Primary MSC culture conditions
The Mayo Clinic Institutional Review Board (IRB) approved protocols for the collection of fat biopsy or bone marrow from consenting donors and clinical trial participants utilized in this study. Adipose- or bone marrow-derived mesenchymal stromal cells were isolated as previously described [Camilleri et. al. 2016; Dudakovic et. al. 2014; Crespo-Diaz et. al. 2011]. Cells were expanded using standard operating procedures used for the culture of MSCs for clinical use. Primary AMSC or BMSCs were maintained in standard culture media composed of Advanced MEM (Gibco) with 5% human platelet lysate (Mill Creek Life Sciences), 2 mM L-glutamine (Invitrogen), 2U/mL heparin (NovaPlus®) and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin) and maintained at 37°C in 5 % CO2. Table 1 describes the patient information of AMSCs utilized in these studies.
Flow cytometry for MSC characterization
AMSCs and BMSCs were harvested from culture for flow cytometry characterization as previously described [Camilleri et. al. 2016]. Briefly, ~1 × 106 cells were pelleted and the supernatant was removed, before incubation with 50μL of mouse serum for 5 min at room temperature. Primary antibody mixes were prepared as described in Supplemental Table 1 and were added to respective tubes, mixed, and incubated for 15 min at room temperature in the dark. Stained cells were washed with a total of 3 ml of PBSFE [PBS with 5 mM NaEDTA (Sigma) and 1% bovine serum albumin (Sigma)], centrifuged, and supernatant was discarded. Cells were resuspended in 200μL of 1% paraformaldehyde (Electron Microscopy Sciences) prior to acquisition. The Beckman Coulter Gallios (Beckman Coulter) flow cytometer was used to acquire 50,000 AMSC events and the Kaluza software (Beckman Coulter) was used to define population gates (% Gated) and mean fluorescence intensity (MFI) values.
Magnetic and Fluorescence Cell Sorting
CD36+ AMSCs were enriched using either magnetic or fluorescence-sorting techniques and specified in the results section. For both isolation techniques, the CD36-APC antibody (A87786, Beckman Coulter) was utilized. Magnetic cell sorting was performed on three AMSC donors (540, 258, 211) at passage four or five. Prior to sorting, cells were maintained in standard culture medium and expanded in T-175 flasks. Cells were collected by TrypLE (Gibco), centrifuged, and washed once with ice cold Advanced MEM with no supplements. After centrifugation, cells were resuspended in ice cold Advanced MEM and filtered using a 40µm cell strainer. Cells were counted and 1x107 cells were transferred to a new tube and loosely pelleted, while the supernatant was discarded. Cells were gently agitated, 50µL of CD36-APC antibody was added, quickly vortexed to mix, and incubated for 15 minutes in the refrigerator in the dark. Following incubation, cells were washed twice with ice cold Advanced MEM. Cell pellets were gently agitated and 20µL of anti-APC microbeads (MACS Miltenyi Biotec) were added per 1x107 cells. After brief vortexing, cells were incubated for 15 minutes at 4°C in the dark. Cells were then washed as described above and up to 1x108 cells were resuspended in 500µL of Advanced MEM. CD36 positive cells were selected using the autoMACS Separator (MACS Miltenyi Biotec) and the negative fraction was also collected. Immediately following magnetic sorting, fractions of cells were analyzed by flow cytometry to evaluate CD36 levels. Cells were incubated with CD90-FITC (BD Pharmingen), CD140b-PE (PDGFRB; BD Biosciences), CD44-PerCP Cy5.5 (BD Pharmingen), and CD36-APC (Beckman Coulter) in the dark for 15 minutes. Following incubation, cells were washed with PBS and centrifuged, while the supernatant was discarded. Antibody labelled cells were resuspended in PBS for analysis on the FACS Calibur (BD Biosciences) flow cytometer. Supplemental table 2 shows the enrichment of CD44+/CD36+ cells for the magnetic separated AMSC populations.
Fluorescence sorting of one AMSC donor (540) was performed. AMSCs at passage five were expanded in vitro in T-175 flasks until 90% confluent. Cells were harvested, filtered with a 40µm cell strainer, counted, then pelleted and resuspended in PBS, and 4x106 cells were transferred to a new tube. Cells were then pelleted and resuspended in 400µL of PBS and stained with CD36-APC (Beckman Coulter) as previously described. Following incubation, cells were washed with PBS and resuspended in 500µL for cell sorting. The BD FACSAria (BD Biosciences) sorter was used to isolate AMSCs based on CD36 staining intensity: negative, dim, and bright. Cells were sorted into growth medium and then expanded as previously described. Supplemental table 2 shows the enrichment of the populations.
AMSCs were seeded onto 6-well plates at 3,000 cells/cm2 in filtered (0.2µm) medium and placed in the Incucyte (Satorius) system maintained at 37°C in 5% CO2. The Incucyte was programmed using the Confluence (v1.5) package to image cells every three hours to assess cell culture confluence. Doubling time was calculated by fitting a line to the slope when cells were between 40-80% confluent with R2 greater than 0.98.
Expression of cell surface markers, CD44 or CD36, on AMSCs were visualized using immunofluorescence staining. Briefly, AMSCs were seeded onto glass coverslips and allowed to adhere until ~90% confluent. Cells were fixed with 4% paraformaldehyde, washed in PBS with 0.2% v/v Tween-20, and blocked with 10% normal goat serum (NGS) in PBS with 0.2% v/v Tween-20 for one hour. Primary antibodies CD44 (BD Pharmingen, 559046) or CD36 (Abcam, ab23680) were diluted in wash buffer (1% NGS in PBS with 0.2% v/v Tween-20) added to coverslips and incubated overnight at 4°C. Coverslips were rinsed with wash buffer and secondary antibodies diluted in wash buffer were added to coverslips, including goat anti-mouse AlexaFluor 488 (ThermoFisher) or donkey anti-mouse AlexaFluor® 555 (ThermoFisher). Coverslips were incubated for one hour at room temperature in the dark, washed with PBS, and mounted with ProLong Gold with DAPI (Invitrogen) onto a microscope slide. Fluorescence images were obtained using an inverted Zeiss LSM 780 laser-scanning confocal microscope (Carl Zeiss).
AMSCs were seeded on 6- or 12-well plates at 3,000 cells/cm2 in standard culture medium until plate was confluent. Upon confluence (day 0), medium was replaced with osteogenic induction medium [standard culture medium supplemented with 50µg/mL ascorbic acid, 4mM beta glycerophosphate, and 10nM dexamethasone] and fresh osteogenic medium was changed every three days. Calcium deposition was detected using Alizarin Red staining at day fourteen as previously described [Dudakovic et al 2015].
AMSCs were seeded on 6- or 12-well plates at 3,000 cells/cm2 in standard culture medium until plate was confluent. Upon confluence (day 0), standard medium was replaced with adipogenic medium that consisted of standard medium supplemented with StemXVivo Adipogenic Supplement (R&D systems). Adipogenic medium was replaced every three days until further analysis. Lipid droplet formation was evaluated at day fourteen using Oil Red O staining as previously described [Dudakovic et al 2015].
Sulfosuccinimidyl oleate sodium (SSO) inhibitor studies
The CD36 inhibitor SSO (Santa Cruz Biotechnology) was reconstituted with DMSO, and DMSO was used as a negative control for these experiments. To optimize the SSO concentration, AMSCs were seeded onto 96-well plates and upon confluence were treated concentrations ranging from 12.5µM to 200µM SSO or DMSO. Viability/metabolic activity was evaluated after 24 hours using CellTite96®AQueous non-radioactive cell proliferation (MTS) assay (Promega). For osteogenic differentiation with DMSO or 200µM SSO, AMSCs were plated onto 12-well plates in standard medium at 3,000 cells/cm2. The following day, medium was replaced with standard medium containing either DMSO or 200µM SSO. After three days (day 0), medium was replaced with osteogenic medium as described above and new osteogenic medium replaced every three days. Alizarin Red staining was performed at day fourteen.
Mass Spectroscopic Analysis of Acylcarnitines and Tricarboxylic Acid (TCA) metabolites
CD36+ enriched, CD36+ depleted, or unsorted AMSCs were established as described above for donor 211 expanded in T-175 flasks. For mass spectroscopic analysis, AMSCs and subpopulations were trypsinized and five cell pellets each containing 5x106 cells were snap frozen in LN2 and stored at -80°C before analysis.
Acylcarnitines analysis was performed as previous described [Chace et. al. 2001]. Briefly, cell pellets were lysed in 1xPBS then internal standard solution was added. Proteins were removed by adding a solution of methanol/acetonitrile (v/v) to the sample mixture. The sample was centrifuged at 12,000 rpm for 10 mins at 4 °C, supernatant transferred to a 1 dram vial, and dried with N2. Samples were reconstituted and analyzed on a Waters Acquity UPLC system coupled with a Thermo Quantiva tandem mass spectrometer in positive (H)ESI mode. Concentrations of carnitine (162.11 to 85.02 m/z), acetylcarnitine (204.12 to 85.02 m/z), propionylcarnitine (218.14 to 85.02 m/z), butyrylcarnitine (232.15 to 85.02 m/z), isovalerylcarnitine (246.17 to 85.02 m/z), octanoylcarnitine (288.22 to 85.02 m/z), lauroylcarnitine (344.28 to 85.02 m/z), myristoylcarnitine 372.36 to 85.02 m/z), palmitoylcarnitine (400.39 to 85.02 m/z), oleoylcarnitine (426.39 to 85.02 m/z), and stearoylcarnitine (438.39 to 85.02 m/z) were measured against a 11-point calibration curve that underwent the same preparation.
Analysis of TCA was performed as described previously [Koek et. al. 2006]. Briefly, cell pellets were washed with 1x PBS twice prior to being lyzed in 1x PBS after spiking in an internal solution containing U-13C labeled analytes. The proteins were removed by adding 250 µL of chilled methanol and acetonitrile solution to the sample mixture. After drying the supernatant in the speed vac, the sample was derivatized with ethoxime and then with MtBSTFA + 1% tBDMCS (N-Methyl-N-(t-Butyldimethylsilyl)-Trifluoroacetamide + 1% t-Butyldimethylchlorosilane) before it was analyzed on an Agilent 5975C GC/MS (gas chromatography/mass spectrometry) under electron impact and single ion monitoring conditions. Concentrations of lactic acid (m/z 261.2), fummaric acid (m/z 287.1), succinic acid (m/z 289.1), oxaloacetic acid (m/z 346.2), ketoglutaric acid (m/z 360.2), malic acid (m/z 419.3), cis aconitic acid (m/z459.3), citric acid (m/z 591.4), and isocitric acid (m/z 591.4), glutamic acid (m/z 432.4) were measured against a 7-point calibration curves that underwent the same derivatization.
Transmission Electron Microscopy
CD36+ enriched, CD36+ depleted, and unsorted AMSCs were seeded onto 6-well plates at approximately 3,000cells/cm2 and allowed to proliferate for three days. AMSC subcellular structures were assessed using digital electron microscopy (Phillips CM10) at the Mayo Clinic Electron Microscopy Core. Briefly, cells were fixed in Trump’s fixative and mounted on mesh grids. Six-eight representative fields of view from each of the populations were randomly selected and visually examined.
Real-time reverse transcriptase quantitative PCR analysis
Total RNA was isolated from AMSCs using Trizol® Reagent (Thermo) and purified using the Direct-zol mini kit (Zymo). The SuperScript III First-Strand Synthesis System (Invitrogen) was used to reverse transcribe RNA into cDNA and used as template for real-time PCR analysis. Real-time reactions were performed with 10 ng cDNA per 10 μl with the QuantiTect SYBR Green PCR Kit (Qiagen) and gene specific primers [Supplemental Table 3]. Reactions were detected using the CFX384 Real-Time System (BioRad). Gene expression levels were normalized to the housekeeping gene, AKT1, and quantified using the 2^(−delta delta Ct) method.
High throughput RNA sequencing
Total RNA from AMSC subpopulations were subjected to RNA-sequencing using the TruSeq RNA Sample Prep Kit v2 (Illumina) and were analyzed using Illumina HiSeq 2000 with TruSeq SBS Kit v3 and HCS v2.0.12 data collection software. Sequence data were processed using MAPRSeq (v.1.2.1) and a bioinformatics workflow (TopHat 2.0.6, HTSeq, and edgeR 2.6.2), where expression data were normalized using the fragments per kilobase per million (FPKM) method. Differential gene expression analysis was performed using gene count data and analyzed with DESeq2 (v1.26.0) package in RStudio (v1.2.5033) with a p-value set at 0.05.
Statistical analyses were performed using Prism 9.2 (GraphPad) software and nonparametric, Mann-Whitney test was used to determine differences in qPCR and metabolomics data. Linear regression was used to analyze Incucyte population doubling data at the exponential phase of cell growth.