IPF patients (group of 5) were identified from Interstitial Lung Diseases Outpatient Clinic by an expert pulmonologist in IPF and other fibrotic diseases of the lung following the ATS/ERS/JRS/ALAT Statement criteria . All aspects of this study involving samples from IPF patients and age and gender matched healthy volunteers were reviewed and approved by the Mayo Clinic Institutional Review Board. All subjects provided written informed consent to participate.
Patients Characteristics for Adipose Tissue collection and MSC isolation
Isolation, Propagation and Identification of Adipose MSCs (aMSCs)
Abdominal wall adipose tissue (approximately 1.5 - 2.5 g) was obtained under sterile conditions from IPF patients and age and gender matched healthy donors in an outpatient surgical suite. Tissues were processed with two hours of procurement. Cells were expended ex vivo according to the protocol based on Standard Operation Procedures for isolation, extraction and expansion of aMSCs analogs for clinical use [30, 31]. In brief, after micro dissection the fat tissue was digested with collagenase Type I at 0.075% w/v, (Worthington Biochemicals, Lakewood, NJ) for 1.5 h at 37°C. Adipocytes were separated from the vascular fraction by centrifugation (400 x g for 5 min, at room temperature). The cell pellet was washed with PBS and passed through cell strainers (70μm followed by 40μm, BD Biosciences, Franklin Lakes, New Jersey). The resulting cell fraction was plated in T-75 cm2 flasks (Thermo Fisher, Waltham, MA) and incubated in a fully humidified incubator supplied with 5%CO2 in PLGold xeno-free media. The xeno-free media named ”PLGold media” consists of: Advanced MEM (Thermo Fisher Scientific) supplemented with 5% (v/v) PLTGold, (Mill Creek Life Sciences, Rochester, MN), 1% (v/v) GlutaMax (Thermo Fisher) and 1% (v/v) antibiotics (100 U/ml penicillin, 100 g/ml streptomycin, HyClone, Logan, UT). Cells were propagated when they were 60-80% confluent using TrypLE (Trypsin-like Enzyme, Invitrogen, Carlsbad, CA) . Cell yield and viability was quantified using Acridine Orange (AO) and Propidium Iodide (PI) nuclear stains for exclusion assay on a Luna-FL Dual Fluorescence Cell Counter (all from Logos Biosystems, Annandale, VA). All aMSCs used in the experimental procedures were between passages two and five.
The proliferation and growth rate of aMSCs, was monitored by adding IncuCyte NucLight Rapid Red Reagent for Nuclear Labeling at 1:500 dilution (Essen Bioscience, Ann Arbor, MI) to the media. After a 30-minute incubation at 370C in a fully humidified incubator supplied with 5% CO2, it was placed in the IncuCyte S3 Live Cell Analysis instrument (Sartorius, Ann Arbor, MI) for fluorescent quantification of cell proliferation. Fluorescent images of red nuclei from sixteen fields in each well were captured at 681nm every six hours with 10x objective. Each cell count was repeated twice in four replicas. The data acquisition, visualization and analysis were done using internal IncuCyte S3 Analyzing Software. The growth rate kinetic and doubling times (td) were evaluated recording the cell proliferation rate by counting the number of red nuclei every six hours for a duration of five days. The population doubling time (td) was calculated by computing the linear regression of N=N0 x ekt, where N is the number of red nuclei count at time t, N0 is red nuclei count at time t = 0, and k is cell growth rate per hour.
Cell phenotype was analyzed by labeling them with primary fluorochrome-conjugated monoclonal antibodies, as previously described [30, 32, 33]. Samples were analyzed using the Beckman Coulter Gallios 3-laser, 10-color flow cytometer and Kaluza 2.1 software (Beckman Coulter, Chaska, MN).
The antibody used, fluorophores, vendors, catalog numbers and dilutions are listed in Table 1.
* The antibodies are added into the samples without dilution, as per manufacturer instructions.
Cells were seeded at 6100 cells/cm2 in a sterile eight well chamber (Cellvis, Sunnyvale, CA) for 48 hours. Fresh media containing 350 nM MitoTracker Red CXMRos (Invitrogen) was added to the cells and incubated 30 minutes in a fully humidified incubator supplied with 5% CO2, followed by washing with PBS. Cells were fixed by adding 10% buffered Formalin (Azer Scientific, Morgantown, PA). The covered glass chambers wrapped in aluminum foil were kept for 30 minutes on a rocker at room temperature. PBS washed cells were permeabilized with 0.3% TRITON X-100 in PBS containing Hoechst 33342 (1:1000 dilution, Thermo Fisher) and AlexaFluorTM 488 Phalloidin (1:1500 dilution, Thermo Fisher). Aluminum foil wrapped covered glass chambers were kept for 30 minutes on a rocker at room temperature. Cells were washed with PBS and kept in PBS to prevent them from drying during imaging.
A laser scanning confocal microscope was used to collect 2D and 3D cell images LSM 780 and ZEN 2010 software (Carl Zeiss, NY). For quantification of the cells mitochondrial volume five individual aMSCs from each cell line [total 25 cells from Healthy Controls (HCaMSCs) and 25 cells from IPF patients (IPFaMSCs)] were imaged under the same acquisition conditions: image size (512 x 512 pixels), number of Z-stack slices (16 slices, 7.692 mm), number of averaging Z-stuck slices (averaging 2 Z-stuck slices), scan zoom (X: 1.0, Y: 1.0), pinhole sizes and laser intensities (1.42 AU for 405 nm laser at 50% intensity, 1.19 AU for 488 nm laser at 50% intensity, and 0.99 AU for 561 nm laser at 40% intensity) using C-Apochromat 63x/1.2W Korr objective.
Detection was carried out at wavelengths of 406-480 nm for Hoechst 33342 (nuclear stain), 499-560 nm for AlexaFluorTM 488 Phalloidin (actin F stain) and 566-696 nm for MitoTracker Red CXMRos (mitochondria stain). For image analysis and mitochondria volume calculations Image Data Management Software Imaris 8 (Oxford Instruments, Abingdon, GB) was used. Unpaired Student t-test analysis was used to determine the statistical difference in mitochondria volumes between aMSCs of the two tested groups.
Adipogenic Differentiation Capacity
Cells, passages 2 and 3, previously cultured in PLGold media, were cultured for two consecutive passages in MSC NutriStem® XF Basal Medium containing MSC NutriStem® XF Supplement Mix (named “MSC NutriStem® XF Medium” Biological Industries, Cromwell, CT) supplemented with 5% (v/v) PLTGold before seeding at 5260 cells/cm2 in a CellBIND 24 well plate (Corning, Corning NY). After 4 days, media in the wells with cells assigned for adipogenesis was replaced with MSCgo™ Adipogenic Differentiation Medium (Biological Industries, Cromwell, CT) containing Adipogenic Differentiation Supplement Mix I and Adipogenic Differentiation Supplement Mix II (Biological Industries). Control cultures were maintained in MSC NutriStem® XF Medium. Cells were kept for six days in the respected media, changing media once before adding fresh respected media containing 1:500 dilution of IncuCyte NucLight Rapid Red Reagent for Nuclear Labeling and 1:1000 dilution of LipidSpotTM 488 Lipid Droplet Stain (Biotium, Fremont, CA). After 30 min incubation at 370C in a fully humidified incubator supplied with 5% CO2 plates were placed in an IncuCyte S3 Live Cell Analysis instrument for red nuclei count and green lipid droplets total green integrated intensity (GCU) imaging using 20 x objective. Fluorescent images of red nuclei (imaged at 681 nm) and green lipid GCU (imaged at 585 nm) from 16 fields in each well were captured every six hours for 24 hours. The data acquisition, visualization and analysis were done using internal IncuCyte S3 Analyzing Software. Each GCU value per well was normalized to the number of cells (red nuclei count) per well.
Secretome Analysis of Resting aMSCs
aMSCs were seeded at 2105 cell/cm2 in 6 well pates with 2.5 ml PLGold media/well. After 48 hours, the media was replaced with 2 ml fresh media/well. One well containing media only was used as a control for media content of analytes, which values were used as a background in secretome analysis.
After four days (96 h), media was collected, spun for five minutes at 750 x g and supernatants were stored at -200C until use. Immediately after collecting the media, 1 ml of fresh media containing 1:500 diluted IncuCyte NucLight Rapid Red Reagent was added to the cells. Cell number (red nuclei count) was counted in an IncuCyte S3 Live Cell Analysis instrument. For determining aMSCs secretome content, a 20plex custom made kit (Human Cytokine/Chemokine, Human Bone and Adipokine Magnetic bead panel, EMD Millipore, Burlington, MA), and Luminex xMAP technology (R&D Systems Inc., Minneapolis, MN) were used. The secretome assay was done in triplicate and was performed following the manufacturer's instructions. The plates were read by the MAGPIX instrument using xPONENT software for acquisition (Luminex, Austin, TX). Data analysis of the Median Fluorescence Intensity (MFI) and Coefficient of Variance (CV%) estimation were done by MILLIPLEX Analyst 5.1 software (EMD Millipore). The analyte concentrations (pg/ml) were normalized to 1x106 cells.
Analysis of aMSCs Senescence Status by Droplet Digital Polymerase Chain Reaction (ddPCR)
For establishing the senescence status of aMSCs we developed a Droplet Digital PCR (ddPCR) protocol for estimating the transcription level of CDKN1, p16INK4A, p53, and RB1 cell cycle inhibitor markers. Total RNA from 1.5 x 106 cell pellets was extracted using RNeasy Mini Kit (Qiagen, Germantown, MD). The reverse transcription reaction was performed with random, Oligo(dT)20, primers using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). For ddPCR reactions fluorescent labeled custom designed primers and probe for p16INK4A:
p16INK4A forward primer: 5’ GCC CAA CGC ACC GAA TAG 3’,
p16INK4A reverse primer: 5’ ACG GGT CGG GTG AGA GTG 3’, and
p16INK4A probe: FAM6-TCA TGA TGA TGG GCA GCG CC-TAMRAIowaBlack, (IDT, Coralville, IA) were used.
For the other tested cell cycle inhibitor markers as well as for TATA Binding Protein (TBP) as reference gene, commercially available fluorescent labeled expression primers and probes were used (Bio-Rad).
The ddPCR reaction setup was as previously described  .The final concentration of primers and probes in the reactions were 900 nmol/L and 250 nmol/L, respectively. Multiwall plates were sealed, vortexed briefly, centrifuged and placed on an automated droplet generator (AutoDG- Bio-Rad). Each sample was partitioned into 15,000-20,000 droplets. PCR amplification was performed on a Veriti Thermal Cycler (Applied Biosystems). The initial heating at 95°C for 10 minutes was followed by 60 cycles of denaturation at 94°C for 30 seconds, annealing and extension at 58°C for 1 minute, and a final extension step at 98°C for 10 minutes. The completed reactions were stored at 4°C until reading them on a QX200 droplet reader (Bio-Rad). Data analysis was performed using 2D Module of the QuantaSoft software (BioRad).
Quantitative Flow Cytometry Assay for Immunoprofiling
A separate group of 15 IPF patients were identified from the Interstitial Lung Diseases Outpatient Clinic by an expert pulmonologist in IPF and other fibrotic diseases of the lung following the ATS/ERS/JRS/ALAT Statement criteria . All aspects of this study involving samples from IPF patients and age and gender matched healthy volunteers were reviewed and approved by the Mayo Clinic Institutional Review Board. All subjects provided written informed consent to participate.
Data are presented as mean ± SD, FVC% predictive = % of Forced Vital Capacity; FEV1% predictive = % of Forced Expiratory Volume in the 1st second, predictive; VCmax % predictive = % Maximal Vital capacity.
“Predictive” means values adjusted for patient age, gender, and race.
To characterize the circulating immune phenotype, peripheral blood samples from 87 healthy volunteers (30 of which age and gender matched) and from a separate group of 15 IPF patients were collected in K2EDTA tubes (Becton Dickinson, Franklin Lakes, NJ) at initial or return visits. Un-manipulated whole blood was stained with antibodies directly within 12 hours of collection. Appropriate antibodies, undiluted (vendors, catalog numbers and amounts added per sample are listed in Additional File 1 and in [35, 36]) were added directly to the blood samples. Quantitative flow cytometry was performed to comprehensively assess 110 leukocyte populations and phenotypes from lymphocytes, monocytes, and granulocytes. All 10-color procedures, antibodies, flow protocols, instrument settings, and gating strategies for peripheral blood flow cytometry have been previously described by Gustafson et al. [35, 36]. The flow cytometry data were analyzed using Kaluza 2.1 software (Beckman Coulter), allowing quantification of the absolute number as well as percent of immune cell subtypes. Fluorescently labeled isotypes were used as a control for each tested cell line.
Results are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 8 software. Intergroup comparisons of parametrically distributed continuous data were done using un-paired two-tailed Student's t- test. Differences were considered significant when p values *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Correlations between IPF patients’ Pulmonary Function Test (PFT) values were established by calculating the Pearson correlation coefficient (r). Flow cytometry data are either represented as percentage of population or number of cells/ml. ddPCR data and presented as mean of three with CV%.