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. 2022 Apr 12;13(1):163.
doi: 10.1186/s13287-022-02839-7.

Every road leads to Rome: therapeutic effect and mechanism of the extracellular vesicles of human embryonic stem cell-derived immune and matrix regulatory cells administered to mouse models of pulmonary fibrosis through different routes

Shengnan Yang #  1   2   3   4 , Peipei Liu #  2   3   4   5 , Tingting Gao #  6   7   8 , Dingyun Song  2   3   4 , Xinyu Zhao  1   2   3   4 , Yupeng Li  1   2   3   4 , Jun Wu  6   7   8   9 , Liu Wang  6   7   8   9   10 , Zai Wang  11 , Jie Hao  12   13   14   15   16 , Chen Wang  17   18   19   20   21 , Huaping Dai  22   23   24
Affiliations

Every road leads to Rome: therapeutic effect and mechanism of the extracellular vesicles of human embryonic stem cell-derived immune and matrix regulatory cells administered to mouse models of pulmonary fibrosis through different routes

Shengnan Yang et al. Stem Cell Res Ther. .

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. Whether extracellular vesicles are effective in treating IPF and what is the optimal administrative route is not clear. Our previous studies have shown that immunity and matrix regulatory cells (IMRCs) derived from human embryonic stem cells can safely treat lung injury and fibrosis in mouse models, and its mechanism of action is related to the paracrine effect. In this study, we investigated the therapeutic effects of IMRC-derived extracellular vesicles (IMRC-EVs) on a bleomycin-induced pulmonary fibrosis mouse model and explored the optimal route of administration.

Methods: To study the biodistribution of IMRC-EVs after administration via different routes, NIR labeled-IMRC-EVs were delivered by intratracheal (IT) or intravenous (IV) route, and in vivo imaging was acquired at different time points. The therapeutic effects of IMRC-EVs delivered by different routes were analyzed by assessing histology, lung function, cytokines levels, and transcriptome profiling. RNA-seq of lung tissues was performed to investigate the mechanisms of EV treatment through IT or IV administrations.

Results: IMRC-EVs mainly reserved in the liver and spleen when administrated via IV route; and mainly retained in the lungs via the IT route. IMRC-EVs administrated via both routes demonstrated a therapeutic effect as attenuated pulmonary fibrosis, improved lung function, and histological parameters. Based on our RNA-seq results, different pathways may be affected by IMRC-EVs administrated via IT or IV routes. In addition, in vitro experiments showed that IMRC-EVs inhibited epithelial-to-mesenchymal transition induced by TGF-β.

Conclusion: IMRC-EVs administrated via IT or IV routes generate different biodistributions, but are both effective for the treatment of bleomycin-induced pulmonary fibrosis. The therapeutic mechanisms of IMRC-EVs administrated via different routes may be different.

Keywords: Biodistribution; Extracellular vesicles; Human embryonic stem cells; IMRC; Mesenchymal stem cells; Pulmonary fibrosis; Route of administration.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Characterization of EVs. a Process of differential ultracentrifugation to purify EVs. b Representative transmission electron microscopy image of EVs. Left scale bar = 2 μm. Right scale bar = 200 nm. c Nanoparticle tracking analysis showed the size and distribution of EVs purified by differential ultracentrifugation. The average size of EVs is 120 nm. d Representative Western blot analysis of the exosomal marker CD63, CD81 and TSG101, and the endoplasmic reticulum marker calnexin in EVs derived from IMRCs and MRC-5 cells. Cellular lysate (CL) was used as a control
Fig. 2
Fig. 2
Therapeutic effect of IMRC-EVs on BLM-induced lung fibrosis mouse model. a Schematic diagram of the in vivo experimental design of the BLM-induced PF mouse model. b Changes in relative body weight (%) of mice receiving different interventions. c Lung coefficient (wet lung weight/total body weight) of all treatment groups. d–f Mouse lung tissues from each group were collected and embedded in paraffin for histological analysis. d Representative histology of lung sections stained with H&E. The small graphs inside the figure represent whole lung from all groups at day 21 post-injury. e Representative Masson staining. myofibers (red), collagen fibers (blue), and nucleus (black-purple). f Representative picrosirius red staining. Scale bar = 100 μm. Collagen I displays an orange-red birefringence under polarized light, whereas Collagen III has a green birefringence. g, h Immunohistochemical (IHC) analysis of Collagen I (col-I) (g), and α-smooth muscle actin (α-SMA) (h) after IMRC-EVs and MRC5-EVs transplantation. Original magnification is ×200, scale = 100 μm. i Quantification of fibrosis by Ashcroft score. Ashcroft score was measured by averaging the score from a blinded and a non-blinded scorer. j Changes in hydroxyproline levels of the lung in different treatment groups. k, l The quantification of relative immunostaining of Col-I (k) and α-SMA (l). Data are represented as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
The distribution of IMRC-EVs delivered by different routes. a Live animal imaging of NIR fluorescence after IMRC-EV injection via different routes. IMRC-derived EVs were labeled with NIR and injected into normal mice and BLM- induced mice via the IT or IV route. Mice in the control group were injected with equal volumes of normal saline. b Representative images of organs (24 h post-injection) from BLM-induced mice injected i.v. with IMRC-EVs. c, d The change trend of the fluorescence intensity signal at different time points after the injection of NIR-labeled IMRC-EVs via the IT route (c) and IV route (d)
Fig. 4
Fig. 4
The effect of IMRC-EVs delivered via the tracheal and intravenous routes on cytokine levels in the acute phase of the BLM-treated mice. Five days after BLM injury, mice were euthanized and cytokine levels in lung lysate were analyzed by ELISA (a–h). Data are represented as the mean ± SD; n = 5. *P < 0.05, **P < 0.01, *** P < 0.001, and ns indicates no difference. TGF-β1 transforming growth factor-β1, IL interleukin, MCP Monocyte chemoattractant protein, TNF-α tumor necrosis factor-α, MIP macrophage inflammatory protein and VEGF vascular endothelial growth factor
Fig. 5
Fig. 5
Inhibition of pulmonary fibrosis by IMRC-EVs delivered via different routes in a mouse model of BLM-induced pulmonary fibrosis. a, b H&E staining (a) and Masson staining (b) micrographs of paraffin sections of mouse lung tissue in each group. The blue filaments represent collagen fibers, indicating interstitial fibrosis. c, d Representative photomicrographs of Col-I (c) and α-SMA (d) immunostained sections from each group. e Semi-quantitative assessment was performed on day 21 using Ashcroft scoring method. f The content of hydroxyproline (HYP) was determined in lung tissues, which is a marker of collagen deposition. g The lung coefficients of mice in each group. h Western blot for Col-I and α-SMA protein expression in lung tissue from each group. Quantification of the relative Collagen I protein expression levels (i) and α-SMA protein expression levels (j). k Representative microCT image 21 days after BLM instillation. l Quantitative CT imaging was used to measure the lung volume (n = 4–6). The data are expressed as the mean ± SD.*P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no difference
Fig. 6
Fig. 6
Analysis of transcriptome changes in BLM-induced pulmonary fibrosis and the EV treatment groups through IT or IV routes. a Principal coordinates analysis (PCA) of transcriptome differences among the Control, BLM, IT_EVs and IV_EVs groups. Colors and shapes indicate different groups. Ellipses indicate the 95% confidence interval. b Comparison of the slopes among BLM, control groups and different pathways of EVs delivery groups. c Differentially expressed mRNAs among the four groups. d Gene expression heatmap across different groups for the DEGs. Heatmap colors indicate normalized gene expression ranging from high (red) to low (blue). e The bubble chart of GO enrichment of pairwise differentially expressed genes. The color of bubbles indicates the P-value, and the radius of bubbles is the gene ratio. Gene ratio, the percentage of DEGs in the enriched gene set
Fig. 7
Fig. 7
A549 cells were co-cultured with IMRC-EVs. A549 cells were incubated with PKH26 fluorescently labeled IMRC-EVs for 4 h. A549 cells were stained for actin (phalloidin, green) and nuclei (DAPI, blue). Scale bar, 50 μm
Fig. 8
Fig. 8
IMRC-EVs inhibit TGF-β-induced EMT in vitro. Immunofluorescence staining of E-cad (a) and Col-I (b) expression in A549 cells with or without the treatment with 2 ng/mL TGF-β and IMRC-EVs for 48 h. Scale bar, 20 μm. c Western blot for E-cad, N-Cad and Fn protein expression in A549 cells, with or without 2 ng/mL TGF-β1 and IMRC-EVs treatment for 48 h. GAPDH was used as a loading control. d–f Quantification of the relative protein expression levels in lung tissues from mice relative to GAPDH. g, h qPCR for the relative expression of E-cad and Col-I mRNA in A549 cells, with or without 2 ng/mL TGF-β1 and IMRC-EVs treatment for 48 h. I The expressions of Smad2/3, active p- Smad2/3 factors, α-SMA and collagen-I were determined by Western blotting. The relative phosphorylation ratio is determined by p-Smad2/3 vs. total Smad2/3 and was presented graphically (j). Quantification of the relative Collagen I protein expression levels (k) and α-SMA protein expression levels (l)
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