Promising Therapeutic Effects of Embryonic Stem Cells-Origin Mesenchymal Stem Cells in Experimental Pulmonary Fibrosis Models: Immunomodulatory and Anti-Apoptotic Mechanisms

DW-MSCs

Embryonic stem cell-derived mesenchymal stem cells (DW-MSCs, passage No. 12) were generously provided by Daewoong Pharmaceutical (Seoul, Korea). These cells originated from embryonic stem cells (SNUhES35) and were produced at a facility that follows the Good Manufacturing Practices. Following expansion and surface marker characterization, the cells were cryopreserved in liquid nitrogen after passage. DW-MSCs demonstrated the potential to differentiate into chondroblasts, osteoblasts, and adipocytes, and their quality was confirmed using various tests (37). Passage-12 DW-MSCs were used for in vivo experiments, whereas passage-13 and 14 DW-MSCs were used for in vitro experiments.

Animals

All animal experiments were conducted in accordance with the procedures approved by the Animal Experimentation Ethics Committee of Gyeongsang National University (GNU-200619-M0038). Male C57BL/6 mice, aged 8–10 wk, were purchased from Central Laboratory Animals (Seoul, Korea) and acclimated to specific pathogen-free conditions for at least 1 wk. All mice were housed in a room with a 12 h/12 h light/dark cycle, 50%±10% humidity, and a temperature of 22±3°C. The mice were provided with standard rodent chow and water ad libitum. The bleomycin-induced pulmonary fibrosis (BPF) mouse model was used as the animal model for IPF. Anesthesia was induced using avertin (500 mg/kg) and bleomycin (BLM; Dong-A ST, Seoul, Korea) was administered intratracheally (50 μl volume [1 U/kg] dissolved in PBS). DW-MSCs were injected into the tail veins of the mice at different doses and at various disease stages as follows:

A collagen-induced arthritis (CIA) mouse model was also used as an animal model of RA. Seven male DBA/1J mice (9-wk-old) were purchased from Central Laboratory Animals. The primary immunization involved a mixture of bovine type II collagen (catalog No. 20022; Chondrex, Redmond, WA, USA) dissolved in 0.05 M acetic acid, at a concentration of 2 mg/ml, with complete Freund’s Adjuvant (catalog No. F5881; Sigma-Aldrich Co., Toluca, Mexico) in a 1:1 ratio. Intradermal injections were administered at the base of the mouse tail. On day 21, booster injections of a mixture of bovine type II collagen and incomplete Freund’s adjuvant (catalog No. 7002; Chondrex) were administered through intradermal tail injection. CIA mice were randomly divided into PBS- (CIA) and DW-MSC-treated (CIA/DW-MSC) groups. DW-MSCs (1×10⁶) were administered as a single intravenous injection on day 22 after CIA induction.

Histological analysis

The lung tissues were fixed in 10% formalin, embedded in paraffin, and sectioned to 5 μm thickness using a microtome (Microm HM 325; Thermo Fisher Scientific, Waltham, MA, USA). The sections were stained with Masson’s Trichrome and observed under a microscope at 200× magnification. Lung fibrosis was assessed at nine different sites in each lung section based on the modified Ashcroft and fibrosis scores (38). Briefly, the grading was scored on a scale of 0 (normal lung) to 8 (total fibrous obliteration of the field) for the Ashcroft score and 0 (no fibrosis) to 4 (fine connective fibrils in 100% of the area) for the fibrosis score. The percentages of collagen and alveolar spaces were measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and the final score of each section was the average of the scores for different sites.

Immunohistochemical and immunofluorescence staining

For immunohistochemistry, we used the VECTASTAIN Elite ABC Peroxidase Kit (catalog No. PK-6102; Vector Laboratories, Burlingame, CA, USA) and followed the manufacturer’s protocol. Lung tissue slides were deparaffinized and stained with the anti-alpha smooth muscle actin (α-smooth muscle actin [SMA] 0.034 µg/ml, catalog No. ab7817; Abcam, Cambridge, UK) and anti-human nucleoli Ab (catalog No. ab190710; Abcam). Slides were incubated with biotinylated secondary Ab (catalog No. BA-9200; Vector Laboratories) and the 3,3′-diaminobenzidine (DAB) system (catalog No. D5697; Sigma-Aldrich, Saint Louis, MO, USA) was employed for color development. The α-SMA-positive area was quantified using the ImageJ software and the staining was normalized based on the average percentage of the α-SMA-positive area in the control group. Slides were analyzed at 200× magnification using the NIS-Elements software (Nikon, Tokyo, Japan) with a microscope imaging system (Nikon).

To conduct immunofluorescence staining for mitochondrial transfer analysis, the lung tissue sections were deparaffinized and incubated with primary Abs against cytochrome c oxidase subunit 4 (COX-4, catalog No. 4850; Cell Signaling Technology, Beverly, MA, USA) and surfactant protein C (SPC, catalog No. sc-518029; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C. The sections were then incubated with the secondary Abs (catalog No. A21206, anti-rabbit Alexa™ Fluor 488 or catalog No. A21203, anti-mouse Alexa Fluor™ 594; Thermo Fisher Scientific) for 2 h at 20–25°C. The sections were mounted using the ProLong^® Gold Antifade Reagent containing 4′,6-diamidino-2-phenylindole (catalog No. P36935; Thermo Fisher Scientific). Representative images of COX-4- and SPC-positive cells were randomly visualized in four fields using a Nikon Eclipse Ni-U microscope (Nikon), following which the COX-4- and SPC-positive cells were counted.

Western blot analysis

Proteins were extracted from tissues and cells using radioimmunoprecipitation assay buffer (catalog No. 8900; Thermo Fisher Scientific) containing a cocktail of protease and phosphatase inhibitors (GenDEPOT, Baker, TX, USA). The protein lysates (20 μg) were separated using 10%–12% SDS-PAGE and transferred (100 volts, 90 min) onto polyvinylidene difluoride membranes (GE Healthcare, Solingen, Germany). Membranes were blocked using 5% skim milk with Tris-buffered saline containing 0.1% Tween 20 for 1 h and then probed with the primary Abs, anti-Miro-1 (catalog No. NBP1-89011; Novus Biologicals, Centennial, CO, USA), anti-Bcl-2 (catalog No. sc-7382; Santa Cruz Biotechnology), and anti-Bcl-2-associated X (Bax) (catalog No. sc-7480; Santa Cruz Biotechnology), overnight at 4°C. The membranes were incubated with horseradish peroxidase-conjugated secondary Abs (anti-rabbit, catalog No. 1706515 or anti-mouse, catalog No. 1706516; Bio-Rad, Hercules, CA, USA) for 1 h at 20–25°C. The signal was developed using an enhanced chemiluminescence western blotting substrate (Bio-Rad). Quantification was performed using the ImageJ software and normalized against β-actin.

Measurement of hydroxyproline levels

Hydroxyproline levels were measured using a Hydroxyproline Assay Kit (catalog No. 6017; Chondrex) according to the manufacturer’s instructions. Briefly, 10 mg of mouse lung tissue was hydrolyzed overnight at 120°C with 100 μl of distilled water and 100 μl of 10 N HCl. The hydrolyzed sample was centrifuged at 9,425 ×g for 3 min, following which 10 μl of supernatant and 100 μl of 1× Chloramine T solution were added and the mixture was incubated for 20 min at 20–25°C. Thereafter, 100 μl of 1× DAB was added to the sample and the mixture was incubated for 30 min. Optical density was measured at a wavelength of 560 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).

Quantitative real-time PCR

Total RNA was isolated from lung tissues using TRIzol™ reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Subsequently, 1 µg of RNA was reverse transcribed into cDNA using the iScript™ cDNA Synthesis Kit (catalog No. 1708891; Bio-Rad). Real-time PCR was performed on a ViiA™ 7 Real-time PCR Detection System (Applied Biosystems, Foster City, CA, USA) using the TaqMan^® Gene Expression Master Mix (catalog No. 4369016; Thermo Fisher Scientific). The relative expression levels of the target genes were determined using the 2^−ΔΔCt comparative method with glyceraldehyde 3-phosphate dehydrogenase as the internal reference gene. Mouse Taqman^® assays (Applied Biosystems) were employed for the following genes: actin alpha 2, smooth muscle (Acta2, Mm00725412_s1), collagen type I alpha 1 chain (Col1a1, Mm00801666_g1), connective tissue growth factor (Ctgf, Mm01192933_g1), transforming growth factor-beta (Tgfb1, Mm01178820_m1), IL-1β (Il1β, Mm00434228 m1), and glyceraldehyde 3-phosphate dehydrogenase (Gapdh, Mm99999915_g1).

Flow cytometric analysis

Single cells from mouse lungs were isolated using a Lung Dissociation Kit (catalog No. 130-095-927, gentleMACS™; Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instructions. Briefly, PBS-washed lungs were transferred to a gentleMACS™ C tube containing the enzyme mix and dissociated using a gentleMACS™ Octo Dissociator with a heating function (catalog No. 130-096-427; Miltenyi Biotec). The resulting cell lysates were filtered through a 100 μm cell strainer and centrifuged at 500 ×g for 5 min. RBCs were removed by incubating the lysates with RBC lysis buffer (catalog No. 420301; BioLegend, San Diego, CA, USA) for 1 min, followed by centrifugation at 500 ×g for 5 min after the addition of PBS. Single lung cells were resuspended in PBS supplemented with 5 mM ethylenediaminetetraacetic acid and 2% FBS. Total cell counts were determined using a TC20 automated cell counter (Bio-Rad).

For flow cytometry analysis, 1×10⁶ cells were incubated on ice with 100 μl of diluted Ab solution for 30 min. The following Abs were used: anti-CD45-BB515 (1:200, clone 30-F11, catalog No. 584590; BD Biosciences, San Jose, CA, USA), anti-CD11b-APC-Cy7 (1:200, clone M1/70, catalog No. 101226; BioLegend), anti-CD11c-BB700 (1:200, clone HL3, catalog No. 745899; BD Biosciences), anti-Ly-6C-BV605 (1:200, clone AL-21, catalog No. 563011; BD Biosciences), anti-CD4-PerCP-Cy5.5 (1:200, clone GK1.5, catalog No. 100434; BioLegend), anti-CD25-PE (1:200, clone PC61.5, catalog No. 12-0257-42; eBioscience, San Diego, CA, USA), anti-FoxP3-APC (1:200, clone FJK-16s, catalog No. 17-5773-82; eBioscience), anti-IFN-γ-FITC (1:200, clone XMG1.2, catalog No. 11-7311-82; eBioscience), and anti-IL-17A-APC (1:200, clone eBio17B7, catalog No. 17-7177-81; eBioscience). Stained samples were analyzed using an LSRFortessa™ X-20 flow cytometer (BD Biosciences) and data were analyzed using FlowJo version 10 (FlowJo, Ashland, OR, USA).

Cell culture and conditions of co-culture

DW-MSCs were cultured in StemPro™ MSC SFM XenoFree medium (Gibco BRL, Grand Island, NY, USA) supplemented with 1% L-glutamine (200 mM) and 1% penicillin-streptomycin. The culture medium was changed every 2–3 days and the cells were maintained under 5% CO₂ at 37°C. A549 cells (CCL-185, human type II AECs; ATCC, Gaithersburg, MD, USA) were cultured in DMEM supplemented with 10% FBS (Gibco BRL), 100 U/ml penicillin, and 100 mg/ml streptomycin. A549 cells were treated with 0 and 400 μM CoCl₂ (SHINYO Pure Chemical, Osaka Japan) for 24 h and co-cultured with or without DW-MSCs. For cell viability assay, western blot analysis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and the mitochondrial respiration assay, DW-MSCs and A549 cells were indirectly co-cultured for 24 h using 0.4 μm Transwell™ inserts. For mitochondrial transfer analysis, DW-MSCs and A549 cells were directly co-cultured for 24 h.

Cell viability

To measure cell viability, we used the Cell Counting Kit-8 assay following the manufacturer’s instructions (catalog No. CK04; Dojindo Laboratories, Kumamoto, Japan). Briefly, cells were incubated with Cell Counting Kit-8 reagent for 1 h and the absorbance was measured at 450 nm using a microplate reader. Each experiment was performed in triplicate.

TUNEL assay

The TUNEL assay was performed using the DeadEnd™ Colorimetric TUNEL System (catalog No. G7131; Promega, Madison, MI, USA) following the manufacturer’s instructions. Briefly, cells were fixed with 4% paraformaldehyde and permeabilized. Subsequently, the cells were incubated with 100 μl of the TdT reaction mixture for 60 min in a humidified chamber maintained at 37°C. The cells were developed with DAB Solution until a light-brown color developed. Stained cells were observed under a light microscope (Nikon) and images of 4–5 random fields were analyzed at an objective magnification of 200×. Cells exhibiting a brown color were counted as apoptosis-positive and their number was expressed as a percentage.

Assessment of mitochondria transfer

A549 cells were treated with 0 and 400 μM of CoCl₂ for 24 h. For immunofluorescence staining of tunneling nanotubes (TNTs), A549 cells and DW-MSCs were stained with CellTrace™ Violet (5 μM, 25 min, 37°C, catalog No. C34557; Invitrogen, Waltham, MA, USA) and MitoTracker™ Green FM (200 nM, 30 min, 37°C, catalog No. M7514; Invitrogen), respectively. The cells were then co-cultured for 24 h at a ratio of 1:1 and photographed under a fluorescence microscope (Nikon). Images were acquired using the NIS-Elements software and a Nikon microscope imaging system (Nikon). For the quantitative mitochondrial transfer assay, A549 cells were treated with 0 and 400 μM of CoCl₂ for 24 h followed by staining with CellTrace™ CFSE (5 μM, 30 min, 37°C, catalog No. C34554; Invitrogen). DW-MSCs were stained with MitoTracker™ Deep Red (5 μM, 30 min, 37°C, catalog No. M46753; Invitrogen). After washing, both cells were mixed at a 1:1 ratio and co-cultured for 0 or 24 h. The cells were then harvested and analyzed using an LSRForetessa™ X-20 flow cytometer. After gating only A549 cells expressing CellTrace™ CFSE, the ratio of A549 cells co-expressing CellTrace™ CFSE and MitoTracker™ Deep Red was analyzed using FlowJo version 10.

Mitochondrial respiration assay

Mitochondrial respiration was analyzed by measuring the oxygen consumption rate (OCR) with a Seahorse XFe96 Extracellular Flux Analyzer and XF Cell Mito Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The co-cultured cells were seeded at a density of 10,000 cells per 96-well Seahorse XF plate. After 24 h, the cells were washed once, and the growth medium was replaced with unbuffered XF DMEM (pH 7.4) supplemented with 25 mM D-glucose, 4 mM L-glutamine, and 1 mM sodium pyruvate. The OCR was measured under basal conditions and after sequential injection of 1.5 µM oligomycin, 2 µM FCCP, and 0.5 µM rotenone/antimycin. Data were analyzed using the Wave software (Agilent Technologies) and expressed as OCR in pmol/min/cell. All OCR values were normalized to the amount of protein obtained from each well after analysis.

Statistical analysis

Data visualization and analyses were performed using Prism versions 7 and 8 (GraphPad, San Diego, CA, USA). All data were analyzed using one-way analysis of variance followed by the Fisher’s least significant difference test. p-values < 0.05 were considered significant. Data are expressed as mean ± SEM.

Establishment of the dosage, timing, and frequency of administration of DW-MSCs to BPF mice

The following experiments were conducted to determine the optimal dose, duration, and frequency of treatment with DW-MSCs to effectively treat pulmonary fibrosis in BPF mice. Fibrosis was assessed histologically in the lung tissues of mice at 4, 7, 14, and 21 days after BLM induction by comprehensively evaluating the Ashcroft score, fibrosis score, percentages of collagen deposition and alveolar space, and indicators of pulmonary fibrosis.

In our previous experiment, from the 4th day after BLM administration, fibrosis was observed in >20% of the total lung area of the mice and pulmonary fibrosis continued for up to 14 days after BLM induction; however, there was no change in the progression of pulmonary fibrosis from day 14 to day 21 (data not shown, unpublished). In particular, collagen deposition, which may reflect irreversible damage, was markedly increased on day 14 after BLM induction. Based on these results, to determine the optimal dose of DW-MSCs, a single injection of 1×10⁵ or 1×10⁶ DW-MSCs was administered on day 7 after BLM induction and pulmonary fibrosis in the mice was evaluated on day 21 (Supplementary Fig. 1A). DW-MSCs alleviated pulmonary fibrosis in BPF mice. BPF mice administered 1×10⁶ DW-MSCs displayed improved pulmonary fibrosis compared with those administered 1×10⁵ DW-MSCs (Supplementary Fig. 1B and C). Next, to determine the frequency of DW-MSC administration, single (on day 7) or serial (on days 7, 11, and 15) injections of 1×10⁶ DW-MSCs were administered after BLM induction, and pulmonary fibrosis in the mice was evaluated on day 21 (Supplementary Fig. 1D). Both serial and single injections were found to improve pulmonary fibrosis in BPF mice to similar extents (Supplementary Fig. 1E and F).

DW-MSCs suppress pulmonary fibrosis in early-, active-, and chronic persistent-phase BPF mice

To investigate the potential therapeutic effects of DW-MSCs on early-phase pulmonary fibrosis in BPF mice, DW-MSCs were administered on the 4th day after BLM induction, following which fibrosis was evaluated in the lung tissue samples obtained from these mice on the 14th day (Fig. 1A). Compared with normal mice, BPF mice exhibited increased Ashcroft scores, fibrosis scores, and collagen content, as well as decreased alveolar space in the lung tissue. However, administration of DW-MSCs resulted in decreased Ashcroft scores, fibrosis scores, and collagen content, as well as increased alveolar space in the BPF mice (Fig. 1B and C). Furthermore, immunohistochemistry for α-SMA, a marker of myofibroblasts that plays an important role in the fibrosis process, revealed an increase in α-SMA-positive cells in BPF mice compared with normal mice. However, administration of DW-MSCs decreased the number of α-SMA-positive cells in BPF mice (Fig. 1B and D).

Figure 1
Effects of DW-MSCs on pulmonary fibrosis in early-phase BPF mice. (A) Experimental schedule schematic. (B) Representative images of the lung tissues from normal and BPF mice with or without DW-MSC treatment stained with Masson’s Trichrome (magnification: 40× and 200×) and of lung sections immunohistochemically stained for α-SMA (magnification: 400×). (C) Semiquantitative assessment of lung fibrosis in terms of the Ashcroft score, fibrosis score, percentages of collagen content, and alveolar space. The data are cumulative results from three independent experiments, with a total of 8–14 mice/group. (D) Semiquantitative assessment of the α-SMA-positive area in the lung. The data are from 3–4 mice/group, where each symbol represents one mouse. Scale bar: 50 μm ± SEM.
Statistical significance was determined using one-way analysis of variance followed by Fisher’s least significant difference. All data are presented as mean ± SEM (^*p<0.05, ^**p<0.01, and ^***p<0.001 vs. normal).

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To investigate whether DW-MSCs improved active-phase pulmonary fibrosis in BPF mice, the mice were administered DW-MSCs on the 7th day after BLM induction, and fibrosis in the lung tissue was evaluated on day 21 (Fig. 2A). Compared with normal mice, BPF mice showed increased Ashcroft scores, fibrosis scores, and collagen content, and decreased alveolar space, which were considerably reversed upon DW-MSC treatment (Fig. 2B and C). Immunohistochemistry and western blot analysis revealed an increase in the number of α-SMA-positive cells in the lung tissues of BPF mice, which was reversed upon DW-MSC treatment (Fig. 2D). In addition, the increased hydroxyproline levels in BPF mice were downregulated by DW-MSCs (Fig. 2E). Next, to confirm the anti-fibrotic and anti-inflammatory roles of DW-MSCs in BPF mice, we examined the gene expression of Acta2, Col1a1, Ctgf, Tgfb1, and Il1β, and confirmed their reversal upon DW-MSC treatment (Fig. 2F). Furthermore, we explored the therapeutic effects of DW-MSCs in chronic persistent-phase BPF mice with repetitive BLM induction (Fig. 3A). BPF mice exhibited increased Ashcroft scores, fibrosis scores, collagen content, and reduced alveolar space, which were markedly ameliorated upon DW-MSC administration (Fig. 3B and C). In chronic persistent-phase BPF mice, a single injection of DW-MSCs improved pulmonary fibrosis, even under repetitive BLM induction. These results indicated that DW-MSCs inhibit pulmonary fibrosis in early-, active-, and chronic persistent-phase BPF mice.

Figure 2
Effects of DW-MSCs on pulmonary fibrosis in active-phase BPF mice. (A) Experimental schedule schematic. (B) Representative images of Masson’s Trichrome-stained lung tissues from normal and BPF mice with/without DW-MSC treatment (magnification: 40× and 200×). (C) Semiquantitative assessment of lung fibrosis in terms of the Ashcroft score, fibrosis score, as well as percentages of collagen content and the alveolar space. (D) Representative images of lung tissue sections immunohistochemically stained for α-SMA (magnification, 200×) and semiquantitative assessment of the α-SMA-positive area in the lung. (E) Semiquantitative assessment of hydroxyproline and (F) expression profiles of pro-fibrotic and pro-inflammatory genes in the lung (n=3–4 mice/group); each symbol represents one mouse. (B-C) Cumulative data from three independent experiments were used for a total of 11–18 mice/group. Scale bar: 50 μm.
Statistical significance was determined using one-way analysis of variance followed by Fisher’s least significant difference (^*p<0.05, ^**p<0.01, and ^***p<0.001 vs. normal).

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Figure 3
Effects of DW-MSCs on pulmonary fibrosis in mice with chronic persistent-phase BPF. Mice were given a total of four doses of BLM every other week for 8 weeks, following which 1×10⁶ DW-MSCs were administered to the mice at 3 wk after BLM induction, and pulmonary fibrosis was assessed 2 weeks after the last BLM induction. (A) Experimental schedule schematic. (B) Representative images of Masson’s Trichrome-stained lung tissues of normal and BPF mice with or without DW-MSC treatment (magnification, 200×). (C) Quantitative assessment of pulmonary fibrosis in terms of the Ashcroft score, fibrosis score, and percentages of collagen content and alveolar space (n=4–8 mice/group). Scale bar: 100 μm.
Each symbol represents a single mouse. ^*p<0.05, ^**p<0.01, and ^***p<0.001 vs. normal.

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DW-MSCs produce an effect that is comparable to that of established anti-fibrotic agents (nintedanib and pirfenidone)

To further establish the efficacy of DW-MSCs, a comparative analysis was performed with nintedanib, an existing treatment for pulmonary fibrosis (Fig. 4A). DW-MSCs alleviated fibrosis in the lungs of BPF mice to a level comparable with that observed with nintedanib; they also downregulated the collagen content more effectively than nintedanib (Fig. 4B and C). Immunohistochemical analysis showed that DW-MSCs reduced the number of α-SMA-positive cells in the lungs of BPF mice to a level similar to that observed with nintedanib (Fig. 4B and D). Furthermore, we confirmed that human nucleoli-positive cells were observed only in the lung tissues of BPF mice administered DW-MSCs (Fig. 4B and C). Next, we compared the efficacy of DW-MSCs to that of pirfenidone (Fig. 4E). DW-MSCs improved pulmonary fibrosis in BPF mice to an extent similar to that observed with pirfenidone (Fig. 4F and G). We found that DW-MSCs reduced the collagen content more effectively than nintedanib or pirfenidone (Fig. 4C and G).

Figure 4
Comparative therapeutic effects of DW-MSCs and NTN or PFD on pulmonary fibrosis in BPF mice. BPF mice were intravenously injected with DW-MSCs (2 × 10⁶) on Day 4 or administered NTN (50 mg/kg) daily for 3 weeks, following which the mice were sacrificed on Day 21. (A) Experimental schedule schematic. (B) Representative images of lung tissues stained with Masson’s Trichrome, α-SMA, and HNA (magnification: 200×). (C) Semiquantitative assessment of lung fibrosis in terms of the Ashcroft score, fibrosis score, and percentages of collagen content and alveolar space. (n=3–8 mice/group). (D) Semiquantitative assessment of the α-SMA-positive area in the lungs of normal and BPF mice with/without DW-MSC or NTN treatment (n=3–5/group). Mice were intravenously injected with DW-MSCs (1×10⁶) on Day 7 or pirfenidone (150 mg/kg/d) was administered daily to the mice for 3 weeks, following which they were sacrificed on day 21. (E) Experimental schedule schematic. (F) Representative images of lung tissues stained with Masson’s Trichrome. (G) Semiquantitative assessment of lung fibrosis in terms of the Ashcroft score, fibrosis score, and percentages of collagen content and alveolar space (n=3–8 mice/group); each symbol represents one mouse. Scale bar: 50 μm.
NTN, nintedanib; PFD, pirfenidone; HNA, human nuclear Ag.

Statistical significance was determined using one-way analysis of variance followed by Fisher’s least significant difference (^*p<0.05, ^**p<0.01, and ^***p<0.001 vs. normal).

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DW-MSCs have a therapeutic effect against pulmonary fibrosis in CIA mice

To investigate whether DW-MSCs improve pulmonary fibrosis in CIA mice, we administered a single dose of 1×10⁶ DW-MSCs on day 22 after CIA induction and evaluated fibrosis in the lung tissues of CIA mice on day 35 (Supplementary Fig. 2A). In our preliminary study, inflammation and fibrosis progression in the entire lung tissue was confirmed on day 22 after CIA induction, which continued to increase until day 29 before decreasing. On day 42, pulmonary fibrosis showed an overall improvement (data not shown). Compared to normal mice, CIA mice displayed increased pulmonary fibrosis, which was significantly reduced upon DW-MSC treatment (Supplementary Fig. 2B and C). Immunohistochemical analysis of α-SMA showed an increase in α-SMA-positive cells in CIA mice compared with that in normal mice, which decreased significantly upon DW-MSC treatment (Supplementary Fig. 2D). These results indicated that DW-MSCs inhibited pulmonary fibrosis in CIA mice.

DW-MSCs suppress lung fibrosis through the regulation of immune cells in BPF mice

To investigate whether DW-MSCs are involved in immune cell modulation in BPF mice, we administered DW-MSCs (1×10⁶) at 4 days after BLM induction and performed flow cytometry analysis of the lung tissues on days 7 and 14 (Fig. 5A). Seven days after BLM induction, the number of B cells (B220⁺ B cells among CD45⁺ cells) in the lungs of BPF mice increased by approximately 20% compared with that in the lungs of normal mice, but decreased upon treatment with DW-MSCs (Fig. 5B). Fourteen days after BLM treatment, an imbalance in Th17 (CD4⁺ IL-17A⁺) and Treg (CD4⁺ FoxP3⁺ CD25⁺) cells was observed in the lungs of BPF mice, which was restored upon treatment with DW-MSCs (Fig. 5C).

Figure 5
Effects of DW-MSCs on immune cell populations in the lung tissues of BPF mice. (A) Experimental schedule schematic. (B) Flow cytometry analysis of lung tissues of BPF mice at 7 d after BLM induction showed an up to 20% increase in the number of B cells (B220⁺ B cells among CD45⁺ cells) compared with that in normal mice; this increase was decreased upon treatment with DW-MSCs. (C) At 14 d after BLM induction, an imbalance in Th17 (CD4⁺ IL-17A⁺) and Treg (CD4⁺ FoxP3⁺ CD25⁺) cells was observed in the lungs of BPF mice, which was restored by treatment with DW-MSCs. The gating strategy is presented in Supplementary Fig. 4 (n=4 mice/group for normal and BPF mice with or without DW-MSC treatment); each symbol represents one mouse.
Statistical significance was determined using one-way analysis of variance followed by Fisher’s least significant difference (^*p<0.05 and ^**p<0.01 vs. normal).

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DW-MSCs mitigate CoCl₂-induced apoptosis in AECIIs

After treatment with 400 μM CoCl₂ for 24 h, the A549 cells displayed significantly lower cell viability than untreated cells; however, co-culture with DW-MSCs significantly increased the viability of A549 cells from 54.6% to 77.2% (Fig. 6A). In western blot analysis, A549 cells treated with 400 μM CoCl₂ showed downregulation of Bcl-2 expression and upregulation of Bax expression. However, co-culture with DW-MSCs reversed these effects, leading to increased Bax expression and decreased Bcl-2 expression (Fig. 6B). Analysis of apoptosis using TUNEL staining revealed a significant increase in apoptotic cells among A549 cells treated with 400 μM CoCl₂ compared to that among untreated A549 cells (0 μM CoCl₂) (Fig. 6C). However, co-culturing with DW-MSCs reduced CoCl₂-induced apoptosis in A549 cells. These results suggested that DW-MSCs exert a protective effect against CoCl₂-induced apoptosis in A549 cells.

Figure 6
Effects of DW-MSCs on CoCl₂-induced apoptosis in A549 cells. CoCl2-induced A549 cells were co-cultured with or without DW-MSCs for 24 h in a hypoxic condition and the cell viability of the AECs and levels of apoptosis in them were assessed. (A) Quantitative analysis of A549 cell viability. (B) Expression of Bax and Bcl-2 proteins in A549 cells. Full-length blots/gels are presented in Supplementary Fig. 5. (C) Representative images of the TUNEL assay and percentage of TUNEL-positive cells (n=3 experiments/group).
^***p < 0.001 vs. A549 cells (without CoCl₂ and DW-MSCs).

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DW-MSCs improve respiratory function via mitochondrial transfer into AECIIs

Using fluorescence microscopy, we observed the migration of mitochondria from DW-MSCs to A549 cells through TNTs (Fig. 7A). Furthermore, we found an increased mitochondrial transfer ratio of DW-MSCs to damaged A549 cells compared to healthy A549 cells (Fig. 7B). A549 cells damaged by CoCl₂ exhibited a significant reduction in basal respiration, maximal respiration, proton peak, and ATP production compared to healthy A549 cells (Fig. 7C). However, co-culture with DW-MSCs significantly improved the overall mitochondrial respiration in the damaged A549 cells, as evidenced by the increase in basal respiration, maximal respiration, proton peak, and ATP production across all indices. We observed the expression of human nuclear-encoded subunit 4 of cytochrome c oxidase (hCOX-4) in SPC-positive AECIIs in BPF mice (Fig. 8), which suggested that the mitochondria of human-origin DW-MSCs migrated to mouse AECIIs.

Figure 7
Modulatory effects of DW-MSC mitochondrial transfer and respiration of hypoxia-induced A549 cells. A549 cells were co-cultured with/without DW-MSCs for 24 h in a hypoxic condition. (A) Representative images showing TNT connections (arrows) between DW-MSCs and hypoxia-induced A549 cells. (B) Representative distribution of co-cultured CFSE-labeled A549 cells and MitoTracker™-labeled DW-MSCs, analyzed using fluorescence-activated cell sorting, and the time-course of the mitochondrial transfer ratio for transfer between DW-MSCs and hypoxia-induced A549 cells (n=3 experiments/group). (C) Mitochondrial respiration was analyzed in hypoxia-induced A549 cells. Overall mitochondrial OCRs were measured after injection of oligomycin (1.5 µM), FCCP (2 µM), and rotenone/antimycin A (0.5 µM). Quantitative assessment of respiration, proton peak, and ATP production (n=6 experiments/group). Statistical significance was determined using one-way analysis of variance followed by least significant difference. Data are presented as mean ± SEM.
FCCP, carbonyl cyanite-4 phenylhydrazone.

^**p<0.01 and ^***p<0.001 vs. A549 cells (without CoCl₂ and DW-MSCs).

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Figure 8
Effects of DW-MSCs on mitochondria transfer to AEC IIs in the lungs of active BPF mice. Representative images of lung tissues stained with hCOX-4 (human specific mitochondria marker; green) and mSPC (ACEII marker; red) n=3 mice/group (normal and BPF mice with/without DW-MSC treatment); each symbol represents one mouse. Statistical significance was determined using one-way analysis of variance followed by least significant difference. Data are presented as mean ± SEM.
mSPC, mouse surfactant protein c/SPC.

^***p<0.001 vs. normal.

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Miro-1 exhibited minimal to negligible expression in bone marrow-MSCs and umbilical cord-MSCs by passage 6. Interestingly, DW-MSCs exhibited robust Miro 1 expression as early as passage 13. Subsequent exposure to IFN-γ stimulation confirmed sustained upregulation of Miro-1 expression persisting through passage 18 (Supplementary Fig. 3).