Therapeutic strategies based on stem cells have been shown to have potential in improving the condition of severe lung diseases. In this study, the suppressive effects of conditioned medium (CM) of induced pluripotent stem cells (iPSCs) on pulmonary fibroblast differentiation were investigated in a series of
Pulmonary fibrosis (PF) is a specific form of chronic fibrosing interstitial pneumonia limited to the lungs, which is characterized by disordered lung function (
Recent evidence that embryonic stem cells (ESCs) or adult stem cells are capable of repairing and regenerating the injured or diseased tissues (
Thus, in the present study, we attempted to evaluate the protective effects of iPSC-conditioned medium (iPSC-CM) against PF through the TGF-β1-related pathway in a mouse model of bleomycin-induced PF. The effects of iPSC-CM on pulmonary morphology were determined by hematoxylin and eosin (H&E) staining and Masson's staining. The regulatory function of iPSC-CM on TGF-β1 and other molecules related to PF was evaluated using enzyme-linked immunosorbent assay (ELISA), reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis.
Antibodies against proliferating cell nuclear antigen [proliferating cell nuclear antigen (PCNA); cat. no. bs-2006R), p-Smad-2 (cat. no. bs-5618R), Smad-2 (cat. no. bs-0718R), p-Smad-3 (cat. no. bs-5459R) and Smad-3 (cat. no. bs-3484R) were all purchased from Bioss, Inc. (Woburn, MA, USA). Antibodies against α-smooth muscle actin (α-SMA; cat. no. BM0002) and collagen I (cat. no. BA0325), were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Antibody against β-actin (cat. no. sc-47778) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Male C57BL/6 mice (8–10 weeks old; weighing 24 to 45 g) were purchased from the Experimental Animal Center of China Medical University. All the animals were maintained at 20–25°C with a constant humidity of 55±5% with free access to food and water. All animal experiments were conducted in accordance with the Institutional Animal Ethics Committee and Animal Care Guidelines of Shengjing Hospital of China Medical University which governed the use of the animals. Previously preserved human lung fibroblasts (HFL1 cell line; cat. no. GNHu28; Cell Bank of Chinese Academy of Sciences, Shanghai, China) were cultured in dulbecco's modified Eagle's medium (DMEM/F-12) medium [10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotics mixture] in 95% air and 5% CO2 at 37°C.
Mouse 3-gene iPSCs were established via the transfection of Oct4/Sox2/Klf4 into C57BL/6 mice as previously described (
Osteogenic induction was performed by incubating the cells in medium consisting of DMEM/F12 (cat. no. SH30023.01B; HyClone, Logan, UT, USA) supplemented with 10% FBS (cat. no. SH30084.03; HyClone), 0.25 mmol/l ASA (cat. no. A8960-5G; Sigma-Aldrich, St. Louis, MO, USA), 10 mmol/l β-glycerolphosphate (cat. no. 201205053; Biosharp, St. Louis, MO, USA), and 10−7 mol/l dexamethasone (cat. no. D1756; Sigma-Aldrich).
Adipogenic induction was performed by incubating the cells in medium consisting of DMEM/F12 (cat. no. SH30023.01B; HyClone) supplemented with 10−7 mol/l dexamethasone (cat. no. D1756; Sigma-Aldrich), 100 nmol/l insulin (Fosun Pharma, Shanghai, China), 0.2 mmol/l indomethacin (cat. no. 17378-5G), and 5% FBS (cat. no. SH30084.03; HyClone).
For Alizarin Red staining, the cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% Alizarin Red (cat. no. A5533-25G; Sigma-Aldrich) for 40 min at room temperature. The results were detected under a microscope at ×400 magnification. For Oil Red O staining, cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.6% Oil Red O (cat. no. O0625-25G; Sigma-Aldrich) for 1 h at room temperature. The results were detected under a microscope at ×400 magnification.
The iPSCs were culture routinely for 24 h before being transferred to serum-free DMEM [containing 2 mM L-glutamine (cat. no. 59202C) and 1×10−4 M non-essential amino acid (cat. no. H7145) (both from Sigma-Aldrich), 1×10−4 M M2-mercapto ethanol (cat. no. 21985-023; Gibio)] and cultured for a further 48 h. The supernatants (supernatant 1) of the iPSC medium were collected and centrifuged for 10 min at 1,500 rpm to separate the supernatants (supernatant 2) from precipitation. For subsequent experiments, the supernatant was employed as iPSC-CM.
To evaluate the inhibitory effect of iPSC-CM on the proliferation of fibroblasts, HLF1 cells were treated with various combinations of iPSC-CM and TGF-β1 as follows: i) the control group, normal HFL1 cells; ii) the TGF-β1 group, HFL1 cells incubated with TGF-β1 (5 ng/ml) for 24 h to induce differentiation into myofibroblasts; iii) the 30% iPSC-CM group, HFL1 cells incubated with 30% iPSC-CM and TGF-β1 (5 ng/ml) for 24 h; iv) the 50% iPSC-CM group, HLF1 cells incubated with 50% iPSC-CM and TGF-β1 (5 ng/ml) for 24 h; v) the 100% iPSC-CM group, HLF1 cells incubated with 100% iPSC-CM and TGF-β1 (5 ng/ml) for 24 h. Each treatment was represented by at least 3 replicates. Moreover, the cytotoxicity of iPSC-CM was assessed and no impairment on the cell normal biological processes was detected (
Upon completion of the 24 h of incubation, MTT assay was performed to determine the viability of the HLF1 cells in the different groups. Briefly, 50
The RNA samples were reverse transcribed into cDNA using Super M-MLV reverse transcriptase (no. RP6502; Bioteke, Beijing, China), and the final reaction mixture of volume 20
Total proteins from the different groups were extracted using the total protein extraction kit according to the manufacturer's instructions (cat. no. WLA019; Wanleibio, Shenyang, China) and the concentration of each sample was determined using the BCA kit (cat. no. WLA004, Wanleibio). β-actin was used as internal reference protein. All the extracts were boiled in loading buffer for 5 min and 20
The HFL1 cells were treated as described above only with the incubation course changed to 48 h. The mRNA and protein expression levels of α-SMA and collagen I were determined using RT-qPCR and western blot analysis as described above.
The HFL1 cells were primarily incubated with various percentages of iPSC-CM as mentioned above for 24 h and were then treated with 5 ng/ml TGF-β1 for a further 30 min. The expression of Smad-2, p-Smad-2, Smad-3 and p-Smad-3 was detected using western blot analysis.
Male C57BL/6 mice were randomly divided into 3 groups (6 in each group) as follows: i) the control group: mice were intratracheally injected with normal saline under anesthesia via an intraperitoneal injection of 80 mg/kg pentobarbital sodium for 24 h; ii) the PF group: mice were intratracheally injected with 5 mg/kg bleomycin sulfate (Merck Millipore, darmstadt, Germany) under anesthesia for 24 h to induce PF before being intravenously injected with 200
Each lung tissue sample was fixed in 4% paraformaldehyde, dehydrated with a graded ethanol series, embedded in paraffin blocks, cut into 3-
To assess the effects of iPSC-CM on collagen accumulation due to PF, Masson's trichrome staining [Aniline blue (cat. no. 229661000), Ponceau (cat. no. p8330) and acid fuchsin (cat. no. 71019360); Sinopharm Group, Beijing, China)] was utilized to demonstrate the changes in the tissue samples which were associated with the formation of collagen according to the method of Flint and Lyons (
The content of TGF-β1 in BALF was determined by ELISA. The expression of TGF-β1 in different lung tissue samples was quantified by RT-qPCR. The production of TGF-β1, Smad-2, p-Smad-2, Smad-3 and p-Smad-3 was assessed by western blot analysis.
All the data are expressed as the means ± SD. ANOVA and post hoc multiple comparisons were conducted using the LSD method with a general liner model with a significance level of 0.05. All the statistical analyses and graph manipulation were conducted using S R language version 3.2.1 (R Foundation for Statistical Computing).
iPSCs were generated by MEFs using mouse 3-gene transfection method. Positive clones were selected and the expression patterns of the genes which were the signature of mouse ESCs were validated by RT-qPCR. The representative image and quantitative analysis of RT-qPCR are shown in
The growth of HFL1 cells was directly assessed by MTT assay and indirectly determined using PCNA western blot analysis. As illustrated in
PF is characterized by the activation of collagen, and myofibroblasts are characterized by the expression of α-SMA. Therefore, the levels of collagen I and α-SMA were both determined at the mRNA and protein level. As shown in
To determine whether the effects of iPSC-CM on PF are exerted through the TGF-β1-mediated signal transduction pathway, the production and activation of Smad-2 and Smad-3 in HFL1 cells were also detected. The overexpression of p-Smad-2 and p-Smad-3 in the HLF1 cells was observed following incubation with TGF-β1 (
The injection of bleomycin led to severe lung injury in mice, as illustrated in the representative image of H&E staining, in which the injured tissues were characterized by neutrophilic alveolitis and patched areas (
The production and distribution of collagen I in different lung samples was determined using ELISA and Masson's staining, respectively. Marked differences in collagen I production were observed between the PF group and PF + iPSC-CM group (
The promoting effect of TGF-β1 on myofibroblast differentiation was evaluated in an
Traditional interventions of PF rely on the specific interference of TGF-β1, a cytokine mediating the differentiation of fibroblasts into myofibroblasts, and the accumulation of collagens in PF. Whereas these schemes have had some achievements, they also lead to certain unexpected side-effects in that TGF-β1 is a key factor involved in multiple biological processes. The arbitrary inhibition of TGF-β1 will certainly result in abnormalities, such as tumors. Fortunately, previous studies have highlighted the therapeutic potential of stem cell-based therapies in improving the outcome of bleomycin-induced PF in animal models (
Compared with the traditional means of using TGF-β1-specific antibodies, stem-based therapies have the advantage of a high treatment efficicay and low tumorigenic risk (
As the intermediate between normal fibroblasts and smooth muscle cells, myofibrolasts have the capability of synthesizing interstitial collagens and the expression α-SMA at the same time. The
To further explore the mechanisms responsible for these treatment processes, we quantified the expression and activation of molecules involved in the TGF-β1-induced myofibroblast differentiation
In conclusion, our study revealed the potential of iPSC-CM as a promising therapy against PF. The administration of iPSC-CM inhibited the differentiation of fibroblasts into myofibroblasts by inhibiting the activation of the TGF-β1/Smad pathway. iPSCs induced from MEFs is a convenient method with which to obtain cells with low tumorigenic potential based on previous studies (
This study was supported by grants from the National Natural Science Foundation of China (no. 81400042) and the Science and Technology Project of Department of Education, Liaoning Province (no. L2013299).
Induced pluripotent stem cell-conditioned medium (iPSC-CM) had no impact on the viability of HFL1 cells. Control group, normal HFL1 cells. HFL1 + transforming growth factor-β1 (TGF-β1) group, HFL1 cells incubated with 5 ng/ml TGF-β1 for 24 h. HFL1 + 100% iPSC-CM, HLF1 cells incubated with 100% iPSC-CM for 24 h.
Induced pluripotent stem cell-conditioned medium (iPSC-CM) had no impact on the protein expression of proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA), collagen I, Smad-2, p-Smad-2, Smad-3 and p-Smad-3. Control group, normal HFL1 cells. HFL1 + transforming growth factor-β1 (TGF-β1) group, HFL1 cells incubated with 5 ng/ml TGF-β1 for 24 h. HFL1 + 100% iPSC-CM, HLF1 cells incubated with 100% iPSC-CM for 24 h.
Generation of induced pluripotent stem cells (iPSCs) using mouse 3-gene transfection. The bars represented the quantitative results of RT-qPCR of genes which were signature of mouse embryonic stem cells (ESCs). Images represent the expression patterns of the target genes compared with mouse embryonic fibroblasts (MEFs) and ESCs. *P<0.05, significantly different from MEFs.
Administration of induced pluripotent stem cell-conditioned medium (iPSC-CM) inhibits the transforming growth factor-β1 (TGF-β1) induced proliferation of HLF1 cells. (A) MTT assay for cell viability; quantitative results are shown. (B) Representative blots and quantitative results of western blot analysis of proliferating cell nuclear antigen (PCNA). aP<0.05, significantly different from the control group; bP<0.05, significantly different from the TGF-β1 group; cP<0.05, significantly different from the 30% iPSC-CM group.
Administration of induced pluripotent stem cell-conditioned medium (iPSC-CM) inhibits the transforming growth factor-β1 (TGF-β1)-induced differentiation of HFL1 cells into myofibroblasts. (A) Representative blots and quantitative results of western blot analysis of collagen I. (B) Quantitative results of RT-qPCR of collagen I. (C) Representative blots and quantitative results of western blot analysis of α-smooth muscle actin (α-SMA). (D) Quantitative results of RT-qPCR of α-SMA. aP<0.05, significantly different from the control group; bP<0.05, significantly different from the TGF-β1 group; cP<0.05, significantly different from the 30% iPSC-CM group; dP<0.05, significantly different from the 50% iPSC-CM group.
Induced pluripotent stem cell-conditioned medium (iPSC-CM) exerts an inhibitory effect on transforming growth factor-β1 (TGF-β1)-induced processes via blocking the Smad signal transduction pathway
Administration of induced pluripotent stem cell-conditioned medium (iPSC-CM) attenuates injury to lung tissues due to pulmonary fibrosis (PF) and reduces collagen accumulation. (A) Representative image of H&E staining (×200 magnification); the nuclei in myocardial tissue were stained blue and cytoplasma were stained red. Green arrow indicates patched areas and yellow arrow indicates neutrophilic alveolitis. (B) Statistical analysis results of ELISA of collagen I. (C) Representative image of Masson's staining, collagen I was stained blue. (D) Statistical analysis results of the detection of hydroxyproline. (E) Representative blots and quantitative results of western blot analysis of α-smooth muscle actin (α-SMA) in model mice. aP<0.05, significantly different from the control group; bP<0.05, significantly different from the PF group.
Administration of induced pluripotent stem cell-conditioned medium (iPSC-CM) reduces the expression of transforming growth factor-β1 (TGF-β1) in model mice. (A) Statistical analysis results of ELISA results of TGF-β1 production in BALF. (B) Representative blots and quantitative results of western blot analysis of TGF-β1 expression in lung tissue. (C) Quantitative results of RT-qPCR of TGF-β1 expression in lung tissue. aP<0.05, significantly different from the control group; bP<0.05, significantly different from the pulmonary fibrosis (PF) group.
Induced pluripotent stem cell-conditioned medium (iPSC-CM) exerts an inhibitory effect on transforming growth factor-β1 (TGF-β1)-induced processes via blocking the Smad signal transduction pathway