Strategies for Measuring Induction of Fatty Acid Oxidation in Intestinal Stem and Progenitor Cells

Abstract

This protocol describes a multipronged approach that we have created to determine transcriptional induction of fatty acid oxidation (FAO) genes in Lgr5^high intestinal stem cells and a subsequent metabolomics-based approach for assessing fatty acid utilization in the mammalian intestinal crypt. More specifically, we describe methods for crypt isolation followed by a FACS-based purification of stem and progenitor populations and RNA-Sequencing analysis. Using this workflow, we can determine both basal gene expression profiles of key metabolic genes, as well as corresponding changes in response to altered metabolic states, such as fasting. Subsequently, we describe a complementarily metabolomics-based approach that we have developed to assess fatty acid uptake and utilization in the crypt using ¹³C stable isotope tracing. Combining these approaches, one can gain a better understanding of substrate utilization and the preceding transcriptional changes that accommodate these reactions in physiologic states of low carbohydrate utilization or during overabundance of dietary lipids.

1. Introduction

Understanding metabolic demands and fuel preferences in stem, progenitor and differentiated cells can help elucidate the role of metabolism in stem cell renewal and differentiation. From numerous previous studies we now appreciate that the metabolic state of adult stem cells can be quite different than that of adjacent progenitor or terminally differentiated cells [1, 2]. One possibility to account for these differences is that adult stem cells may have unique metabolic gene expression signature or metabolic flexibility, resulting in different metabolic flux compared to differentiated cells [3]. These dissimilarities could also be the consequence of higher proliferation in comparison to more terminally differentiated and post-mitotic cells. In addition, environmental factors and paracrine signaling from adjacent niche cells can also play a significant role in somatic stem cell nutrient sensing and metabolism [4].

Measuring metabolic activities of adult stem cells in vivo remains a significant challenge due to i) the low analyte abundances and limited sample volumes of the rare cell populations and ii) the small observation window of rapid metabolic changes. As cells regulate gene expression in response to environmental changes, gene-expression profiles of metabolic pathways have been used as the surrogate measurements of metabolic rearrangements in response to physiological status. Population RNA-Sequencing (RNA-Seq), that can amplify the transcript sequences of the rare cells, therefore, becomes a popular method to study metabolic features of adult stem cells. However, despite a strong association between metabolic gene expression and metabolic phenotypes, experimental evidence shows that gene expression levels alone cannot accurately predict metabolic activities [5]. Therefore, subsequent validation by stable isotope labeling using stem-cell enriched tissue is crucial for providing a complementary metabolomic insight.

In this chapter, we describe the workflow (Fig. 1) that we have developed to identify and validate upregulation of fatty acid oxidation genes (FAO) in intestinal stem cells following a 24-hour fasting protocol [1]. Understanding how these metabolic states in adult stem cells become altered in response to environmental signals or in diseased states can potentially lead to therapeutic opportunities in regenerative medicine, as well as diseases such as cancer [3, 6-8].

Fig. 1.

Flowchart for isolation and dissociation intestinal crypts followed by transcriptional and mass spec-based analysis of FAO induction.

Ad libitum control mice or fasted mice were sacrificed and small intestine was dissected. Crypts were isolated following chemical and mechanical dissociation and further dissociated into a single cell suspension. Flow based cell sorting enriches for intestinal stem and progenitor cells which are collected for RNA-Seq analysis. In parallel, purified crypts, which are enriched for intestinal stem and progenitor cells, are used for stable isotope tracing experiments to assess fatty acid oxidation and contribution to TCA cycle intermediates.

2. Materials

2.1. Crypt isolation materials

1. 1x PBS (without calcium and magnesium)

2. 15 cm cell culture plates

3. Glass microscope slides

4. 20 mL luer syringe

5. Gavage needle (18G)

6. Sterile surgical tools

7. 70-micron mesh

8. 0.5 mM EDTA pH 8.5

9. Inverted Microscope

10. 48 well cell culture plates

2.2. Dissociation Methods

1. TrypLE™ Express Enzyme (1x)

2. S-MEM

3. 40 Micron mesh

2.3. Antibody and Viability Staining

1. Antibody cocktail:

   CD31-PE (2 ul)(0.2 mg/mL)

   CD45-PE (2 ul)(0.2 mg/mL)

   TER119-PE (2 ul)(0.2 mg/mL)

   CD326-APC (Epcam) (0.2 mg/mL)

   CD24-pacific blue (3μL) (0.2 mg/mL)

   CD117-APC-Cy7 (3μL) (0.2 mg/mL)

2. 7-Aminoactinomycin D (7-AAD)

3. S-MEM

4. 1000 μL filter pipette tip

5. FACS tube: Falcon Round-Bottom Tubes with Cell Strainer Cap, 5 mL

2.4. Flow-sorting Lgr5^high intestinal stem cells

1. Low-binding 1.5 mL microcentrifuge tubes

2. TRIzol™ Reagent

3. Aria3: 4 laser, 11 color sorter running BDFACS Diva software on XP. Can sort into 1.5 mL, 5 mL, 15 mL, or 96 well plates. Can sort up to 4 populations at one time.

2.5. RNA purification of flow sorted cells for RNA-sequencing

1. Chloroform

2. Glycoblue

3. 75% Ethanol

4. nuclease-free water (not DEPC-treated)

5. Centrifuge with temperature control (Eppendorf Microcentrifuge 5430 R)

6. Chemical fume hood

2.6. Clustering analysis and heatmap generation for FAO gene expressions

Software:

1. R 3.4.0

2. javaGSEA

3. Qlucore Omics Explorer 3.2

2.7. Isotope labeling of purified crypts

1. RPMI

2. Murine EGF 40 ng mL⁻¹

3. Recombinant Murine Noggin 200 ng mL⁻¹

4. R-spondin 500 ng mL⁻¹

5. N-acetyl-L-cysteine 1 μM

6. N2 100X

7. B27 50X

8. Chir99021 10 μM

9. Y-27632 10 μM

10. Sterile Saline prepared with LC-MS grade water

11. 13-C Palmitate

12. 80% methanol (LC/MS grade) containing internal standards (909 nM each of 17 isotopically labeled amino acids)

13. 4 °C centrifuge with 15 mL tube canisters

14. Tissue culture incubator

15. Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer

16. Nitrogen dryer

3. Methods

3.1. Crypt Dissociation Protocol

1. Chill 1x PBS to 4 °C.

2. Mice are euthanized in designated chambers using fixed rates of carbon dioxide flow.

3. Following euthanasia, abdominal side is sprayed with 70% ethanol Surgical tools are prepared, including 20 mL syringe and gavage needle (Fig. 2a).

4. After abdominal incision, small intestine was dissected and placed in ice cold sterile 1xPBS (Fig. 2a). Intestine is then flushed several times with 20 mL of ice cold 1x PBS and cut sagittally to remove any digested food debris (Fig. 2b-d).

5. Once the intestine is opened up, the mucus layer is cleaned gently using the index finger (Fig. 2e).

6. Next, the intestinal tissue is placed 49 mL of ice cold 1x PBS and 1 mL 0.5 mM EDTA for a final concentration of 0.01 mM EDTA in PBS and incubated on ice for 45 min.

7. Following the incubation, intestinal tissue and solution are returned to a 15 cm plastic dish on ice and are scraped using a sterile glass microscope slide. Tissue slurry is passaged through 70-micron mesh continuously to separate villi from dissociated crypts. (Fig. 3a-c). (see Note 1,2).

8. Flow through and crypt fraction is assessed for purity and presence of villi contamination. (see Note 3).

9. The filtrate is spun down at 4 °C at 300 x g in 50 mL conical tube.

Fig. 2.

Crypt isolation procedure (a). 20 mL luer lock syringes fitted for 18G gavage needles are assembled and used in this procedure. (b). Following dissection of the small intestine, tissue is placed in 1x ice cold PBS. (c). Intestine is cleaned by applying pressure and passaging ice cold 1xPBS using the leur lock syringe and gavage needle. (d). The intestine is cut sagittally and mucus and debris are further removed from intestinal lining (e). Following an incubation in PBS containing EDTA on ice, a glass microscope slide is used to gently scrape the intestinal lining and remove villi and crypts (f). Tissue slurry is further filtered through 70 micron mesh to separate crypt fraction from villi. Purified crypts are then either used in metabolomic based analysis for fatty acid utilization using stable isotope tracing or further dissociated and used for gene expression analysis of intestinal stem and progenitor cells using RNA-seq.

Fig. 3.

Gating strategy and workflow described in section 3.4.

3.2. Isolation of Lgr5^high intestinal stem cells (ISCs)

1. To prepare samples for cell sorting by flow-cytometry, intestinal crypts pellets isolated from step 3.1.10 should be transferred to a Falcon® 15 mL centrifuge tubes with 500 μL TrypLE per sample.

2. Crypts will become dissociated after for 1 min incubation with TrypLE at 32 °C.

3. To remove TrypLE, wash cells with 10 mL ice cold S-MEM, invert twice and centrifuged at 300g at 4 °C for 5 min (see Note 4).

3.3. Antibody and viability staining

1. After removing of the supernatant form step 3, resuspend each cell pellet in 250-400 μL of the following antibody cocktail: CD31-PE, CD45-PE, TER119-PE, CD326-APC (Epcam), CD24-pacific blue CD117-APC-Cy7 per 1 mL S-MEM.

2. Pipet vigorously up and down using a 1000 μL filtered pipette tip until most of the cells are dissociated and suspension (see Note 5).

3. Incubate the cell mixture in the antibody cocktail for 15 min on ice.

4. Next, quench and wash cell mixture with 10 mL ice cold S-MEM, invert twice and then centrifuge at 300 x g, 4 °C, 5 min.

5. Resuspend the pellet in 7AAD solution containing 10 μL of 7AAD reagent in 1 mL of S-MEM (amount may vary depending on 7AAD stock concentration). Cell suspension should be approximately 3x the volume of the cell pellet.

6. Next, filter the resuspended cell mixture through 40-micron mesh before transferring the cells through cell strainer cap to a FACS tube (see Note 6).

3.4. Flow-sorting Lgr5^high intestinal stem cells

1. Gating strategy of Lgr5-eGFP^hi, Lgr5-eGFP^low and CD24⁺C-kit⁺ Paneth cells is described below (Fig. 3).

2. First, gate cells for singlets (SSC-A vs. FSC-A, SSC-A vs. SSC-H and FSC-A vs. FSC-H) and further gate for live cells by excluding 7AAD+ dead cells (PerCP-Cy5.5).

3. Next, use the following surface markers to further isolate different cell populations of 7AAD− live cells. Typically, Lgr5+ stem cells and progenitor cells are CD326/ Epcam+ (APC)+, CD24− (Pacific Blue) and GFP^hi (stem) and GFP^low (progenitor) cells. Paneth cells are CD24+(Pacific blue), c-kit+ (APC-Cy7) and SSC^high cells.

4. Select the stem cell (Epcam+/CD24−/GFP^high), progenitor cell (Epcam+/CD24−/GFP^low) and Paneth cell populations (Epcam+/GFP−/CD24+/c-Kit+/SSC^high) populations and sort 25K cells per population into the assigned collection tube (low-binding tubes containing 400 μL TRIzol™ Reagent) with the setting of 4-way purity on the flow cytometry.

5. Following the completion of each 25K sort, vortex and place the collected sample on ice until all sample sorting has been completed (see Note 7).

3.5. RNA purification of flow sorted cells for RNA-sequencing

1. Start this protocol by thawing cells at room temperature from step 3.4.5. (see Note 8).

2. To the 400 μL of Trizol containing 25K of stem cells, add 200 μL chloroform.

3. Vortex vigorously for 15 sec and incubate at RT for 10 min.

4. Centrifuge at max speed (~12,000g) at 4 °C for 15 min.

5. Transfer aqueous phase to a clean, labeled tube.

6. Add 6 μL glycoblue to ~600 μL of the aqueous phase.

7. Add 600 μL Isopropanol to the glycoblue + aqueous samples, mix and store at −20 °C overnight or longer.

8. On the following day, remove the supernatant from the tube, leaving only the RNA pellet (blue).

9. Wash the pellet with 1 mL of 75% ethanol and centrifuge at maximum speed at 4 °C for 15 min.

10. Carefully remove the supernatant and air dry the pellet for 5 min in a chemical fume hood.

11. Resuspend and pool the samples (RNA from 200K cells) in 20 μL nuclease-free water.

3.6. Clustering analysis and heatmap generation for FAO gene expression

1. Normalize aligned reads (against the mm10 murine genome assembly, with ENSEMBL 88 annotation) using the “geometric means” scaling method mplemented in the DESeq R package [9].

2. Analyze potential enrichment of FAO-related gene sets using the command-line version of the GSEA tool developed by Broad Institute.

3. Rank gene sets according to their log2 (FoldChange) values and analyzed using the “pre-ranked” mode of the GSEA software using the following parameters: -norm meandiv -nperm 5000 -scoring_scheme weighted -set_max 2000 -set_min 1 -rnd_seed timestamp.

4. Generate a merged gene expression matrix with the normalized reads and the metadata with sample annotations (column: sample ID/age/diet/cell population) and variable annotations (row: ENSEMBL ID/gene symbol). Save the matrix as Tab Delimited Text file.

5. Prepare the FAO-related gene list by exporting FAO-related gene sets from GSEA database.

6. Combine multiple gene lists and eliminate duplicated genes using R (unique()) or excel (=IFERROR(LOOKUP).

7. Save the merged unique gene list as a variable list (VL_FAO genes.txt file).

8. In Qlucore, open the gene expression matrix (.txt file) with wizard.

9. Select samples “Young_AL_hi” for ISC from ad libitum fed mice (Ctrl), “Young_Fast_hi” for ISC from fasted mice.

10. Import the variable list VL_FAO genes.txt.

11. Click “VL_FAO genes” in the window of “Variable” to show only the normalized reads of genes listed in VL_FAO genes. Under Data, chose “Heat” as method.

3.7. Isotope labeling of purified crypts (see Note 9)

1. 1. Isolated crypts from step 3.1 were divided in 4 equal fractions (50 mL fraction divided in 12.5 mL) and spun down at 200 x g, 4°C for 5 minutes.

2. Next, three of the fractions per each biological replicate are resuspended in 1ml volume of 30 mM ¹³C-Palmitate in RPMI and placed in a 6 well plate.

3. 6 well plates containing crypts and ¹³C labeled Palmitate are incubated at 37 °C in tissue culture incubators for 60 minutes or appropriate time point.

4. Following the incubation, crypts are spun down and washed once with saline.

5. Following the second spin and saline removal, the crypts are resuspended in LC/MS grade 80% methanol solution containing internal standards (909 nM each of 17 isotopically labeled amino acids and vortexed for 10 min). (see Note 10).

6. Samples were then spun down and dried in a vacuum dryer or dried under a stream of nitrogen and can be stored at −80 °C at this point. (see Note 11).

7. Next, samples were resuspended in 100 μL LC/MS grade water and analyzed by LC/MS as described in [10].

8. 2 mL of each sample was injected onto a ZIC-pHILIC 2.1 3 150 mm (5 mm particle size) column.

9. Buffer A is 20 mM ammonium carbonate, 0.1% ammonium hydroxide; buffer B is acetonitrile.

10. The chromatographic gradient was run at a flow rate of 0.150 mL/min as follows: 0–20 min.: linear gradient from 80% to 20% B; 20–20.5 min.: linear gradient from 20% to 80% B; 20.5–28 min.: hold at 80% B.

11. The a QExactive orbitrap mass spectrometer instrument was operated in full-scan, polarity switching mode with the spray voltage set to 3.0 kV, the heated capillary held at 275 °C, and the HESI probe held at 350 °C. The sheath gas flow was set to 40 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 1 unit.

12. MS data acquisition is performed in a range of 70–1000 m/z, with the resolution set at 70,000, the AGC target at 10^6, and the maximum injection time at 80 msec.

13. Relative quantitation of polar metabolites was performed with XCalibur QuanBrowser 2.2 using a 5 ppm mass tolerance and referencing an in-house library of chemical standards.

14. The fraction m+2 acetylcarnitine was calculated as the raw peak area of m+2 acetylcarnitine, divided by the sum of raw peak areas of unlabeled acetylcarnitine, m+1 acetylcarnitine, and m+2 acetylcarnitine.

15. The same calculations are was used for M+2 citrate and other TCA metabolites.