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Adipose-derived mesenchymal stem cell therapy for reverse bleomycin-induced experimental pulmonary fibrosis – Scientific Reports

Republished By Plato
Republished By Plato

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Animal ethics approval

C57BL/6 mice were obtained from the Fudan University Animal Center.

All animals were placed in stainless steel cages in a well-ventilated room maintained at an ambient temperature of 24–26 °C with controllde relative humidity. Throughout the experiment, the animals under trial were provided with diet pellets and tap water. At the end of experimental period, all animals were euthanized under mild anesthesia by 20 mg/kg of ketamine and 3 mg/kg of xylazine. All animal experiments were performed with the approval of the Scientific Investigation Board of Fudan University (2019-JS-011). The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). All methods were performed in accordance with the relevant guidelines and regulations.

Bleomycin-induced pulmonary fibrosis model

Female or male 8-week-old C57BL/6 mice (Shanghai, China) were anesthetized with isoflurane and received a single endotracheal dose of bleomycin sulphate (50 μL, 3 U/kg) at day 0 to induce pulmonary fibrosis. Bleomycin naive mice (control) received an endotracheal dose of saline (50 μL). At day 14, the treated group received a single intravenous (tail vein) dose of MSC (200 μL; dose, 5 × 106 MSCs). The control group received an equivalent volume of phosphate-buffered saline (PBS). Mice were assessed at day 14 and/or at day 21 or day 28 for cytometric, histological, and/or quantitative PCR (qPCR) analysis.

Isolation and culture of MSCs and lung fibroblasts

MSCs and lung fibroblasts were obtained from C57BL/6 mice. All procedures for the isolation and culture these cells were performed with the approval of the Scientific Investigation Board of Fudan University.

The male mice was euthanized under mild anesthesia by 20 mg/kg of ketamine and 3 mg/kg of xylazine. Adipose tissue was obtained from the epididymis using surgical manipulation, and blood vessels were separated from the fat and cut into 1 mm3 on a cold plate. The tissue was then digested using in 1 mg/mL collagenase I (Biosharp, China) at 37 °C for 0.5 h to obtain a single-cell suspension. The suspension was centrifuged at 2000 rpm for 5 min, and the cells were plated in a 100 mm dish. The culture medium consisted of Dulbecco’s modified Eagle’s medium, which contains 10% FBS, 100 mM non-essential amino acids, and 100 mM sodium pyruvate, 20 mM sodium pyruvate, β-mercaptoethanol L-glutamine, 20 μg/mL EGF, 100 IU/mL penicillin and 100 μg/mL streptomycin (all from Gibco, Carlsbad, CA). After that, the MSCs were cultured at 37 °C and 5% CO2 for about 5 days. When the cells reach 70–80% confluence, they were harvested using 0.05% trypsin (Gibco) and subcultured in the same medium. All experiments were performed using MSCs at the third generation.

The lung tissue of mice was cut into 1 mm3 piece under a sterile environment, and then placed in a H-glutamine, 20 μg/mL EGF, 100 IU/mL penicillin, and 100 μg/mL streptomycin (all from Gibco, Carlsbad, CA). After that, Lung tissue was cultured at 37 °C and 5% CO2 for about 7 days. When the cells reach 80% confluence, they were harvested using 0.05% trypsin (Gibco) and subcultured in the same medium. All experiments were performed using lung fibroblasts at the third generation.

Flow cytometry analysis

To confirm the identity of the MSCs, flow cytometry analysis was performed according to the guidelines set by the International Society for Cellular Therapy (ISCT). has defined that the MSCs can express the surface markers CD29, CD44, CD73, and CD90 but lack of expression of CD34, CD45, CD11b24,25,26. MSCs at the third passage were trypsinized, washed twice with PBS and every 1 × 105 cells were stained separately with human monoclonal antibodies, including CD29-FITC, CD44-FITC, CD90-FITC, CD45-FITC and CD11b-FITC (BD Biosciences, Franklin Lakes, NJ). Cells were incubated with the antibodies at 4 °C in the dark for 20 min, along with a fluorescein isothiocyanate (FITC) isotype control. After washing, the cells were suspended in 500 μL of FACS buffer and analyzed by Flow Cytometer (Accuri C6, BD Biosciences).

MSC cell differentiation identification

To determine the multi-lineage differentiation potentiality of the MSCs, adipogenic, osteogenic, and chondrogenic differentiation experiments were performed using the Mesenchymal Stem Cell Functional Identification Kit (R&D Systems Inc., Minneapolis, MN). The induction processes for the three lineages were carried out according to the manufacturer’s instructions, with differentiation media changed every two days. After 10 days, the adipogenic, osteogenic and chondrogenic differentiation were sssessed using oil red staining, Alizarin Red-S staining, and alcian 8GX blue staining (Sigma-Aldrich, St. Louis, MO), respectively.

Cell fluorescent labeling

For cell visualization, the MSCs were labeled with PKH26 Red Fluorescent Cell Connection Kit (Tanon; Tanon Science & Technology, Shanghai, China) to stain the cell membrane, and DAPI was used to stain the nucleus. A total of 2 × 107 MSC cells were stained following the instructions provided with the kit. Cell images were captured with a laser scanning confocal microscopy (Olympus, Tokyo, Japan).

RNA extraction and qRT-PCR analysis

Total RNA was extracted from tissues or cultured cells as previously described27. Total RNA was extracted by trizol according to kit instructions (Ambion, Foster City, CA). Then cDNA was synthesized using the All-In-One RT MasterMix (ABM). Forward and reverse primer sequences used in reverse transcriptase PCR (Table 1) were designed by Primer 5.0 software (PREMIER Biosoft International, Palo Alto, CA), based on mRNA information of each gene obtained from the National Center for Biotechnology Information (NCBI) database. Real-time PCR was performed using EvaGreen 2 × qPCR MasterMix (ABM) on a Light Cycler 480 II (Roche Diagnostics, Basel, Switzerland) with the designed forward and reverse primers.

Table 1 Reverse transcriptase PCR primers.
Full size table

Western blotting analysis

A Western blot assay was performed as described28. For Western blot analysis of whole-cell extracts, the following primary antibodies were used: TGF-β1 (1:5000; Cell Signaling Technology, Danvers, MA, USA), α-SMA (1:5000; Abcam,), and Col I (1:5000; Abcam). Smad2 (1:1,000 dilution, Protein-Tech, China), phospho-Smad2 (1:1,000 dilution, Protein-Tech, China), Smad3 antibody (1:1,000 dilution, Protein-Tech, China), phospho-Smad3 (1:1,000 dilution, Protein-Tech, China), p62 (1:1,000 dilution, ProteinTech, China), α-tubulin (1:1,000 dilution, Affinity, China). The proteins were separated using SDS-PAGE and transferred onto a membrane, followed by incubation with horseradish peroxidase-conjugated anti-IgG (1:5000; Santa Cruz Biotechnology, Dallas, TX, USA). Protein bands were visualized using an enhanced chemiluminescence kit (Tanon; Tanon Science & Technology, Shanghai, China). To ensure accurate quantification, the Results were normalized with GAPDH (1:2000; Abcam).

In vitro migration of lung fibrotic cells

To analyze the migratory and invasive properties of lung fibrotic cells, wound healing and transwell migration assays were performed, based on previously published methods27. To assess the migration capability of lung fibrotic cells, cell migration experiments were performed using a 24-well transwell plate (PET membrane 8 μm pore sizes, Corning Incorporated Costar, Corning, NY). Lung fibrotic cells (2 × 105) suspended in 200 μL serum-free medium were seeded in the upper well of the transwell chambers, individually. In each group, one bottom was filled with 600 μL 0.5% serum medium as control, while in the experimental group, the bottom was filled with 4 × 105 MSCs suspended in the same medium. After 24 h of incubation, non-migratory cells on the upper surface of the membrane were removed with a cotton swab, and the cells on the lower surface were fixed with methanol and stained with 0.1% crystal violet for 20 min. The stained cells were observed and counted using a fluorescence inverted microscope (Olympus, Tokyo, Japan). 10 photographs were captured in a tenfold objective lens to count the number of stained cells migrating through the membrane pore.

Histology

Mice were euthanized and their right lungs were embedded in paraffin and sectioned for H&E or Masson’s trichrome staining29. The left lung was either snap frozen in liquid nitrogen and used for RNA and protein isolation or used fresh for collagen quantification or cytometric analysis. Randomly selected areas (10–15 fields) from 5 μm thick lung sections were examined under a Nikon Eclipse 80i microscope (Nikon) at × 100 and × 200 magnification. For histologic quantification, the Ashcroft score was used in a blinded fashion30. The Ashcroft score ranges from 0 to 8, with higher scores indicating more severe fibrosis. Scores of 0–1 represented no fibrosis, scores of 2–3 represented minimal fibrosis, scores of 4–5 were considered as moderate fibrosis, and scores of 6–8 indicated severe fibrosis.

Sircol collagen assay

Collagen quantification was performed using the Sircol collagen assay following the manufacturer’s protocol (Bicolor, Life Science Assays)31. Briefly, the left lung was homogenized overnight at 4 °C in 5 mL of 0.5 M acetic acid with 0.6% v/v pepsin. A total of 1 mL of dye reagent was added to 100 μL of transparent supernatant, and the samples were vortexed for 30 min. The residual pellet was washed by acid-salt wash buffer to eliminate unbound collagen, and the pH was normalized with alkalization buffer. Absorbance was measured at a wavelength of 550 nm in a microplate reader. The collagen content was determined by comparing it to a standard curve, represented as mg/mL of left lung homogenate.

Statistical analysis

All experimental data were expressed as the means ± standard deviation (SD). Statistical analysis was performed using the Student’s independent t-test via Prism 9 software (GraphPad Software, La Jolla, CA, USA) between experimental groups. The significance level was set at *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001.

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