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Umbilical cord-derived mesenchymal stem cells preferentially modulate macrophages to alleviate pulmonary fibrosis

Stem Cell Research & Therapy volume 15, Article number: 475 (2024) Cite this article

Abstract

Background

Idiopathic Pulmonary Fibrosis (IPF) is a type of interstitial lung disease characterized by chronic inflammation due to persistent lung damage. Mesenchymal stem cells (MSCs), including those derived from the umbilical cord (UCMSCs) and placenta (PLMSCs), have been utilized in clinical trials for IPF treatment. However, the varying therapeutic effectiveness between these two MSC types remains unclear.

Methods

In this study, we examined the therapeutic differences between UCMSCs and PLMSCs in treating lung damage using a bleomycin (BLM)-induced pulmonary injury mouse model.

Results

We showed that UCMSCs had a superior therapeutic impact on lung damage compared to PLMSCs. Upon cytokine stimulation, UCMSCs expressed higher levels of inflammation-related genes and more effectively directed macrophage polarization towards the M2 phenotype than PLMSCs, both in vitro and in vivo. Furthermore, UCMSCs showed a preference for expressing CC motif ligation 2 (CCL2) and C-X-C motif chemokine ligand 1 (CXCL1) compared to PLMSCs. The expression of secreted phosphoprotein 1 (SPP1), triggering receptor expressed on myeloid cells 2 (Trem2), and CCAAT enhancer binding protein beta (Cebpb) in macrophages from mice with the disease treated with UCMSCs was significantly reduced compared to those treated with PLMSCs. Conclusions: Therefore, UCMSCs demonstrated superior anti-fibrotic abilities in treating lung damage, potentially through inducing a more robust M2 polarization of macrophages than PLMSCs.

Introduction

Idiopathic pulmonary fibrosis [1] (IPF) is a chronic and progressive pulmonary disease characterized by lung injuries, including diffuse alveolar damage (DAD) [2], which can manifest as an acute response to various insults during acute respiratory distress syndrome (ARDS) [3]. ARDS typically progresses through distinct phases, starting with the exudative phase, followed by the proliferative phase, and ultimately culminating in the fibrotic phase [4]. The duration of these phases can vary, with the exudative phase typically lasting around 7 days, the proliferative phase extending for approximately 14 days, and the fibrotic phase emerging after about 21 days post-injury, often persisting for an extended period [5]. At the cellular level, the occurrence of DAD results in damage to endothelial and alveolar epithelial cells. This damage leads to the diffusion of edema into the lung interstitium and alveoli, triggering inflammatory responses [6]. This includes infiltration and activation of innate immune cells, notably macrophages, which accumulate and release various cytokines, further prompting alterations in other cell types [7]. Among these changes, fibroblasts play a crucial role as they become activated to produce extracellular matrix, ultimately leading to pulmonary fibrosis.

Macrophages are essential for innate immunity and host defense, playing important roles in initiating and maintaining inflammatory responses including lung inflammation and repair [8]. During the early stage of ARDS, macrophages secrete various inflammatory cytokines to recruit neutrophils and monocytes, activate alveolar epithelial cells and T cells, and lead to systemic inflammation and tissue damage [9] Three types of macrophages including bronchial macrophages (BMs), alveolar macrophages (AMs), and interstitial macrophages (IMs) reside in the lung tissue under normal physiological conditions [10]. AMs are the most abundant innate immune cells in the distal lung and reside on the surface of the alveolar space, where they first encounter pathogens and harmful substances from the environment and initiate immune responses in the lung [11]. AMs are mobile and maintain alveolar homeostasis by chemotaxis, with an ability to capture and clear off bacterial pathogens [12]. On the other hand, IMs, resided in the interstitium, are thought to be antigen-presenting macrophages [13,14,15]. While macrophages protect the body against the bacterial pathogens, they polarize into different forms. Currently, M1 and M2 macrophages have been identified and their roles appear quite opposite. M1 is considered to be a population with an ability of promoting inflammation but M2 is reported to own an anti-inflammatory effect. Intriguingly, M2 cells are strong inducers for the activation of fibroblasts, which induced fibrosis in the damaged lung.

Mesenchymal stem cell (MSC) therapy is a promising approach for treating inflammatory diseases like idiopathic pulmonary fibrosis (IPF) [16, 17]. Preclinical and clinical studies have shown that MSCs migrate to lung injury sites, reduce inflammation, and aid in recovery from pulmonary fibrosis [18,19,20]. However, clinical outcomes of MSC treatments vary across diseases [21], potentially likely due to differences in MSC sources and culture conditions. Evidence suggests that MSCs from different tissues are in preference to treatments of specific diseases [22]. However, further investigation is needed to determine if MSCs from different sources retain their efficacy across various diseases. Among MSCs, umbilical cord (UCMSCs) and placenta-derived (PLMSCs) cells have attracted a great attention, yet their comparative effectiveness in treating the lung injury is still unclear.

This study aims to address the preference of MSCs by evaluating the therapeutic potential of UCMSCs versus PLMSCs, intending to achieve optimized cell-based therapies for the lung injury treatment. Our ultimate goal is to identify the most promising cell type for future clinical applications by comparing UCMSCs and PLMSCs. This comparison could guide decisions on prioritizing specific cell sources in the development of lung injury treatments, thereby accelerating the translation of stem cell therapies from research to clinical practice. Additionally, since the mechanisms by which MSCs regulate inflammation in various diseases are not yet fully understood, we determined to reveal that UCMSCs preferentially polarize macrophages to reverse fibrosis. We propose that UCMSCs may be more suitable for the therapy of IPF.

Materials and methods

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 phosphate buffered saline (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 Dulbecco's modified eagle medium (DMEM) supplemented with 5% knockout serum replacement (KOSR), 1% Ultroser G, 1 × L-glutamine, 1 × non-essential amino acids (NEAA), 10 ng/mL basic fibroblast growth factor (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 g 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 Computed Tomography (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 Bleomycin (BLM), mice were anesthetized by intraperitoneal injection of Tribromoethanol (Avertin, Sigma). The dosage of Avertin ranged from 192 to 384 mg/kg [23, 24], equivalent to 100–200 μl/10 g, 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 [25], Syno, Cat: NSC125066) dissolved in saline under light anesthesia. Intravenous administration of UCMSC and PLMSC was performed on day 3, 9 or 15 post-injury. Mice were euthanized at day 21 or 29 following BLM-induced injury [26]. 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, Thermo, USA), supplemented with 2 mM L-glutamine, 5% fetal bovine serum (FBS, Gibco, Thermo, USA), 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco, Thermo, USA), and cytokines (epidermal growth factor, EGF, bFGF, platelet-derived growth factor, PDGF, and insulin like growth factor, 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, Thermo, USA) and sub-cultured in the aforementioned medium.

To cultivate bone marrow-derived macrophages (BMDMs), 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 (Roswell Park Memorial Institute) 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 RNA-sequencing data was analyzed using Hisat2 (version 2.1.0) and Cufflinks (version 2.2.1). Either the human reference genome (UCSC hg19) or the mouse reference genome (UCSC 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 (Fragments Per Kilobase of exon model per Million mapped fragments) 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 Gene Set Enrichment Analysis (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 Cytometry (Beckman, USA) and associated software (CytExpert, Beckman, USA). The antibodies used for flow cytometry were as follows: APC-conjugated mouse anti-mouse F4/80 (Cat. 123,115), Percp-cy5.5-conjugated mouse anti-mouse CD11b (Cat. 301,417), PE-conjugated mouse anti-mouse CD11c (Cat. 117,307), APC-Cy7-conjugated mouse anti-mouse CD206 (Cat. 321,119), PE-conjugated mouse anti-mouse Siglec-F (Cat. 155,505), FITC-conjugated mouse anti-mouse CD11c (Cat. 101,205), APC-conjugated mouse anti-mouse CD206 (Cat. 141,707), PE-conjugated mouse anti-mouse CD206 (Cat. 141,705), Percp-cy5.5-conjugated mouse anti-mouse CD45 (Cat. 103,131), FITC-conjugated mouse anti-mouse CD11c (Cat. 123,115), PE-conjugated mouse anti-mouse CD64 (Cat. 161,003), PE-Cy7-conjugated mouse anti-mouse CD127 (Cat. 135,013), APC-conjugated mouse IgG1 (Cat. 405,308), Percp-cy5.5-conjugated mouse IgG1 (Cat. 405,314), PE-conjugated mouse IgG1 (Cat. 405,307), APC-Cy7-conjugated mouse IgG1 (Cat. 405,316), FITC-conjugated mouse IgG1 (Cat. 406,001), PE-Cy7-conjugated mouse IgG1 (Cat. 405,315) (all from Biolegend, San Diego, CA, USA). The dilution ratio of the antibody for flow cytometry is 1:40.

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, 120,237). 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 base pair (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 Uniform Manifold Approximation and Projection (UMAP) algorithm was used to facilitate non-linear dimensional reduction. Visualization of the results was accomplished using the DimPlot and VlnPlot functionalities within the R toolkit (Seurat).

Cell culture with interleukin (IL)−1β, IL-6, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α 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) (285-IF, R&D systems, USA), IL-1β (10 ng/mL) (4130–50, Biovision, USA), TNF-α (5 ng/mL) (1050–10, Biovision, USA), and IL-6 (10 ng/mL) [27,28,29] (4143–100, Biovision, USA). 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 polymerase chain reaction (PCR)

Total RNA was extracted with TRIzol (Invitrogen, USA) and reverse-transcribed using the Quantscript RT Kit (TIANGEN Biotech, China). For quantitative polymerase chain reaction (qRT-PCR) analysis, the Talent qPCR PreMix (SYBR Green) Kit (TIANGEN Biotech, China) 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 Additional file 1: Table S1.

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, Denmark). Sections were then incubated in protein block solution (Dako, Denmark) for 10 min, followed by an overnight incubation at 4 °C with primary antibodies. The primary antibodies 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) were purchased from Abcam, UK.

For immunohistochemistry, after primary antibody incubation, sections were treated with a horseradish peroxidase (HRP)-conjugated rabbit/mouse secondary antibody solution from Dako, followed by color development using diaminobenzidine (DAB) (Dako, Denmark). Hematoxylin solution (03971, Sigma, USA) 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 (8890s, 1:1000, CST, USA) and TRITC-conjugated anti-mouse (ab6718, 1:1000, Abcam, UK) secondary antibodies in a 2% BSA solution for 60 min at room temperature in the absence of light. Nuclei were counterstained using 4',6-diamidino-2-phenylindole (DAPI) (D8417, Sigma, USA) for 8 min. For Hematoxylin and Eosin (H&E) and Gomori's Trichrome 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 Radio Immunoprecipitation Assay (RIPA) Lysis Buffer (strong) containing protease inhibitors (4,693,124,001, Roche, Switzerland). A total of 40 µg proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to Polyvinylidene fluoride (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 (ab2413, 1:1000, Abcam, UK). The membranes were washed with Tris buffered saline with Tween-20 (TBST) for 3 times and incubated for 1 h with a secondary antibody, anti-rabbit IgG antibody (ZB2301, 1:10,000, ZSGB-BIO, China).

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 ± standard error of the 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 Analysis of Variance (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. (Animal Research: Reporting of In Vivo Experiments).

Results

UCMSCs preferentially mitigated lung injury and lessened fibrosis

To evaluate the therapeutic efficacy of MSCs for pulmonary fibrosis, we administered UCMSCs or PLMSCs to bleomycin (BLM)-induced C57BL/6 mice three times during the onset of inflammation [30] (Fig. 1A). Throughout the experimental period, mice challenged with BLM exhibited a significant decrease in body weight, whereas control mice maintained an upward trend in body weight (Fig. 1B). Notably, mice treated with UCMSCs exhibited better recovery in terms of body weight compared to those treated with PLMSCs (the blue and green curves in Fig. 1B). Furthermore, Kaplan–Meier survival analysis revealed that UCMSC treatment significantly extended both the overall survival rate and median survival time (BLM group: 12.5 days vs. PLMSC: 14.5 days vs. UCMSC: 21 days) in mice with BLM-induced lung injury (Fig. 1C). These findings suggest that UCMSC treatment outperforms PLMSC treatment in mitigating BLM-induced lung injury.

Fig. 1
figure 1

UCMSCs demonstrated a superior therapeutic efficacy for lung injuries. A A schematic demonstration for the animal experiments. Mice were used to receive intratracheal bleomycin (BLM; 2 mg/kg body weight) or the same amount of saline at day 1. At day 3, two groups of BLM-challenged mice were subject to an intravenous (I.V.) injection of UCMSC or PLMSC (1 × 106), via the caudal vein, while one group of BLM-challenged mice and the group of control mice (without BLM-challenge) received the same volume of saline treatment. Mice were randomly grouped (n = 5 per group). B Body weight was examined for the mice (n = 5) subjected to various treatments. **: p < 0.01. C Kaplan–Meier survival curves of mice receiving different interventions. D Representative micro-CT images of lungs from mice in the experiment. The images were taken at 18th day post-injury. Three repeats from 5 mice were shown. E Lung volumes evaluated based on the three-dimensional reconstruction data from micro-CT; n = 5, p values were calculated. F Pulse distention, G Breath distention and H Oxygen saturation levels were measured by the MouseOx Small Animal Vital Signs Monitor. P values were calculated by two tail t-test; n = 5

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To decipher the physiological alteration of lung injury, we performed a micro-CT analysis. The result showed that mice treated with UCMSCs gained decreased tissue density, traction bronchiectasis, and septal thickening compared with the mice with saline treatment or PLMSC therapy (Fig. 1D). Of note, although PLMSC therapy demonstrated an improved imaged alteration, UCMSC therapy showed a significant regression of the fibrosis progression as demonstrated by the fibrotic dash area in the lung (Fig. 1D). To demonstrate the lung function, we performed a three-dimensional reconstruction based on the micro-CT images. The result showed that the lung volume was dramatically decreased when the mice were challenged with BLM (Fig. 1E, column 2 vs 1). However, UCMSC treatment notably restored lung volume to a greater extent than PLMSC therapy in mice challenged with BLM (Fig. 1E, column 3 and 4 vs 2). Consistently, we observed that the pulse distention (Fig. 1F), breath distention (Fig. 1G) and oxygen saturation (Fig. 1H), were much significantly improved by the UCMSC therapy, with a better effect than the PLMSC therapy.

To confirm the role of UCMSCs, we further treated the mice with secretomes from these cells (Additional file 11: Fig. S1A). Consistently, we observed that UCMSC secretomes outperformed those from PLMSCs in promoting the recovery of lung injury, as indicated by changes in body weight (Additional file 11: Fig. S1B), survival rate (Additional file 11: Fig. S1C), micro CT imaging (Additional file 11: Fig. S1D), and lung volumes (Additional file 11: Fig. S1E). These findings collectively suggest that UCMSCs offer distinct advantages over PLMSCs in reducing lung fibrosis and improving lung function in response to BLM challenge.

Pathological evaluation of the therapy on lung injury and fibrosis induced by Bleomycin

To investigate the therapeutic effects of UCMSCs and PLMSCs, we aimed to examine the alterations in lung tissues of mice sacrificed on 21th day at the pathological level. An anatomical analysis revealed that lungs subjected to the BLM challenge exhibited hemorrhagic necrosis, but both UCMSC and PLMSC treatments mitigated its severity (Fig. 2A). Hematoxylin–eosin staining analyses indicated that BLM challenge induced diffuse pneumonic lesions characterized by loss of normal alveolar architecture, septal thickening, enlarged alveoli, and increased infiltration of inflammatory cells in the interstitial and bronchiolar areas (Fig. 2B, upper panel). However, the MSC treatment, in particular the UCMSC treatment, dramatically reduced the pathological alterations (Fig. 2B, upper panel, compare UCMSC and saline). A Masson staining experiment showed that the pathological alterations of the lung occurred accompanied with the accumulation of collagenous fibers, which was significantly reduced by the UCMSC treatment (Fig. 2B, bottom panel). A statistical analysis showed a significantly reduced Ashcroft score (Fig. 2C) and collagen fiber area (Fig. 2D) in the lung tissues from the UCMSC treatment. Consistently, an immune-histochemical staining analysis showed that Col1a1, Col3a1 and ACTA2, markers of fibrosis, were highly expressed in the BML-challenged lungs but were significantly reduced by both UCMSC and PLMSCS (Fig. 2E). Of note, the UCMSC treatment showed better effects than PLMSC (Fig. 2E, compare UCMSC with PLMSC).

Fig. 2
figure 2

UCMSCs effectively alleviated the symptoms of fibrosis induced by Bleomycin. A Representative macroscopic views and H&E stain (scale bar, 500 μm) of whole lungs from mice at 21-day post-injury. B Representative histological lung sections from mice at 21 days post-injury stained with H&E and Masson’s trichrome. Scale bars, 50 μm. C Quantitative evaluation of fibrotic severity with the Ashcroft score in lungs of mice receiving different interventions. The Ashcroft scores were calculated based on the H&E staining. The severity of fibrotic alterations in each section was assessed as the mean score in the observed microscopic fields. Ten fields per section were selected and the scores were marked by two evaluators and averaged as the final values. D Quantitative collagen volume fraction (CVF). The CVF represents the percentage or proportion of collagen volume in tissues stained using Masson's trichrome. Three slides stained were assessed for each group. E Representative images of immunostaining against Col1a1, Col3a1 and ACTA2 in serial sections of lung tissues (n = 5). Bronchiolar regions are demarcated with an arrowhead in control sections. F–H Quantitative PCR analyses for the relative mRNA levels of Col1A1, Col3A1, and Fibronectin in the lung tissues of mice. I Western blots of Fibronectin in the lungs (n = 3). GAPDH was used as a loading control. Molecular weight was labeled. (* p < 0.05, ** p < 0.01, *** p < 0.001)

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To verify the pathological results, we performed an RT-PCR analysis. We observed that the expression of Col1a1 and Col3a1 was dramatically decreased by both USMSCs and PLMSCs (Fig. 2H, I). In particular, it appeared that the level of Col3a1 was recovered to the basal level as presented in the control group (Fig. I, compare UCMSC with control). Furthermore, we observed that fibronectin, another marker of late fibrosis, was dramatically decreased by the UCMSC treatment at both mRNA and protein levels (Fig. 2I, J). All these results suggest that the USMSC treatment is effective to reduce the fibrosis process after the lung damage.

UCMSCs showed superior therapeutic efficacy in treating lung injury and fibrosis induced by Bleomycin, when compared to PLMSCs

To investigate whether MSCs could halt the fibrosis process, we aimed to treat mice with BLM-induced fibrosis, spanning from the proliferative phase to the fibrosis phase. To achieve this, we initially induced fibrosis in mice by administering BLM and assessed fibrosis occurrence using micro-CT on day 11 (Fig. 3A). We then categorized mice based on the severity of the disease and initiated therapies at days 12, 18, and 24. Notably, all mice treated with MSCs survived, whereas 40% (3/7) of mice in the saline treatment group died (data not shown). We evaluated changes in fibrosis across different lung sections using micro-CT on day 28 (Additional file 11: Fig. S3A). The results revealed that fibrosis continued to progress in BLM-challenged mice treated with saline (Fig. 3B, indicated by the fibrotic foci in yellow), whereas it notably regressed in mice receiving UCMSC or PLMSC therapy (Fig. 3B, indicated by red arrows). Notably, fibrotic foci observed in various lung regions, including the bottom, middle, and top sections, decreased following MSC therapy across different experimental repeats (Fig. 3B, comparing day 28 with day 11). Furthermore, 3-D reconstruction analysis (Additional file 11: Fig. S3A) illustrated a significant reduction in lung volumes in all mice following BLM challenge on day 11, which were substantially restored with MSC treatments (Fig. 3C). We calculated the changes in lung volumes before and after therapy. The results indicated a decrease in lung volumes in mice treated with saline, while an increase was observed in mice receiving MSC therapy (Fig. 3D). Importantly, UCMSC therapy exhibited superior effectiveness in terms of lung volumes (Fig. 3C, blue vs. green columns) and volume alteration compared to PLMSC therapy (Fig. 3D, blue vs. green columns). These findings suggest that UCMSCs effectively halted the progression of fibrosis induced by BLM challenge. Overall, our results strongly indicate that UCMSCs are particularly effective in mitigating BLM-induced fibrosis in mice.

Fig. 3
figure 3

UCMSCs partially reversed pulmonary fibrosis and demonstrated a superior efficacy compared to PLMSCs. A A schematic map to show the animal experiments. Arrows indicate the indicated events. BLM was used at a dosage of 2 mg/kg. Mice were randomly grouped (n = 5 per group). B Representative micro-CT images on the same cross-section of lungs from all mice at the 11th day post-injury and at the 28th day after BLM-challenge. Two repeats were presented. C Lung volumes evaluated based on the three-dimensional reconstruction data from micro-CT images on the 28th day post injury (n = 5). D An illustration of the alterations in lung volumes observed in response to therapeutic interventions. Lung volumes were calculated both before (baseline) and after treatment. A negative value indicates a decrease in lung volume post-treatment. (n = 3). E A representative immunofluorescent image of lung sections against ACTA2 from the mice is shown

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We proceeded to analyze the pathological changes in mice with late-stage fibrosis. Consistently, we observed that UCMSC treatment significantly enhanced lung volume and ameliorated fibrosis in the lungs (Additional file 11: Fig. S3B). Histological analysis further revealed that UCMSC treatment reversed fibrosis (Additional file 11: Fig. S3C, S3D). Finally, we conducted an immunofluorescence assay using an antibody against ACTA2, a marker of myofibroblasts. The results demonstrated that UCMSC treatment reduced the severity of fibrosis, which appeared to worsen during the late stage of fibrosis (Fig. 3E). Overall, these findings suggest that both UCMSCs and PLMSCs can effectively mitigate fibrosis induced by lung injury, with UCMSCs exhibiting superior efficacy over PLMSCs.

The genetic expression features of UCMSCs and PLMSCs

To reveal the feature of UCMSCs and PLMSCs in the regulation of fibrosis, we performed a scRNA-Seq analysis (Additional files 2, 3: Tables S2 and S3). The results revealed that both UCMSCs and PLMSCs could be categorized into six subgroups through UMAP dimensionality reduction via cluster analysis (Fig. 4A, 4B). Interestingly, subgroup distribution exhibited a preference between UCMSCs and PLMSCs, with a higher proportion of cells in subgroups 2, 3, and 5 in UCMSCs and subgroups 1 and 4 in PLMSCs (Fig. 4C, 4D). Further analysis unveiled that the highly expressed genes in subgroups 2, 3, and 5 were associated with pathways related to Nuclear Factor-kappa-B (NF-kB), IL17, Tumor Necrosis Factor (TNF)-α, and Transforming Growth Factor (TGF)-β, while subgroups 1 and 4 were associated with cell behaviors including cell cycle, focal adhesion, and senescence (Fig. 4E). The detailed gene expression patterns revealed that the most abundantly expressed genes in subgroups 1 and 4 are related to the extracellular matrix, including Febronectin1 (FN1) and matrix metallopeptidase (MMP1/3), whereas those in subgroups 2, 3, and 5 are associated with inflammation, including CCL2, IL1B, and CXCL1/6 (Fig. 4F). Additionally, both UCMSCs and PLMSCs exhibited a similar small proportion of subgroup 6, characterized by genes related to stem cell features, such as Mesoderm specific transcript (MEST), Insulin like growth factor binding protein 2 (IGFBP2), Tissue inhibitor of metalloproteinase 3 (TIMP3), Actin gamma 2 (ACTG2), and Thy-1 cell surface antigen (THY1) (Fig. 4C, 4F, bottom). Detailed analysis of gene expression in different subgroups demonstrated consistency with the grouped genes (Fig. S4A).

Fig. 4
figure 4

The features of UCMSCs and PLMSCs. A UMAP projection of single cells analysis for the UCMSC and PLMSC mixture. UCMSCs were represented in green, while PLMSCs were depicted in orange (B) Six clusters were identified in the UMAP projection. The color corresponding to each subpopulation was delineated in the figure notes. C The percentage of UCMSCs and PLMSCs in each subpopulation. D The proportion of each subpopulation in UCMSCs and PLMSCs. E An enrichment analysis of highly expressed genes across six subpopulations of UCMSC and PLMSC from single-cell clustering. F The top 10 highly expressed genes and their preference of each subpopulation were categorized. G The highly expressed secreted proteins in UCMSCs and PLMSCs were shown in a heatmap. H The quantification of the expression levels of highly secreted proteins in UCMSCs and PLMSCs was shown in each group via a violin diagram. I–J Highly expressed secreted proteins CCL2 (I) and CXCL1 (J) amongst UCMSCs and PLMSCs in UMAP projection of single cells analysis were shown

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On the other hand, we examined the changes in genes encoding secretory proteins between UCMSCs and PLMSCs. We found that 18 genes encoding secretory proteins were preferentially highly expressed in UCMSCs, while 6 genes were abundantly expressed in PLMSCs but not in UCMSCs (Fig. 4G). Interestingly, the expression of these genes aligned with the subgroup features (Additional file 11: Fig. S4B). In other words, we attribute the expression of secretory proteins to the subgroups of MSCs. Notably, we observed that Wnt5A/B and EGF1/5 were expressed at low levels in UCMSCs but were abundant in PLMSCs (Fig. 4G, bottom). We concluded with a fluorescence-activated cell sorting (FACS) analysis to validate the expression of genes in the subgroups (Fig. 4H, I). As expected, the expression of CCL2 was detected in subgroups 2, 3, and 5 (Fig. 4H), while CXCL1 expression was observed in subgroup 2, 3, and 5 (Fig. 4I). Similarly, the expression of other genes encoding secretory proteins was confirmed in different subgroups (Additional file 11: Fig. S4C). Notably, MMP1 expression was found to be differentially upregulated in UCMSCs and PLMSCs (Additional file 11: Fig. S4D). Taken together, these findings suggest that UCMSCs maintain subpopulations to regulate inflammation, while PLMSCs possess subpopulations to regulate the extracellular matrix. We speculate that the different subgroups of cells might contribute to the preference of MSCs in ameliorating the lung injury.

The response of gene expression to cytokines in UCMSCs and PLMSCs

The analyses conducted on the intrinsic features of UCMSCs and PLMSCs prompted us to investigate whether these cells could alter their phenotypes under in vivo conditions in an inflammatory environment. To address this question, we performed bulk RNA-seq analysis on the cells under inflammatory cytokine challenges (Additional file 4: Table S4). Initially, we confirmed that the results from the bulk RNA-seq analysis were consistent with those from previous RNA-seq analyses (Additional file 11: Fig. S5A). Next, we compared the up-regulated genes in response to inflammation challenges. We used four cytokines (4-F), IL-1β, IL-6, IFN-γ, and TNF-α, reported as the main components of inflammation storm, to prime the MSCs in culture dishes. The results revealed that 764 and 545 genes were elevated by the cytokines (Fig. 5A, 5B). Heat map analysis indicated that more up-regulated genes were induced in UCMSCs than in PLMSCs by 4-F (Fig. 5C). Specifically, 2159 genes were specifically induced in UCMSCs, while 1185 genes were up-regulated in PLMSCs by 4-F (Fig. 5D, top panel). Notably, 1962 genes were induced in both UCMSCs and PLMSCs (Fig. 5D, top middle). To elucidate the functions of MSCs under inflammatory conditions, we focused on secreted proteins, as we observed that the supernatants from the MSCs remained effective (see Additional file 11: Fig. S1 and S2). We identified that 41 secretory proteins were specifically increased in UCMSCs, while 16 secretory proteins were induced in both UCMSCs and PLMSCs upon 4-F priming (Fig. 5D, bottom panel). Heat map analysis further demonstrated five groups of secretory proteins from the MSCs in response to 4-F (Fig. 5E, Additional file 5: Table S5). Notably, group 1 represented genes specifically decreased in UCMSCs, group 2 represented genes specifically increased in PLMSCs, group 3 represented genes specifically decreased in UCMSCs, group 4 represented genes specifically increased in MCMSCs, and group 5 represented genes increased in both UCMSCs and PLMSCs upon 4-F priming (Fig. 5E). We focused on the proteins in groups 4 and 5 as those induced in UCMSCs, which showed a better effect than PLMSCs in the recovery of lung injury. Subsequently, we performed a Gene Ontology (GO) analysis to address the targets of the secretory proteins on cells in the lung. The results indicated that the majority of the proteins retained the ability to regulate macrophages (Fig. 5F). Consistently, we demonstrated that the upregulated proteins possessed the ability to activate different signaling pathways, including type I interferon response and viral response (Additional file 11: Fig. S5B). Detailed heat map analyses demonstrated that cytokines (Additional file 11: Fig. S5C), inflammatory regulator genes (Additional file 11: Fig. S5D), anti-inflammatory cytokines (Additional file 11: Fig. S5E), and receptors (Additional file 11: Fig S5F) were significantly induced by 4-F. Overall, these findings suggest that UCMSCs can secrete factors under inflammatory conditions, seemingly able to regulate macrophage responses.

Fig. 5
figure 5

The gene expression of UCMSCs and PLMSCs upon inflammation challenge. A A scatter plot displaying the differentially expressed genes (DEGs) between 4 factors stimulate UCMSCs (4-F) and UCMSCs (Ctrl). 764 genes were upregulated. 4 factors (4-F) were 4 cytokines, IL-1β, IL-6, IFN-γ, and TNF-α. B A scatter plot displaying the differentially expressed genes (DEGs) between stimulate PLMSCs (4-F) and PLMSCs (Ctrl). 545 genes were upregulated. C A heatmap illustrating the expression of total genes in UCMSCs and PLMSCs, before and after priming with 4 cytokine D Venn diagrams illustrating overlapped upregulated genes between UCMSCs and PLMSCs upon 4-F priming. Green circle represents 2159 genes that were individually upregulated after 4-F priming. Purple circle signifies 1185 genes that were individually upregulated after 4-F priming. Overlapping regions indicates 1962 genes co-upregulated. The bottom Venn diagrams displays the secreted genes upregulated post stimulation. E A heatmap illustrating the expression of secreted proteins in UCMSCs and PLMSCs, before and after 4-F priming. F A heatmap depicting the gene enrichment in macrophage-related GO terms for secreted proteins. The functions of the protein groups were annotated

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UCMSCs exhibited a higher efficiency in stimulating the M2 polarization of macrophages compared to PLMSCs

To investigate whether UCMSCs preferentially regulate macrophages, we determined to study the phenotype alteration of monocytes under an in vitro condition. To this end, we isolated monocytes from mouse bone marrow (Additional file 11: Fig. S6A) and co-cultured with MSCs. Intriguingly, we observed that the monocytes dramatically emerged in an M2 phenotype when co-cultured with MSCs (Additional file 11: Fig. S6B). Of note, more M2 macrophages were observed under the culture with UCMSCs than PLMSCs (Additional file 11: Fig. S6B, UCMSCs vs PLMSCs). This result suggests that MSCs may induce the macrophage polarization.

To characterize the features of macrophages regulated by MSCs, we induced monocyte differentiation towards the M1 phase, recognized as a polarized subpopulation that promotes inflammation [31]. The results demonstrated that co-cultured MSCs significantly decreased the population of M1 macrophages (Additional file 11: Fig. S6C) but increased the subpopulation of the M2 phase (Fig. 6A, top panel, 6C, left columns), despite the monocytes being initially induced under conditions to promote M1 macrophage polarization (100 ng/ml Lipopolysaccharides, LPS + 20 ng/ml IFN-γ for 36 h) [32]. This result suggests that MSCs maintain an ability to shift M1 macrophages into M2 macrophages. Conversely, we cultured isolated monocytes under conditions to induce M2 macrophages (10 ng/ml IL-4 for 36 h) [33] using the conditioned medium from MSCs. The results showed that the monocytes were polarized into the M2 phase, and both UCMSCs and PLMSCs significantly enhanced the population (Fig. 6B, 6C, right columns). Of note, UCMSCs exhibited a greater capacity to sustain the M2 phenotype compared to PLMSCs (Fig. 6A-6C, UC vs. PL). These findings indicate that MSCs, particularly UCMSCs, possess the ability to enhance the polarization of macrophages into M2 more effectively than PLMSCs in vitro.

Fig. 6
figure 6

UCMSCs induced macrophage polarization to M2. A Representative contour plots showing expression of F4/80, CD11b, CD11c, CD206 in macrophages stimulated with LPS (100 ng/ml) and IFNγ (20 ng/ml) and co-cultured with UCMSC and PLMSC for 36 h. The numbers indicate the percentage of cells within the gates. B Representative flow cytometry plots showing expression of F4/80, CD11b, CD11c, CD206 in macrophages stimulated with IL-4 (20 ng/ml) and co-cultured with UCMSC and PLMSC for 36 h. The numbers indicate the percentage of cells within the gates. C Proportions of M2 macrophages co-cultured with UCMSC and PLMSC. D Representative flow cytometry pseudo color plots showing expression of Siglec-F, CD11b, CD206 in alveolar macrophages from different groups of mice on day 3 post BLM challenge. E Proportions of Siglec-F + , CD11b + macrophage from bronchoalveolar lavage fluid on the 3rd and 7th day. F Representative FACS pseudo color plots showing expression of Siglec-F, CD11b, CD206 in alveolar macrophages from different groups of mice on day 7 post BLM challenge. G Proportions of Siglec-F + , CD11b + CD206 + M2 macrophages from bronchoalveolar lavage fluid on the 3rd and 7th day. H Immunofluorescence images for F4/80 and CD206 expression in the lung tissues from BLM-induced mice after UCMSC and PLMSC treatment on the 7th day. I Relative proportions of M2 macrophages that are double-positive for F4/80 and CD206

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To address if MSCs regulate macrophage polarization in vivo, we investigated the populations of macrophages during the therapy of the BLM-induced acute pneumonia in mice. To this end, we collected bronchoalveolar lavage fluid (BAL) from mice challenged with BLM and treated with saline, UCMSCs, and PLMSCs via caudal intravenous injection at different time points. FACS analyses showed that total macrophages (characterized by Siglec-F + CD11C +) [34] were decreased when the mice were challenged with BLM under saline treatment (Fig. 6D, top panel, saline vs control), but were recovered when the mice were treated with UCMSCs and PLMSCs within 3 days (Fig. 6D, top panel, UC and PL vs control and saline). Of note, CD206 + macrophages, representing the M2 phenotype, exhibited consistent decreases under the BLM-challenge condition but were restored by both UCMSCs and PLMSCs (Fig. 6D, bottom panel, 6E). Similar alterations in macrophage populations were observed after 7 days of treatment (Fig. 6F), with a more pronounced recovery of both total macrophages and CD206 + macrophages in mice treated with UCMSCs compared to PLMSCs. Quantitative analysis further confirmed the recovery of M2 macrophages in BLM-challenged mice treated with MSCs (Fig. 6G). These analyses indicate that MSCs promote the recovery of M2 macrophages during the pathological process of BLM-induced lung injury.

To demonstrate the accumulation of M2 macrophages in lung tissue (acknowledged as interstitial macrophages), we conducted an immunostaining experiment using an antibody against CD206 [35]. The results revealed an increase in CD206 + cells in the lung tissues from BLM-challenged mice treated with UCMSCs and PLMSCs on day 7 (Fig. 6H). Quantitative analysis showed a higher presence of CD206 + cells in the lung tissue from mice treated with UCMSCs compared to PLMSCs (Fig. 6I). Interestingly, we observed that while the total macrophage count in the BAL was low in the saline group, the level of CD206 + macrophages remained highest in this group after 21 days of BLM challenge (Additional file 11: Fig. S6D). Taken together, these results suggest that both UCMSCs and PLMSCs regulate the polarization of macrophages into the M2 phase from acute pneumonia to chronic pneumonia.

Changes in gene expression in macrophages from diseased mice treated with UCMSCs and PLMSCs

To identify critical genes expressed in macrophages polarized by UCMSCs and PLMSCs, we conducted transcriptome sequencing analyses. Initially, we assessed the gene expression alterations of macrophages under co-culture conditions with MSCs (Additional file 6: Table S6). Subsequently, we classified the genes according to the definition of M1 and M2 features. Interestingly, we observed that UCMSCs upregulated 41 genes (Fig. 7A, Group 1 and Group2), whereas PLMSCs upregulated 36 genes (Fig. 7A, Group 1 and Group 5). Significantly, these 41 genes were also suppressed by PLMSCs, although the reduction level was not as pronounced as that by UCMSCs (Fig. 7A, Additional file 7: Table S7). Interestingly, we observed that UCMSCs suppressed the expression of 13 genes that are reported to be upregulated in M2 macrophages (GSEA M14515) (Fig. 7A, Group 3). Reciprocally, we noted that UCMSCs repressed 30 genes associated with the M1 phenotype (Fig. 7B, Additional file 8: Table S8). Additionally, for these feature genes, we observed that PLMSCs upregulated 29 genes (Fig. 7B, bottom right). These findings collectively suggest that UCMSCs polarize macrophages towards an M2-like phenotype, while PLMSCs exhibit a partial effect. This alteration may explain our earlier observations indicating that UCMSCs possess a stronger ability to facilitate lung injury recovery compared to PLMSCs.

Fig. 7
figure 7

Macrophages educated by MSC demonstrated a dynamic gene expression profile. A–B Heat maps showing the transcriptomes of M2 vs M1 featured genes (A) or M1 vs M2 featured genes (B), in macrophages co-cultured with medium, UCMSC and PLMSC. C A schematic representation of the experimental protocol. Mice were administered intratracheally with BLM or saline on day 1. UCMSC or PLMSC treatment was performed for BLM-challenged mice on day 2. Saline was given to either the control mice or the BLM-challenged mice. Bronchoalveolar lavage fluid was collected, cells were flow-sorted by an antibody against CD45, and mRNAs were sequenced. Mice were randomly grouped (n = 3 per group). D–F The expression of SPP1 (D) and Trem2 (E) and Cebpb (F) was shown. FPKM (fragments per kilobase of exon per million reads mapped) values, represented as mean ± SEM, were calculated from the RNA-seq data. G–I Streamgraphs illustrating the expression patterns of alveolar macrophage-associated genes in the macrophages from lung bronchoalveolar lavage fluid in mice. Different genes were presented in colors, and the samples of mice received UCMSC or PLMSC treatment at day 3 or 7 were analyzed. PBS treatment was used a control to demonstrate the spontaneous alteration of gene expression

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To further address the alteration of gene expression in the macrophages under the pathological condition, we isolated the alveolar macrophages during the recovery of BLM-induced lung injury under the therapy by UCMSCs or PLMSCs (Fig. 7C). A PCR experiment showed that the M2-macrophage marker genes including Cxcl10, Entpd1, Mrc1, Fizz1, Tgfb, Cd36 and Klf3 [36] were significantly up-regulated by both UCMSCs and PLMSCs, but it appeared that UCMSCs remained of a stronger effect than PLMSCs, at day 3 after BLM challenge, with one dosage of therapy by MSCs (Fig. S7A). We then performed RNA-seq analyses on the alveolar macrophages at day 3 and 7 after BLM challenge, when the mice were treated with MSCs in two times (Additional file 9: Table S9). In particular, we observed that genes including SPP1, Trem2 and Cebpb, which were reported to be upregulated in the macrophages with the process of fibrosis [37,38,39], were reduced by UCMSCs at both day 3 and 7 while changed slightly by PLMSCs (Fig. 7D-7F). Finally, we examined the dynamic alteration of genes featured for macrophages. The results showed that UCMSC treatment induced the gene expression quite differentially from PLMSC treatment (Fig. 7G–I). Notably, CD127 and Arginase 1 (ARG1) were significantly altered differently by UCMSCs compared to PLMSCs. The expression of these two genes increased during inflammation and decreased during fibrosis (Fig. 7G–I, brown and blue). We verified the alteration of CD127 + macrophages by a FACS analysis. The result showed that CD45+CD11C−F4/80+CD64+CD127+ macrophages were dramatically induced by UCMSCs at day 3 (Additional file 11: Fig. S7B). Consistently, we found that CD127 + macrophages were induced by UCMSCs but not by PLMSCs under a culture condition (Additional file 11: Fig. S7C). Taken together, all the results suggest that UCMSCs induce the macrophages towards a phenotype to ameliorate the fibrosis.

Discussion

Idiopathic pulmonary fibrosis (IPF) remains of a thread to human health. To date, no efficient treatment is available to cure this chronic inflammation related disease. In this study, we evaluated the therapeutic effect of UCMSCs and PLMSCs on the lung injury and pulmonary fibrosis. We have provided evidence that UCMSCs exhibited a better ability to effectively ameliorate the BLM-induced lung injury and fibrosis than PLMSCs. Intriguingly, we observed that UCMSCs were able to terminate the process of fibrosis caused by inflammation. Our findings shed a light for the application of UCMSCs on the therapy of chronic lung injury. As many clinical trials have been reported for the application using MSCs for therapy [22], our study defined a preference of MSCs in the inflammation-induced fibrosis. Obviously, we propose to use UCMSC for its tendency to polarize M2 macrophages and to affect the fibrosis process (Fig. 8). We expect that this therapy could be useful in the clinical practice in human patients in the near future.

Fig. 8
figure 8

A cartoon to illustrate the mechanism of UCMSC’s preference on the therapy of lung-injury induced fibrosis. UCMSCs preferentially respond to the inflammation environments and secret factors such as CCL2 and CXCL1 upon cytokine priming. UCMSCs polarize the macrophages into the M2 phase, by down-regulating the expression of genes related to fibrosis, including SPP1, Trem2, and Cebpb, but up-regulating the genes of CD127 and Arg1. UCMSCs maintain a better ability to ameliorating the lung injury induced fibrosis

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We have characterized the features of UCMSCs and PLMSCs to unravel the preference in the regulation of macrophages. These features are deciphered as preferred expression of genes and mutual expression of genes. Based on the intrinsic difference of the gene expression, we further revealed the different expression of genes under an inflammation-challenge condition. We used IL-1β, IL-6, IFN-γ, and TNF-α, 4 cytokines for the inflammation storm, to prime both UCMSCs and PLMSCs. This is a mimic of the environment of infection or damages of the lung in human. Intriguingly, we identified that 41 secreted proteins were specifically upregulated in UCMSCs and 16 factors were upregulated in mutually in UCMSCs and PLMSCs upon the cytokine priming. We considered that these factors are the regulators from MSCs to reverse or terminate the process of inflammation-induced fibrosis. Basically, we propose that UCMSCs, better than PLMSCs, response to the inflammation-challenge and then secret factors to regulate the fibrosis process.

We attributed the role of MSCs to the regulation of macrophage polarization. We provided evidence that both UCMSCs and PLMSCs possessed an ability to induce macrophages into the M2 phase rather than M1 phase. As M1 macrophages are the major sources of factors to promote fibrosis, we considered that the presence of M2 macrophages is beneficial for the recovery of the inflammation. Our conclusion is supported by several previous studies reporting that MSCs remained potential to transform macrophages into an anti-inflammatory/immunosuppressive phenotype [40]. In another study, Nakajima and Honglong Zhou observed that MSCs were able to recover spinal cord injuries by shifting macrophages from the M1 to the M2 subtype [41, 42]. Consistently, a study in the therapy of MSCs in the acute liver injury and fibrosis confirmed that MSCs suppressed pro-inflammatory M1 cells and promoted anti-inflammatory M2 cells [43, 44]. Similar findings were reported in the therapy of diabetes using MSCs [45, 46]. All these studies support our notion that MSCs regulate macrophage polarization to inhibit inflammation and inflammation-induced fibrosis.

We considered that the role of MSCs on the regulation of macrophages was through the secreted factors. We identified 41 specific factors and 16 mutual factors. Our bioinformatics analyses revealed that these factors are the major force to regulate macrophages. However, we could not exclude the possibility that MSCs might directly regulate macrophages through cell–cell interactions. Indeed, several studies validated that MSCs modulated macrophage polarization through direct cell-to-cell interactions [47]. This might be through the receptors on the MSCs. Indeed, in our RNA-seq results, we observed that several receptors on the MSCs were upregulated upon the 4-F stimulation (Additional file 11: Fig. S5F). While we agree the role of the receptors of MSCs on the macrophage regulation, it appeared that the secreted factors take a majority of alteration in MSCs upon the cytokine challenge. Therefore, we speculate that MSCs regulate macrophages mainly through the secreted factors.

Another role of MSCs on the regulation of inflammation-induced fibrosis might be through a direct regulation on other cells such as fibroblasts. One of such an effector might be MMP1/3. In our results, we observed that MMP1/3 was induced, in particular, by UCMSCs. As MMP1 plays a role in the regulation of extracellular matrix, which is critical for the fibrosis [48], we speculate that its induction by MSCs is beneficial for the termination of fibrosis. Therefore, in the late stage of the recovery of the lung injury, MMP1 may play an important role in the remodeling of the extracellular matrix so that the fibrous tissue could be eliminated. Indeed, in our animal experiments, we observed that the images of the fibrosis nodules were reduced by the UCMSC therapy (see Fig. 3). This could not be explained by the reduced inflammation as fibrosis nodules are hard to be reduced as observed in the clinical practice. In line with our hypothesis, a study reported that transplanting human MMP1-overexpressing bone marrow-derived mesenchymal stem cells mitigated CCL4-induced liver fibrosis in rats [49]. Therefore, it is possible that induction of MMP1/3 remodeled the fibrosis nodules to ameliorate the lung function.

The preference of UCMSCs on the inflammation-induced lung injury and fibrosis is of great interest. Our experiments demonstrated that UCMSCs always performed a better effect on the lung injury recovery than PLMSCs. This might be due to the specifically secreted factors as identified. We have attributed these factors to polarize M2 macrophages. Also, we found that UCMSCs produced MMP1 more abundantly than PLMSCs, as we discussed in aforementioned results. However, other specific features of UCMSCs might also contribute to the merit on the recovery of fibrosis. One critical characteristic might be the low immunogenicity of UCMSCs. Recent studies have reported that MSCs are typically detected and cleared by the immune system in the body [50], although previous studies neglected their immunogenicity and observed no immune privileges [51, 52]. It is worth investigating whether UCMSCs have a lower immunogenicity than PLMSCs, which allows them to reside in the body for a longer duration. To address this question, we should clarify the distribution and duration of MSCs in different organs. As many studies reported that MSCs mainly distributed in the lung, with a duration of about 2 weeks [53, 54], we consider that UCMSCs might reside more in the lung, as well as other organs, than PLMSCs. This needs to be addressed further in our future study. On the other hand, it is also possible that UCMSCs might be more sensitive to chemokines, which direct them to migrate into the injury tissues. Indeed, in our results, we observed that UCMSCs expressed more chemotactic genes than PLMSCs (see Additional file 11: Fig. S5). We speculate that UCMSCs remain of a better chemotactic ability than PLMSCs. Nevertheless, we speculate that the preference role of UCMSCs on the inflammation-induced lung injury and fibrosis is a comprehensive read-out of the features in this population of the MSCs. As the MSCs currently used in the clinical trials are of a notable heterogeneity [38], we speculate to purify the subpopulation of the cells with the features on the regulation of macrophages, or the production of MMP1, will improve the efficiency of the therapy in the clinical practice.

Conclusions

In summary, our results demonstrated that UCMSCs displayed preference of response to cytokine stimulation and on the polarization of M2 macrophages. These intrinsic differential features of MSCs defined that UCMSCs were better than PLMSCs in therapeutic efficacy against lung injury and fibrosis.

Availability of data and materials

The data and materials supporting the findings of this study are available within the article. Additional data are made available in supplementary tables of this manuscript. The relevant experimental data are available from a repository, namely zenodo, and can be accessed through the following link (https://doiorg.publicaciones.saludcastillayleon.es/10.5281/zenodo.10906087 for single RNAseq, and https://doiorg.publicaciones.saludcastillayleon.es/10.5281/zenodo.10906224 for bulk RNAseq).

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Acknowledgements

This work was supported by the Tsinghua University Spring Breeze Fund (20201080606), Vanke Special Fund for Public Health and Health Discipline Development, Tsinghua University (2022Z82WKJ008), China.

Funding

This work was supported by the Tsinghua University Spring Breeze Fund (20201080606), Vanke Special Fund for Public Health and Health Discipline Development, Tsinghua University (2022Z82WKJ008). The roles of the funding body in the design of the study encompass providing financial support, offering input on research questions and methodologies, and assisting in defining objectives and scope.

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Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, School of Basic Medical Sciencese, Institute of Precision Medicine, Tsinghua University, Beijing, 100084, China

    Meng Li, Ying Wang, Guancheng Jiang, Hanguo Jiang, Mengdi Li, Fangli Ren, Yinyin Wang & Zhijie Chang

  2. Heya Pharmaceutical Technology Company, Beijing, 100176, China

    Jun Li

  3. First Medical Center, Chinese PLA General Hospital, Beijing, 100853, China

    Ziying Zhu & Muyang Yan

Authors
  1. Meng Li
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  2. Jun Li
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  11. Zhijie Chang
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Contributions

ZJC, MYY conceived the project and supervised the experiments. ML completed majority of the experiments and data analysis. ML, ZJC wrote the manuscript with help from all the authors, JL, YW, GCJ, HGJ, MDL, ZYZ, FLR, YYW participated in the experiments and data analysis. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Muyang Yan or Zhijie Chang.

Ethics declarations

Ethics approval and consent to participate

All experimental designs and protocols involving animals were approved by the Institutional Animal Care and Use Committee of Tsinghua University (Title of the approved project: Umbilical cord-derived mesenchymal stem cells preferentially modulate macrophages to alleviate pulmonary fibrosis. Approval Form ID: THU-LARC-2024-008. Date of IACUC approved: 2020/3/20) and complied with the recommendations of the academy’s animal research guidelines. The animal quality test batch number is BJVRL-WBKH-20200407A4. The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Ethics Committee of Seventh Medical Center of Chinese PLA General Hospital (Title of the approved project: Umbilical cord and placental tissues and derivatives used in scientific studies of chronic inflammation in laboratory animals. Approval Form ID: 202200016. Date of IACUC approved: Jan 31, 2020). The patient(s) provided written informed consent for the use of samples. The inclusion and exclusion criteria for clinical umbilical cord and placenta samples are described in detail in the Additional file 10: Table S10.

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The authors declare no competing interests.

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Li, M., Li, J., Wang, Y. et al. Umbilical cord-derived mesenchymal stem cells preferentially modulate macrophages to alleviate pulmonary fibrosis. Stem Cell Res Ther 15, 475 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04091-7

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04091-7

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512