Isolation and culture of UCMSCs and PLMSCs
Healthy full-term human umbilical cord and placental samples were collected following the guidelines of the Ethics Committee of Seventh Medical Center of Chinese PLA General Hospital in Beijing, China. Written informed consent was obtained from all donors before this study was initiated. All samples were utilized in compliance with the approved standard experimental protocols set forth by the Animal and Medical Ethics Committee of Tsinghua University, Beijing, China. In brief, umbilical cords and placenta from full-term newborns were obtained from the clinic and rinsed with PBS to eliminate any residual blood. Subsequently, after removing the artery and vein, the cords were cut into approximately 2 mm segments. These segments were then placed directly into 10 cm2 culture flasks containing DMEM supplemented with 5% KOSR, 1% Ultroser G, 1× L-glutamine, 1× NEAA, 10 ng/mL bFGF, and 10 mg/L L-ascorbic acid. The cells were cultured in an environment with 5% CO2 at 37°C. UCMSCs were sub-cultured when reached about 80% confluence. Following four passages in culture, cells were harvested for further characterization.
Experimental animals
Male C57BL/6 mice, aged between 6 to 8 weeks under specific pathogen-free (SPF) criteria, were acquired from the Tsinghua University's Laboratory Animal Resources Center in Beijing, China. This study encompassed a total of 196 mice. To ensure consistency, both experimental and control mice were weight-matched, between 20 to 25 grams per mouse. Mice were housed in the Laboratory Animal Resources Center, Tsinghua University and kept under SPF conditions at a room temperature ranging from 20–24°C and a humidity level of 35–55%, following a 12 h light and 12 h dark cycle. Mice had unrestricted access to food and water and were regularly monitored for the overall health, fur quality, activity levels, and weight, adhering to institutional protocols. When necessary, euthanasia was performed humanely using CO2 inhalation at specified time points. The laboratory animal facility has been accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International) and the IACUC (Institutional Animal Care and Use Committee) of Tsinghua University approved all animal protocols used in this study.
Animal anaesthesia
For micro CT scans, mice were anesthetized using inhaled isoflurane delivered via a face mask and were positioned on a heated pad to ensure thermoregulation. For intratracheal induction with saline or BLM, mice were anesthetized by intraperitoneal injection of Tribromoethanol (Avertin). The dosage of Avertin ranged from 192 to 384 mg/kg, equivalent to 100–200 µl/10g, administered via intraperitoneal injection (IP) into the right abdomen.
Cell therapy using the BLM-induced pulmonary fibrosis mouse model
To establish the BLM-induced pulmonary fibrosis model, mice were subjected to intratracheal injections of Bleomycin Sulfate (2 mg/kg, Syno, Cat: NSC125066) dissolved in saline under light anesthesia. Intravenous administration of UCMSC and PLMSC was performed on day 3, 9 or 16 post-injury. Mice were euthanized at day 21 or 29 following BLM-induced injury. After perfusion with saline, the left lungs underwent morphometric analyses, while the right lungs were excised for further examination.
Lung coefficient
The lung tissue was entirely excised and weighed using an electronic balancer. The lung coefficient was determined as wet lung weight (g) divided by total body weight (g).
Cell culture
UCMSCs were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY), supplemented with 2 mM L-glutamine, 5% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco), and cytokines (EGF, bFGF, PDGF, and IGF). The cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. The adherent spindle-shaped cells, when reached at 80% confluence, were trypsinized using 0.25% trypsin (Gibco) and sub-cultured in the aforementioned medium.
To cultivate bone marrow-derived macrophages, mice were humanely euthanized and briefly immersed in 75% ethanol for sterilization. Tibia and femur bones were then carefully extracted and then underwent a gentle flush with 10 mL of sterile RPMI medium (HyClone, USA), fortified with 10% fetal bovine serum (FBS, HyClone, USA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (HyClone, USA), utilizing a 27½ gauge needle. Subsequently, 5×106 bone marrow cells were introduced into 10 cm2 tissue culture dishes and nurtured in 10 mL of RPMI medium, supplemented with murine macrophage colony-stimulating factor (M-CSF) (50 ng/mL) (315-02-100, PeproTech, USA), 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The cultured cells were maintained at 37°C in a 5% CO2 environment. A medium refreshment was carried out on the fourth day. By the 7th day, BMDMs were delicately detached from the dishes, quantified, and subsequently transferred to fresh plates for further experimentation.
RNA-seq library preparation and data analyses
Total RNA from cells was extracted using Trizol reagent (Invitrogen, Waltham, MA, USA; Catalog No. 15596018). Subsequent RNA-seq library preparations were carried out using the NEBNext® UltraTM RNA Library Prep Kit designed for Illumina® sequencing platforms. The libraries were sequenced on an Illumina HiSeq X-Ten sequencer, utilizing a 150 bp paired-end sequencing protocol. The bulk RNA-Seq data for hESCs was retrieved from the GEO database.
The RNA-sequencing data was analyzed using Hisat2 (version 2.1.0) and Cufflinks (version 2.2.1). Either the UCSC hg19 or mm10 annotations with default configurations was employed. For subsequent analyses, transcript reads were mapped to unique genomic locations and genes displaying at least 1 FPKM in a minimum of one sample were considered. A two-fold change threshold was set to identify differentially expressed genes (DEGs).
Data visualization and interpretation involved clustering, heatmap generation, Venn diagrams, and scatterplots were done by the Hierarchical Clustering and Heatmap.2 available in the R software environment. Additionally, the Pearson correlation coefficient was calculated using the “cor.test” function in R software. Lastly, gene set enrichment analyses were conducted using GSEA.
Flow cytometry
Cells were harvested and blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich, B2064) for 20 min at room temperature. Then, the cells were stained with fluorescein-conjugated antibodies for 40 min at room temperature in 1% BSA. After incubation, cells were washed 3 times and analyzed with MoFlo (Beckman, USA) and associated software (CytExpert, Beckman, USA). The antibodies and the reagents used for flow cytometry were as follows: APC-conjugated mouse anti-mouse F4/80 (Biolegend, San Diego, CA, USA; 123115), Percp-cy5.5-conjugated mouse anti-mouse CD11b (Biolegend, San Diego, CA, USA; 301417), PE-conjugated mouse anti-mouse CD11c (Biolegend, San Diego, CA, USA; 117307), APC-Cy7-conjugated mouse anti-mouse CD206 (Biolegend, San Diego, CA, USA; 321119), PE-conjugated mouse anti-mouse Siglec-F (Biolegend, San Diego, CA, USA; 155505), FITC-conjugated mouse anti-mouse CD11c (Biolegend, San Diego, CA, USA; 101205), APC-conjugated mouse anti-mouse CD206 (Biolegend, San Diego, CA, USA; 141707), PE-conjugated mouse anti-mouse CD206 (Biolegend, San Diego, CA, USA; 141705), Percp-cy5.5-conjugated mouse anti-mouse CD45 (Biolegend, San Diego, CA, USA; 103131), FITC-conjugated mouse anti-mouse CD11c (Biolegend, San Diego, CA, USA; 123115), PE-conjugated mouse anti-mouse CD64 (Biolegend, San Diego, CA, USA; 161003), PE-Cy7-conjugated mouse anti-mouse CD127 (Biolegend, San Diego, CA, USA; 135013), APC-conjugated mouse IgG1 (Biolegend, San Diego, CA, USA; 405308), Percp-cy5.5-conjugated mouse IgG1 (Biolegend, San Diego, CA, USA; 405314), PE-conjugated mouse IgG1 (Biolegend, San Diego, CA, USA; 405307), APC-Cy7-conjugated mouse IgG1 (Biolegend, San Diego, CA, USA; 405316), FITC-conjugated mouse IgG1 (Biolegend, San Diego, CA, USA; 406001), PE-Cy7-conjugated mouse IgG1 (Biolegend, San Diego, CA, USA; 405315).
Single-cell RNA-seq library preparation & sequencing
UCMSCs and PLMSCs were collected and suspended in PBS. Subsequently, the cell suspensions were loaded into the Chromium Single Cell Controller (10× Genomics) to produce individual Gel Beads-in-Emulsion (GEMs) utilizing the Single Cell 30 Library and Gel Bead Kit V2 (10× Genomics, 120237). Upon lysis of the cells, the liberated RNA underwent barcoding via reverse transcription within separate GEMs. After the reverse transcription process, cDNAs bearing both barcodes underwent amplification. Libraries for each sample were then crafted using the Single Cell 30 Reagent Kit (v2 chemistry) according to the manufacturer's guidelines. Sequencing was done on an Illumina NovaSeq 6000 System using the 2 × 150 bp paired-end sequencing mode. Subsequently, raw data was filtered with Cell Ranger employing default mapping parameters. For gene expression profiling and cell type categorization, Seurat V3.1 was utilized. After normalization and quality assessment, the UMAP algorithm was used to facilitate non-linear dimensional reduction. Visualization of the results was accomplished using the DimPlot and VlnPlot functionalities within Seurat.
Cell culture with IL-1b, IL-6, IFN-γ, and TNF-a priming
Cells from passage 4 were enzymatically dissociated and subsequently plated into 6-well dishes. Both UCMSCs and PLMSCs were inoculated at a concentration of 3 × 105 cells per well. Following a 24 h incubation period for cell adherence, the culture medium was supplemented with IFN-γ (20 ng/mL) (R&D systems, 285-IF), IL-1b (10 ng/mL) (Biovision, 4130-50), TNF-a (5 ng/mL) (Biovision, 1050-10), and IL-6 (10 ng/mL) (Biovision, 4143 − 100). Post a 24 h exposure to these cytokines, both cells and their corresponding conditioned media were harvested for subsequent qPCR and RNA sequencing analyses.
Real-time quantitative PCR
Total RNA was extracted with TRIzol (Invitrogen) and reverse-transcribed using the Quantscript RT Kit (TIANGEN Biotech). For quantitative polymerase chain reaction (QRT-PCR) analysis, the Talent qPCR PreMix (SYBR Green) Kit (TIANGEN Biotech) was utilized on a Roche instrument under the specified condition: initial denaturation at 95°C for 5 s, followed by annealing at 60°C for 10 s, and extension at 72°C for 15 s. The primer sequences employed for the qRT-PCRs are detailed in Supplementary Table 9.
Histology
Mouse tissues were fixed using a 4% paraformaldehyde solution, and subsequently embedded in paraffin. Sections of 4 µm thickness were deparaffinized using xylene and then gradually hydrated with alcohol. For antigen retrieval, tissue sections were treated with sodium citrate buffer and quenched by a peroxidase-blocking solution (Dako). Sections were then incubated in protein block solution (Dako) for 10 min, followed by an overnight incubation at 4°C with primary antibodies. The primary antibodies used included mouse anti-col1a1 (ab6308, 1:200), mouse anti-col3a1 (ab7778, 1:200), mouse anti-ACTA2 (ab7817, 1:200), mouse anti-F4/80 (ab6640, 1:200), and mouse anti-CD206 (ab64693, 1:200).
For immunohistochemistry, after primary antibody incubation, sections were treated with an HRP-conjugated rabbit/mouse secondary antibody solution from Dako, followed by color development using DAB (Dako). Hematoxylin solution (Sigma, 03971) was utilized to stain the nuclei. Bright-field images of the sections were captured using a slide scanner. For immunofluorescence, sections were treated with 594-conjugated anti-mouse (CST, 8890s, 1:1000) and TRITC-conjugated anti-mouse (abcam, ab6718, 1:1000) secondary antibodies in a 2% BSA solution for 60 min at room temperature in the absence of light. Nuclei were counterstained using DAPI (Sigma, D8417) for 8 min. For H&E, Gomori's Trichrome, and Hematoxylin and Eosin staining, sections embedded in paraffin were employed. The Hematoxylin and Eosin-stained sections were utilized for Ashcroft scoring, which was determined by averaging the scores assigned by one blinded and one non-blinded evaluator.
Western blot
Tissues from mice were harvested in RIPA Lysis Buffer (strong) containing protease inhibitors (Roche, 4693124001). A total of 40 µg proteins were separated by SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked at room temperature with milk for 1 h and incubated overnight at 4°C with primary antibody, rabbit Fibronectin antibody (abcam ab2413). The membranes were washed with TBST for 3 times and incubated for 1 h with a secondary antibody, anti-rabbit IgG antibody.
Lung function assessment
Pulse distention, breath distention and Oxygen saturation levels were measured by the MouseOx Small Animal Vital Signs Monitor (STARR, USA) following the manufacturer’s instructions.
Micro-CT
Mouse CT scans (Quantum GX, USA) were performed according to the manufacturer’s instructions.
Statistics
The data were presented as mean ± SEM. Survival curves were constructed using the Kaplan-Meier method and evaluated using the generalized Wilcoxon test. Statistical evaluations were conducted using GraphPad Prism 8.0 software (San Diego, CA, USA). For comparisons among multiple groups, Tukey's multiple comparison test in ANOVA was employed. A p-value less than 0.05 was deemed statistically significant.
Statement
The work has been reported in line with the ARRIVE guidelines 2.0.