash the cells by centrifuging again at 600 x g for 5 min at 4 °C. Discard the supernatant use the pelleted cells.
16. Resuspend the pelleted cells in the appropriate volume of gonadal APC culture media (for cell culture) or appropriate buffer for subsequent analysis.

3. Immunomagnetic separation of adipogenic and non-adipogenic fractions

1. Isolate SVF from gonadal white adipose tissue following steps 1.1-1.7.
2. Aspirate the supernatant and resuspend the SVF pellet in 10 mL of 1x commercially available MS Buffer.
3. Filter the cell suspension through a 40 µm cell strainer.
4. Centrifuge at 600 x g for 5 min at 4 °C.
5. Aspirate the supernatant and resuspend pelleted cells in 100 µL of 1x MS Buffer.
6. Perform CD31⁺ and CD45⁺ cell depletion as discussed below (Figure 2A, Step 1). This is done to remove endothelial and hematopoietic lineage cells.
   1. Count cells using a cell counter and adjust the concentration to ≤ 1 x 10⁸ cells/mL.
   2. Add the biotin conjugated CD31 and CD45 antibodies to the cell suspension, each at a concentration of ≤ 0.25 µg per 10⁶ cells and incubate on ice for 15 min.
   3. Wash cells by adding 4 mL of 1x MS Buffer.
   4. Centrifuge at 600 x g for 5 min at 4 °C.
   5. Aspirate the supernatant and resuspend the pellet with 100 µL of 1x MS buffer.
   6. Add 10 µL of streptavidin nanobeads and incubate cells on ice for 15 min.
   7. Wash cells by adding 4 mL of 1x MS buffer.
   8. Centrifuge at 600 x g for 5 min at 4 °C, then aspirate the supernatant.
   9. Resuspend cells in 2.5 mL of 1x MS buffer and transfer to a 5 mL polypropylene tube.
   10. Place the tube in the magnet rack for 5 min.
   11. 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.
   12. Remove the tube from magnet and repeat steps 3.6.9. to 3.6.11. two more times for a total of 3 washes.
7. Separation of adipogenic and non-adipogenic fractions (Figure 2A, Step 2)
   1. Continue working with unlabeled fraction (i.e., CD45^- CD31^- fraction).
   2. Centrifuge the 15 mL tube containing unlabeled cells at 600 x g for 5 min at 4 °C.
   3. Aspirate the supernatant and resuspend the pellet in 100 µL of 1x MS buffer.
   4. Add biotin conjugated LY6C and CD9 antibodies, respectively, at a concentration of ≤ 0.25 µg per 10⁶ cells and incubate on ice for 15 min.
   5. Follow steps 3.6.3. to 3.6.12. to complete the second separation.
   6. Collect unbound fractions. Unlabeled cells (LY6C^- CD9^-) represents adipogenic fraction containing APCs (Figure 2A, Step 3).
   7. 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).
   8. Centrifuge labeled and unlabeled fractions at 600 x g for 5 min to pellet cells at 4 °C.
8. Proceed immediately to cell culture (step 6) or use undifferentiated precursors for preferred analyses (step 4 and/or step 5).

4. Assess purity of adipogenic and non-adipogenic fractions by flow cytometry

1. Resuspend bead-isolated cell fractions in 200-400 µL of 2% FBS/PBS containing Fc block (1:200).
2. Incubate at 4 °C for 10 min.
3. Transfer the cell suspension to 1.5 mL tubes and add antibodies. Antibody concentrations are listed in the Table of Materials.
   1. Prepare separate control tubes for 1) unstained cells, 2) single color controls, and 3) FMO controls.
   2. To the samples, add CD45, CD31, CD9, and LY6C antibodies.
4. Incubate at 4 °C for 15 min protected from light.
5. Centrifuge at 600 x g for 5 minutes at 4 °C.
6. Aspirate supernatant and resuspend cells in 400 µL of 2% FBS/PBS.
7. Centrifuge the suspension at 600 x g for 5 min at 4 °C.
8. Aspirate the supernatant and resuspend cells in 400-800 µL of 2% FBS/PBS. Filter through 40 µm filter caps into 5 mL polystyrene round-bottom tubes for analysis by flow cytometry.
9. Flow cytometry analysis to assess purity
   1. Use unstained and single-color controls for compensation.
   2. Use FMO controls to set experimental gates.
   3. Use the gating strategy for live and single cells as shown in Figure 2B.
   4. Perform the gating as shown in Figure 2B for CD45, CD31, LY6C, and CD9.
   5. Use flow cytometry software to evaluate the frequencies of CD45^- CD31^- LY6C^- CD9^- cells (APCs) and CD45^- CD31^- LY6C⁺ cells (FIPs) within isolated fractions.

5. Gene expression analysis using quantitative PCR to assess purity of FIPs and APCs

1. Extract mRNA from magnetically or FACS isolated cells using commercially available extraction kit (see Table of Materials) following the manufacturer’s instructions.
2. Use 1 µg of RNA to synthetize cDNA using a cDNA Reverse Transcriptase kit (see Table of Materials) by following the manufacturer’s instructions.
3. Determine relative mRNA levels of APC and FIPs-selective genes (Table 1) by quantitative PCR using SYBR green PCR master mix.
   1. Set up the sample reaction for PCR as follows: 5 µL of 2x SYBR green dye, 1 µL of cDNA (~50 ng/ µL), 0.5 µL of each forward and reverse primer (10 µM) and 3 µL of water.
   2. Use the following standard PCR conditions for the quantitative PCR run: hold stage: 50 °C for 2 min, 95°C for 10 min; PCR stage (40 cycles): 95 °C for 15 s, 60 °C for 1 min.
4. Normalize values to house-keeping gene (Rps18) levels by calculating ΔΔ-Ct.
5. To evaluate statistical significance, perform unpaired Student’s t-test.

6. Cell culture and differentiation

1. Centrifuge magnetically- or FACS- isolated cells at 600 x g for 5 min at 4 °C.
2. Resuspend the pellet in 500 µL of gonadal APC culture media and plate 40K cells/well from each fraction in a 48 well culture plate.
3. 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.

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Wyniki

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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Dyskusje

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 manner^19,20,21,

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Materiały

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