Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Summary

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

Abstract

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 – including single-cell RNA-sequencing and single-cell proteomics – 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.

Introduction

Adipose tissue plays a key role in energy metabolism. Excess energy is stored in the form of lipids, and adipose tissue is capable of significant expansion or retraction depending on nutritional status and energetic demand. Expansion of adipose tissue can result from an increase in adipocyte size (hypertrophy) and/or from an increase in adipocyte number (hyperplasia); the latter process tightly regulated by proliferation and differentiation of adipose progenitor cells^1,2. During obesity, adipose tissue excessively expands, and tissue dysfunction – including hypoxia, inflammation, and insulin resistance – often develops^3,4. These complications are risk factors to many chronic diseases including hypertension, diabetes, cardiovascular diseases, stroke, and cancer⁵. Hence, limiting uncontrolled adipose tissue expansion and mitigating adipose tissue pathologies are top biomedical research priorities. During adipose tissue expansion, resident adipose tissue-derived stem cells (ASCs) proliferate and differentiate sequentially into preadipocytes (committed progenitor cells) and then into mature adipocytes⁶. Recent single-cell RNA-sequencing (scRNA-seq) studies show that these adipose progenitor cell (APC) populations (ASCs and preadipocytes) exhibit substantial molecular and functional heterogeneity^{7,8,9,10,11,12}. For example, ASCs display a reduced adipogenic differentiation capacity, while also exhibiting higher proliferation and expansion capabilities, compared to preadipocytes⁷. Further molecular differences are reported within ASC and preadipocyte populations, although the functional relevance of these differences remains unclear⁷. Together, these data highlight the complexity of the adipose progenitor cell pool and underscore the need to develop and standardize tools to better understand and manipulate these critical cell populations.

This protocol details the isolation of high viability Sca1⁺ adipose progenitor cell populations from mouse epididymal fat pads that are suitable for sensitive downstream analyses, including single-cell studies (scRNA-sequencing) and cell culture. Isolation and dissociation of epididymal fat pads was performed as previously described^7,13 with slight modifications that improve the viability of isolated APCs. In brief, dissociated cells from epididymal fat pads are stained with antibodies against Sca1, a marker for both ASCs and preadipocytes^6,7, and other lineage (Lin) markers: Ter119 (erythroid cells), CD31 (endothelial cells), and CD45 (leukocytes). Viable Sca1⁺/Ter119^-/CD31^-/CD45^-/DAPI^- cells are then sorted by fluorescence activated cell sorting (FACS). Importantly, this protocol was validated by successful isolation and analysis of viable Sca1⁺/Lin^- adipose progenitor cells reported in a recent single cell RNA sequencing study that identified functionally heterogeneous subpopulations within ASCs and preadipocytes⁷.

Protocol

All animal experimental procedures were performed under approval by the Mayo Clinic Institutional Animal Care and Use Committee.

1. Solution preparation

1. Prepare collagenase 2% (w/v) solution by dissolving collagenase II 2 g in 100 mL Hanks' balanced salt solution (HBSS). Aliquot 200 µL each for each use.
2. Prepare neutralization medium by mixing 84 mL F-12 medium, 15 mL horse serum, and 1 mL penicillin/streptomycin.
3. Prepare flow cytometry buffer by dissolving 500 mg of bovine serum albumin (BSA) and 400 µL of 0.5 M EDTA in 100 mL of Dulbecco's phosphate-buffered saline (DPBS).

2. Dissection of epididymal fat pad and tissue dissociation

1. Humanely euthanize (isoflurane/cervical dislocation) one male mouse. In this protocol, a four-month-old FVB mouse was used.
2. Apply/spray 70% ethanol on the abdomen and expose the lower abdominal cavity using clean scissors and forceps.
3. Locate epididymal fat pad (proximal/attached to testes). Hold the junction between testes and epididymal fat pad with blunt forceps and pull gently to liberate the epididymal fat pad.
4. Remove testes by cutting using scissors and incubate epididymal fat pad in 5 mL of HBSS supplemented with 3% BSA in a 50 mL conical tube at room temperature for 15 min.
5. Centrifuge at 150 x g for 7 min at 4 °C.
6. Take floating epididymal fat pad from the 50 mL conical tube and finely mince the tissue using clean scissors.
7. Add 200 µL collagenase 2% solution into 3.8 mL of HBSS in a 13 mL culture tube. Add the minced tissue and incubate in a rotating incubator at 5 rpm for 1 h at 37 °C. Instead of a rotating incubator, cell culture incubator at 37 °C can be alternatively used with occasional agitations.
8. Transfer entire contents into a 50 mL conical tube and add 10 mL of neutralization medium. Mix gently by inverting the tube 2-3 times.
9. Prepare another 50 mL conical tube fitted with a 70 µm cell strainer. Filter the digested tissue into the new tube using the cell strainer.
10. Transfer the flow-through to a 15 mL conical tube and centrifuge at 350 x g for 10 min at 4 °C.
11. Carefully remove the supernatant and resuspend cell pellets with 5 mL of DPBS. Centrifuge at 350 x g for 10 min at 4 °C.
12. Carefully remove the supernatant and add 50 µL of flow cytometry buffer. Mix well by pipetting gently and keep cells on ice. Due to the volume of the cell pellets and small amount of residual supernatant, total volume of cells in the flow cytometry buffer will be greater than 50 µL (approximately 100 µL).

3. Antibody labeling and fluorescence activated cell sorting (FACS)

1. After mixing the cells well with gentle pipetting, transfer 54 µL of the cell suspension into a 1.7 mL microcentrifuge tube. Add 6 µL of FcR blocking reagent and mix well by pipetting gently. Incubate at 4 °C for 10 min. Save any leftover cells after taking 54 µL and keep them in the ice to use as an unstained control in step 3.5 and step 3.6.
2. During the incubation in step 3.1, prepare an antibody cocktail by combining 11 µL each of anti-Sca1-APC, anti-Ter119-FITC, anti-CD31-FITC, and anti-CD45-FITC antibodies in a microcentrifuge tube. Mix well by gentle pipetting and keep on ice protected from light.
3. After incubation with FcR blocking reagent for 10 min, add 40 µL of the antibody cocktail and mix well by gentle pipetting. Incubate at 4 °C for 10 min.
4. Add 500 µL of flow cytometry buffer supplemented with 1 µg/mL of DAPI into the stained cells. Mix well by gentle pipetting and filter cells using a 5 mL test tube with cell strainer snap cap. Keep cells on ice.
5. Add 500 µL of flow cytometry buffer to remaining cells in step 3.1 and mix well by gentle pipetting. Filter cells using a 5 mL test tube with cell strainer snap cap. Keep these cells on ice and use as an unstained control in the following step.
6. Take the stained cells and unstained control to FACS analysis or sorting instrument.
   1. Identify and isolate the APC⁺/FITC^-/DAPI^- population with gating strategies shown in Figure 1. These cells represent viable Sca1⁺/Ter119^-/CD31^-/CD45^- cells. Debris and cell aggregates are depleted using forward (FSC) and side scatter (SSC) plots. Then, DAPI^- population is gated, followed by gating of APC⁺/FITC^- population. Unstained controls should be used to aid in setting gating parameters.
   2. Collect the cells into 500 µL of flow cytometry buffer in a 1.5 mL microcentrifuge tube. Moving forward, all procedures should be performed under sterile conditions to minimize a contamination. Approximately 50,000 – 100,000 cells are collected from one mouse.
7. Count cells using a hemocytometer to evaluate cell viability. Since cell aggregates can interfere with further downstream analyses, the presence of cell aggregates is also evaluated to ensure a minimal presence (<5% of viable cell number) of these aggregates.

Results

Four-month-old male FVB mice were used in this experiment. After exclusion of debris and doublets using FSC/SSC plots, viable cells (DAPI^- population) were gated, followed by the selection of APC⁺/FITC^- population (Figure 1). DAPI, APC, and FITC gates were drawn based on the unstained control. Gating strategies are shown in Figure 1.

After 1 h of sorting, the quality of isolation was quantitatively eva...

Discussion

Single cell RNA sequencing (scRNA-seq) is rapidly gaining traction as a powerful tool to simultaneously study diverse cell populations at the single cell level. Due to high costs associated with sample preparation and high throughput sequencing, it is imperative to optimize cellular inputs (high viability and purity) to increase the likelihood of experimental success. Some cell preparation protocols rely on removal of dead cells and debris using low-spin washes and column-based separation without FACS sorting

Materials

Name	Company	Catalog Number	Comments
1.7 mL microcentrifuge tube	VWR	87003-294
13 mL culture tube	Thermo Fisher Scientific	50-809-216
15 mL conical tube	Greiner Bio-one	188 271
5 mL test tube with cell strainer snap cap	Thermo Fisher Scientific	08-771-23
50 mL conical tube	Greiner Bio-one	227 261
70 µm cell strainer	Thermo Fisher Scientific	22-363-548
Anti-CD31-FITC antibody	Miltenyi Biotec	130-102-519
Anti-CD45-FITC antibody	Miltenyi Biotec	130-102-491
Anti-Sca1-APC antibody	Miltenyi Biotec	130-102-833
Anti-Ter119-FITC antibody	Miltenyi Biotec	130-112-908
BSA	Gold Biotechnology	A-420-500
Collagenase type II	Thermo Fisher Scientific	17101-015
DAPI	Thermo Fisher	D1306
Dulbecco's phosphate-buffered saline (DPBS)	Thermo Fisher Scientific	14190-144
F-12 medium	Thermo Fisher Scientific	11765-054
FcR blocking reagent	Miltenyi Biotec	130-092-575
Hanks' balanced salt solution (HBSS)	Thermo Fisher Scientific	14025-092
Horse serum	Thermo Fisher Scientific	16050-122
Penicillin-streptomycin	Thermo Fisher Scientific	15140-122
Propidium iodide solution	Miltenyi Biotec	130-093-233