hiPSC culture and differentiation

hiPSCs (passage 9) procured from ATCC (American Type Culture Collection) were plated at 2 × 104 cells/cm2 in 6-well plates coated with 2 mg/mL Matrigel Matrix (Corning). hiPSC culture and differentiation were carried at 37 °C, under atmospheric oxygen tension (21% O2) and in 5% CO2. 10 ng/mL Rock Inhibitor Y27632 (Tocris Bioscience) was initially added to the medium (NutriStem) for 24 h to improve survival. Cells without Rock Inhibitor were then cultured for an additional 2–3 days until 70–80% confluency was reached. hiPSCs were passaged using ReLeSR (StemCell Technologies), seeded on 12-well Matrigel-coated plates, and differentiated towards a distal lung phenotype (diPSCs), following our lab’s previously published protocol [22]. Briefly, hiPSCs were exposed to growth factors to induce Definitive Endoderm (DE), Anterior Foregut Endoderm (AFE) and Lung Progenitor (LP) differentiation over 25 days. Following AFE induction phase, cells were split using TrypLE (ThermoFisher) to remove uncommitted cells and improve purity as previously described [22]. Cell characterization during the differentiation process is shown in Additional file 1: Fig. 1.

Collection of cell-conditioned media and EV isolation

hiPSC-conditioned media was collected from cells after 48 h of culture. diPSC-conditioned media was collected following the 25-days differentiation process outlined above; at that time, cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, ThermoFisher) supplemented with human serum replacement 50x (Millipore Sigma), penicillin/streptomycin solution 1×, Glutamax 1×, Non-essential Amino Acids 1× (Gibco, ThermoFisher), recombinant human fibroblast growth factors FGF2, FGF7, FGF10, and bone morphogenetic protein BMP4 (all growth factors from Peprotech, used at a final concentration of 10 ng/mL).

EVs were isolated from hiPSC- and diPSC-conditioned media by ultrafiltration (UF) combined with size-exclusion chromatography (SEC). Starting with 12 mL of cell culture supernatant, each sample was centrifuged sequentially at 500×g for 10 min to remove cellular debris, 10,000×g for 10 min to remove large microvesicles, then 3260×g for 12 min using Amicon Ultra-15 Centrifugal Filter Unit with a 50-kDa molecular weight cutoff (Millipore Sigma). In the last step, EVs were recovered in a total volume of 500 µL. Once the chromatography column (qEV original 35 nm, IZON) was equilibrated to room temperature and flushed with phosphate buffered saline (PBS), the concentrated EV sample was loaded, and the run-through collected. The buffer volume was first discarded, then three purified EV fractions (500 µL each) were collected. Fractions were characterized as follows below, then stored in PBS at − 20 °C. The second and third fractions (F2 and F3) were found to have the highest EV yield and were subsequently used in the lung explant experiments.

EV characterization

The isolated EVs were characterized using four modalities: transmission electron microscopy (TEM), imaging flow cytometry (IFC), nanoparticle tracking analysis (NTA), and mass spectrometry (MS).

Transmission electron microscopy (TEM)

Carbon-coated copper grids were used for EV adsorption. Glow discharge was performed using a Denton Vacuum 502B to enhance adhesion, then 5 µL of EVs were deposited on each grid and allowed to adsorb for 2 min. After three brief washes with distilled water, grids were negatively stained with two droplets of 1% Uranyl Acetate and air-dried. Imaging was carried out using a Hitachi H-7650 transmission electron microscope at 80 kV and AMT Image Capture Engine.

Imaging flow cytometry (IFC)

EV samples were stained with the following labels and monoclonal antibodies: CFDA-SE (carboxyfluorescein diacetate succinimidyl ester, ThermoFisher) at 2 µM, antiCD81-FITC (ThermoFisher) at 0.75 µg/mL (1:40 dilution), and antiCD63-PE (ThermoFisher) at 0.31 µg/mL (1:80 dilution).

CFDA-SE is a pan-EV label used to assess EV integrity. When CFDA-SE crosses intact membranes, the acetate group is cleaved by intra-vesicular esterases, resulting in the highly fluorescent CFSE molecule. CD81 and CD63 are tetraspanins present on the surface of some, but not all, EVs [23].

Monoclonal antibodies were first centrifuged at 17,000×g for 30 min (using centrifugal filter units with a 0.22-µm pore size, Millipore), to eliminate clumps or aggregates [24]. Antibody titrations were then performed using a series of dilutions in PBS (1:5, 1:10, 1:20, 1:40, 1:80, 1:160) to identify the optimal concentration that results in the highest signal-to-noise ratio. Fluorescent labels were added to 7.5 µL of EVs at the concentrations mentioned above to achieve a final volume of 30 µL per sample. Negative controls of buffers and antibodies (PBS alone, PBS with CFDA-SE, PBS with antiCD81-FITC, PBS with antiCD63-PE) were run to ensure the purity of PBS and the absence of antibody clumps. Single-stained samples were used to create compensation matrices. Double-staining of EVs with CFDA-SE/antiCD63-PE and antiCD81-FITC/antiCD63-PE were performed. Since significant spectral overlap exists between CFSE and FITC, co-staining with these labels was not done.

Multispectral imaging acquisition was performed using Amnis ImageStreamX MKII (EMD Millipore) [25, 26]. Calibration beads were run with each sample to verify optimal instrument performance. Fluidics were set to low speed, sensitivity to high, and magnification to 60×. High gain mode was turned on. Controls and EV samples were acquired over one minute each. Compensation matrices were created in the absence of brightfield illumination and side-scatter. Data analysis was performed using Image Data Exploration and Analysis Software (IDEAS).

Nanoparticle tracking analysis (NTA)

EV concentration and size distribution were measured by our collaborators at the University of Vermont (Weiss Lab) using a ZetaView NTA (Particle Metrix). All samples were diluted in Nanopure water to a final volume of 1 mL. 100-nm uniform beads were run as a positive control and for quality check before loading the samples. PBS run through the chromatography column was also tested for the presence of contaminants from the column. The captured videos were analyzed using ZetaView software.

Protein quantification and mass spectrometry (MS)

EV samples were lysed using radioimmunoprecipitation assay (RIPA) buffer and sonication. BCA assay (bicinchoninic acid assay kit, ThermoFisher) was performed to quantify proteins in the samples. Lysed EVs were then incubated overnight in 33% TCA (trichloroacetic acid) for protein denaturation. The next day, samples were centrifuged at 20,000×g for 20 min. The pellet was re-suspended in 500 µL of Acetone, and centrifuged again at 20,000×g for 20 min. The precipitated proteins were air-dried for 5 min then re-suspended in 1 M urea for mass spectrometry analysis.

Liquid chromatography–Mass Spectrometry (LCMS) was performed at the University of Connecticut proteomic core facility. Submitted samples were reduced with 5 mM dithiothreitol at room temperature, and alkylated with 10 mM iodoacetamide at room temperature, protected from light. Modified porcine trypsin protease (Promega #V5113) was added at a ratio of 1:20 w/w enzyme: protein at 37 °C and incubated overnight, and the digestion was quenched with formic acid. Peptides were then desalted by reverse-phase chromatography using Pierce peptide desalting spin columns (Thermo Fisher part #89870), dried completely, and resuspended in Solvent A (0.1% formic acid in Fisher Optima LC/MS grade water). Samples were quantified by A280 absorbance and total injection amount was normalized to 500 ng across samples.

Peptides were subjected to mass analysis using a Thermo Scientific Ultimate 3000 RSLC nano ultra-high performance liquid chromatography (UPLC) system coupled to a high-resolution Thermo Scientific Q-Exactive HF mass spectrometer. Each sample was injected onto a 25 cm C18 column held at 50 °C and separated by reversed-phase UPLC using a gradient of 4–90% Solvent B (0.1% formic acid in Fisher Optima LC/MS grade acetonitrile) over a 60-min gradient at 300 nL/min flow, followed by a 10-min wash and 20-min column re-equilibration. Peptides were eluted directly into the QE-HF using positive mode nanoflow electrospray ionization. MS1 scan parameters were set to 60,000 resolution, 1e6 AGC target, maximum ion time of 60 ms, and a mass range of 300–1800 m/z. MS2 data were acquired in data-dependent Top15 mode using the following parameters: 15,000 resolution, maximum ion time of 40 ms, isolation window of 2.0 m/z, 30 s dynamic exclusion window, normalized collision energy of 27, a scan range of 200–2000 m/z, and charge exclusion “on” for all unassigned, +1, and > + 8 charged species.

Peptides were identified using MaxQuant (v1.6.10.43) and its embedded Andromeda search engine and quantified by label-free quantification [27]. The raw data were searched against both the complete UniProt Homo sapiens reference proteome (identifier UP000005640, accessed 11Jan2022) and the MaxQuant contaminants database. Variable modifications allowed oxidation of Met, acetylation of protein N-termini, deamidation of Asn/Gln, and peptide N-terminal Gln to pyroGlu conversion. Carbamidomethylation of Cys was set as a fixed modification. Protease specificity was set to trypsin/P with a maximum of 2 missed cleavages. All results were filtered to a 1% false discovery rate at the peptide and protein levels using the target-decoy approach; all other parameters were kept at default values. MaxQuant output files were imported into Scaffold (Proteome Software, Inc.) and IPA (Ingenuity Pathway Analysis, Qiagen) for data visualization and subsequent analyses.

Fetal lung tissue explant model

All animal experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Connecticut Health Center (Protocol number AP-200408-0224). C57/BL6 mice were bred and monitored for the presence of a plug. The day of vaginal plug visualization was designated as E0.5 (embryonic day 0.5). Dams were euthanized at E17.5 using CO2 narcosis, and laparotomy was performed to extract the fetuses. E17.5 corresponds to 24–26 weeks of human gestation, where lungs are at the late canalicular or early saccular stage of their development [10, 28,29,30]. Given the relative resistance of fetuses to the effects of CO2, cervical dislocation was also performed on fetuses following their extraction to ensure death prior to thoracotomy and lung tissue collection. Dams and fetuses’ death was confirmed by the cessation of respiration and loss of reflexes.

Fetal lung lobes were collected and washed in PBS and Hanks Balanced Salt Solution (HBSS). Ex vivo culture was carried at an air–liquid interface using an adapted protocol for culture of murine embryonic lungs [31]. Briefly, lung explants were placed over a trans-membrane insert and embedded in 15 µL of 8 mg/mL Matrigel solution (Corning). Plates were incubated at 37 °C for 2 h to allow for Matrigel gelation. DMEM:F12 medium (80:20) supplemented with 10% FBS (fetal bovine serum), penicillin/streptomycin solution 1×, Glutamax 1×, FGF2 at 25 ng/mL, FGF7 at 10 ng/mL and FGF10 at 25 ng/mL, was then added below the inserts. These growth factors concentrations were previously shown to promote alveolarization and angiogenesis in in vitro fetal lung models [32,33,34,35]. Culture in Matrigel at an air–liquid interface at 37 °C, as described, preserved the viability of the explant model for up to 7 days, as shown with Calcein-Ethidium tissue staining (Additional file 1: Fig. 2).

Experimental designExposure to hyperoxia

After 4 h of in vitro culture in Matrigel under atmospheric oxygen tension (21% O2, 5% CO2) at 37 °C, plates were transferred into a modular incubator chamber MIC-101 (Billups-Rothenberg). Two petri dishes filled with sterile water were also placed inside the chamber to allow for humification. The chamber was then sealed and purged with a gas mixture of 95% O2/5% CO2 at 7 L/min for 13 min at 2 psi. Flushing this volume allows for a complete gas exchange. Once the purge was completed, the inlet port was disconnected from the gas source, and both inlet and outlet ports were clamped. The chamber was then placed back at 37 °C. The MIC-101 is designed to maintain constant gas levels for a minimum of 72 h. Explant models were exposed to 24 h of 95% O2 to induce airspace disruption and enlargement.

Following 24 h of hyperoxia exposure, the plates were taken out of the modular incubator chamber, and the media below the membrane inserts was replaced and supplemented with either hiPSC-EVs or diPSC-EVs at 5 × 106 particles per mL of media. Particle abundance in the EV preparations was determined by NTA. All samples were maintained during the treatment phase for 48 h at 37 °C, in 21% O2 and 5% CO2. The experimental design is outlined in Fig. 1. Controls included explants that remained in 21% O2 since their collection, and explants that were exposed to hyperoxia but left untreated. Histologic and genotypic assessment was performed on day 3 of in vitro culture.

Fig. 1

Experimental design. Fetal pups were randomly assigned to one of the 4 listed groups. The principal investigator was aware of the group allocation at different stages of the experiment (no blinding was performed)

Histologic assessment

Three samples from each group (normoxia, hyperoxia without treatment, hyperoxia followed by hiPSC-EV treatment, and hyperoxia followed by diPSC-EV treatment) were assessed histologically on day 3. Tissues were first vacuum-inflated to preserve their architecture during sectioning. Briefly, lung explants were transferred into labeled Eppendorf tubes filled with 10% formalin. The tubes were then placed in a vacuum desiccator, and negative pressure was applied for 20 min. This degassing technique has been reported in the literature [36, 37], and results in homogeneous lung inflation.

Following their inflation, tissues were embedded in OCT (Optimal Cutting Temperature compound, Fisher Healthcare) and stored at − 80 °C until sectioning. 10 µm slices were sectioned using a LEICA-CM3050S Cryostat, fixed in methanol/acetone mixture, stained with Hematoxylin and Eosin (H&E), and imaged on a Zeiss Observer inverted microscope at 10× magnification. Brightfield images were acquired, then processed stereologically using ImageJ software. Briefly, an 80-point grid system was applied to each image, and the following data were extracted: number of points hitting nonparenchymal (vascular or bronchial) structures, number of points hitting septal surfaces, and number of intercepts between septal structures and grid lines. The Mean Linear Intercept (MLI) was then derived from this data, as described in the literature [38].

Immunohistochemistry was performed on additional tissue sections to detect alterations in vasculature structure. The markers used were DAPI (4’,6-diamidino-2-phenylindole, for nucleic acids staining) and PECAM (platelet and endothelial cell adhesion molecule, for endothelial cell staining). Stained sections were also imaged on a Zeiss Observer inverted microscope at 10× magnification.

Genotypic assessment

Gene expression was assessed on day 3 using qRT-PCR. Tissues were lysed in buffer RLT (Qiagen), homogenized using a Bead Mill (Fisher Scientific), then centrifuged at 20,000× g for 3 min. The supernatant was then collected and stored at − 80 °C until RNA was extracted. An RNeasy Kit (Qiagen) was used for RNA isolation. RNA quality and concentration were checked with a Nanodrop One Spectrophotometer (ThermoFisher). Following DNAse treatment (BioRad) and reverse transcription (T100 Thermal Cycler, BioRad) PCR reactions were prepared, using the following primers: Prdx5 (peroxiredoxin-5, BioRad), Nfe2l2 (nuclear factor erythroid 2-related factor 2, also known as NRF2, BioRad), and VEGFa (vascular endothelial growth factor A, BioRad). The housekeeper GAPDH (BioRad) was used as the internal control gene. PCR reactions were run using a CFX96 Real-Time System (BioRad), and results processed with BioRad CFX Maestro software.

Statistical analysis

Sample size was based on our initial observations that, following hyperoxia exposure, hiPSC-EVs, added to the medium at a concentration of 5 × 106 particles/mL for 48 h, ameliorate airspace enlargement by around 30%. To achieve a study power of 80% with 5% significance for detecting a true improvement, a minimum of three samples per group were required. For statistical analysis, we used one-way Analysis of Variance (ANOVA) and Sidak Multiple Comparison Test (GraphPad). A P-value ≤ 0.05 was considered significant.

EV proteomic data was analyzed using Scaffold Q+ as described above. Differentially expressed proteins were determined by applying Mann–Whitney Test with unadjusted significance level p ≤ 0.05.