1 Extraction, differentiation and identification of adipose-derived MSC
First, we isolated and cultured MSCs from adipose tissue in C57BL/6 mice. The adipose tissue from the epididymis of C57BL/6 mice was collected in a sterile environment and digested with type I collagenase to remove impurities mature adipocytes. The primary adipose stem cells were obtained and purified by subsequent adherent culture and passage to the third generation[21]. The morphology of MSCs gradually changed after the adherent culture and passage, showing a long spindle shape comparable to a fibroblast-like morphology (Fig 1a).
We then used a specific differentiation medium to identify the differentiation ability of the MSCs. In the differentiation into adipocytes experiment, large lipid droplets were seen in the cytoplasm of the induced group, which could be stained with oil red O and then stained red, indicating that the cells could be induced to differentiate into adipocytes. In the osteogenic experiment, the cells were stained with Alizarin Red. The induced group changed and light brown calcium nodules were formed, indicating that the MSCs can be induced to differentiate into osteoblasts. In the differentiation into chondrocyte experiment, the cells aggregated into clumps after induction, and were dyed blue-green by Alcian Blue after being sliced, indicating that our MSCs can be induced to differentiate into chondrocytes (Fig 1b).
Finally, we performed flow cytometry analysis on the third-generation MSCs (Fig 1c). CD45 and CD11b antigen/cell marker are negatively expressed; while CD29, CD44, CD90 and other antibodies are positively expressed. This result is in line with the International Cell Association’s Identification criteria for the phenotype of adipose-derived MSCs.
2 Bleomycin promotes lung fibrosis in mice
We used the murine bleomycin–induced experimental model of pulmonary fibrosis to assess the therapeutic capacity of MSCs[22]. We used bleomycin to model C57BL/6 mice by nasal inhalation, with a control group receiving the same amount of PBS solution. Three weeks later, the success of the model was evaluated by gross observation of animal lung tissue, pathological section analysis of lung tissue, and determination of cytokine content. Observing the lung tissues of the two groups in general, the surfaces of the organs in the drug group are smooth, with no lesion or necrosis, while the surface of the tissue and the deep tissue of the control group have a large amount of white spot-like material (Fig 2b).
After the lungs were collected, hematoxylin-eosin (HE) staining was performed (Fig 2c). The alveolar structure of the control group was normal without obvious inflammation or fibrosis. In the drug group, the alveolar structure showed obvious deformation and hyperplasia, the alveolar interval was significantly widened, the alveolar wall of the lung tissue was significantly thickened, the alveolar cavity was edematous and significantly reduced with substantial inflammatory cell infiltration. Masson staining of the lung tissues of the two groups showed that the alveolar structure of the control group was normal without obvious inflammation or fibrosis. In the drug group, the alveolar structure showed severe hyperplasia, with prominent blue collagen fiber deposition. Bleomycin-induced pulmonary fibrosis has a significant increase in collagen deposition (Fig 2d). Accordingly, compared with the control group, animals that received bleomycin presented with a greater Ashcroft score (Fig 2e).
Finally, we extracted genomic RNA from lung tissue to identify the level of related factors. The results are shown in Figure 2f, transforming growth factor β (TGF-β) and hydroxyproline (HYP; which can indicate the degree of pulmonary fibrosis), Tumor Necrosis Factor α (TNF-α), Interleukin 6 (IL-6) gene expression levels in the drug group were significantly higher than those in the control group. This shows that bleomycin can form a stable pathological state of pulmonary fibrosis (Fig 2f).
3 MSC therapy reverses bleomycin-induced pulmonary fibrosis
MSCs were injected into the pulmonary fibrosis animals through the tail vein for treatment. Firstly, the cell fluorescent dyes DAPI and PKH26 were used to stain MSCs to trace the cell's residence in the body. Seven days later, the peripheral blood of the mice was taken for study under a fluorescence microscope. Fluorescently labeled active mesenchymal cells were present in the blood of mice, indicating that MSCs can run with the blood to the whole body (Fig3b).
After 7 and 14 days of injection of MSCs, lung tissues were taken for frozen sections and studied under a fluorescence microscope. MSCs were still present in the lung samples (Fig 3c). This indicates that MSCs can migrate to lung tissue after injection, and therefore are poised to play a therapeutic effect.
Finally, after cell therapy in pulmonary fibrosis mice, lung tissues of different treatment times were selected for Masson staining. The results showed that the collagen content in lung tissues was significantly reduced after cell treatment for one week, and the blue collagen tissues gradually decreased with time after treatment (Fig 3d). This showed that MSCs could significantly reduce the level of collagen deposition in lung tissue, reduce the degree of lung fibrosis, and ultimately repair the biological function of lung tissue. At the same time, animals treated with MSCs gradually showed a lower Ashcroft score (Fig 3e).
Next we studied the expression of lung fibrosis-related factors in both the control group and the therapy group. The gene expression of hydroxyproline (HYP), matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-2 (MMP-2) was significantly lower in the treatment group compared to the control group, showing that the presence of MSCs can reverse the pathological state of pulmonary fibrosis and reduce the level of fibrotic lesions (Fig 3f-h).
4 Construction of a cellular model of pulmonary fibrosis
Our studies have shown that MSCs can reverse pulmonary fibrosis in mice. At the same time, we studied the effect of MSCs on pulmonary fibrosis cells at the cellular level. First we obtained a large number of fibroblasts from the lung tissue of newborn C57 mice. (Fig 4b). Subsequently, TGF-β1 was used to construct a cellular model of pulmonary fibrosis. After the lung fibroblasts were treated with TGF-β1 (10 mg·L), cell morphology continued to maintain a long spindle-shaped state without aging (Fig 4c).
We then identified whether a pulmonary fibrosis cell model was formed by studying the level of related factors. The results showed that the level of related genes in the TGF-β1 group was significantly increased compared to the control group (Fig 4d), including transforming growth factor β1 (TGF-β1), α-smooth muscle actin (α-SMA) and type I collagen. However no significant difference was found between the groups in the content of E-cadherin. This outcome shows that the treatment of lung fibroblasts with TGF-β1 can cause intracellular collagen deposition and fibrotic changes.
5 MSC supernatant promotes the migration and proliferation of fibrotic lung cells in vitro.
After the successful modelling of lung fibroblasts, the supernatant of MSCs was then used to explore its effect on the proliferation and migration of the lung fibrosis cell model. The results of cell wound healing experiments show that the addition of MSC supernatant can significantly increase the migration level of lung fibrotic cells (Fig 5b). This indicates that the cytokines secreted by MSCs into the supernatant may have a positive effect on fibrotic lung cells.
A transwell experiment was used to explore the effect of MSC supernatant on the migration level of fibrotic lung cells (Fig 5c). After the intervention of MSC supernatant, the number of cells passing through the membrane increased significantly. This shows that MSCs have a certain tropism towards pulmonary fibrotic cells and can promote their migration.
Finally, the CCK-8 experiment was used to explore the effect of MSC supernatant on the proliferation of fibrotic lung cells. As the culture time increased, the addition of MSC supernatant significantly increased the OD value (Fig 5d). Furthermore, MSC supernatant increased cell proliferation in a dose dependent manner (Fig 5d).
6 MSCs promote the migration and proliferation of lung fibrotic cells in vitro
We also directly co-cultured MSCs and lung fibrotic cells to explore the effect of MSCs on the biological function of lung fibrotic cells (Fig 6a). The results of cell wound healing experiments showed that, compared with the control group, a co-culture of MSCs and lung fibrotic cells can speed up wound healing (Fig 6b-c). This may be attributable to the fact that MSC can accelerate the migration rate of lung fibrotic cells.
The results of the Transwell experiment are shown in Figure 6 c&d. When the lower chamber contains MSCs, the number of lung fibrotic cells passing through the chamber increases significantly (Fig 6d-e), showing that MSCs can promote the migration level of lung fibrotic cells.
7 MSC supernatant reduces expression of fibrosis-related genes and protein levels of pulmonary fibrosis cells.
Finally, we studied the expression levels of pulmonary fibrosis cell-related fibrosis genes and proteins after application of the MSC supernatant.
Firstly, we used qRT-PCR to detect the gene levels of pulmonary fibrosis-related factors. The results showed that the gene levels of TGF-β1, α-SMA and type I collagen in the MSC supernatant treated group were significantly lower than those in the control group. This shows that MSC supernatant can change the level of collagen deposition in pulmonary fibrotic cells. This may be related to the active substances secreted by MSCs that improved the fibrosis of the cells (Fig 7b).
Then we detected the protein levels of pulmonary fibrosis-related factors by Western blot. We used high and low concentrations of MSC supernatant to treat lung fibroblasts, and found that the more MSC supernatant, the lower the protein expression levels of TGF-β1, α-SMA and type I collagen. This shows that cytokines and other active substances contained in the MSC supernatant can change the level of collagen deposition in lung fibroblasts and reduce the formation of collagen (Figure 7c-d).