Predifferentiated amniotic fluid mesenchymal stem cells enhance lung alveolar epithelium regeneration and reverse elastase-induced pulmonary emphysema
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Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD). Emphysema progression attributed not only to alveolar structure loss and pulmonary regeneration impairment, but also to excessive inflammatory response, proteolytic and anti-proteolytic activity imbalance, lung epithelial cells apoptosis, and abnormal lung remodeling. To ameliorate lung damage with higher efficiency in lung tissue engineering and cell therapy, pre-differentiating graft cells into more restricted cell types before transplantation could enhance their ability to anatomically and functionally integrate into damaged lung. In this study, we aimed to evaluate the regenerative and repair ability of lung alveolar epithelium in emphysema model by using lung epithelial progenitors which pre-differentiated from amniotic fluid mesenchymal stem cells (AFMSCs).
Pre-differentiation of eGFP-expressing AFMSCs to lung epithelial progenitor-like cells (LEPLCs) was established under a modified small airway growth media (mSAGM) for 7-day induction. Pre-differentiated AFMSCs were intratracheally injected into porcine pancreatic elastase (PPE)-induced emphysema mice at day 14, and then inflammatory-, fibrotic-, and emphysema-related indices and pathological changes were assessed at 6 weeks after PPE administration.
An optimal LEPLCs pre-differentiation condition has been achieved, which resulted in a yield of approximately 20% lung epithelial progenitors-like cells from AFMSCs in a 7-day period. In PPE-induced emphysema mice, transplantation of LEPLCs significantly improved regeneration of lung tissues through integrating into the lung alveolar structure, relieved airway inflammation, increased expression of growth factors such as vascular endothelial growth factor (VEGF), and reduced matrix metalloproteinases and lung remodeling factors when compared with mice injected with AFMSCs. Histopathologic examination observed a significant amelioration in DNA damage in alveolar cells, detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), the mean linear intercept, and the collagen deposition in the LEPLC-transplanted groups.
Transplantation of predifferentiated AFMSCs through intratracheal injection showed better alveolar regeneration and reverse elastase-induced pulmonary emphysema in PPE-induced pulmonary emphysema mice.
KeywordsPredifferentiation Amniotic fluid mesenchymal stem cell Elastase-induced pulmonary emphysema
Amniotic fluid mesenchymal stem cells
Clara cell secretory protein
Chronic obstructive pulmonary disease
Intercellular adhesion molecule 1
Inducible nitric oxide synthase
Lung epithelial progenitor-like cells
Monocyte chemotactic protein-1
Modified small airway growth medium
Porcine pancreatic elastase
Small airway basal medium
Surfactant protein C
Transforming growth factor-beta 1
Thyroid transcription factor 1
Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling
Vascular endothelial growth factor
Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation and irreversible lung structure damage, leading to steadily increasing mortality rates . Emphysema is one of the major components of COPD, which is characterized by persistent and chronic inflammation, alveolar walls destruction, and permanent enlargement of air spaces. This leads to progressive disability and death in COPD patients worldwide .
Inhalation of noxious substances, such as cigarette smoke, is the major risk factor for developing emphysema. Years of noxious particle exposure causes the infiltration of inflammatory cells, especially neutrophils and macrophages, into the airways. This results in the activation of various proteolytic enzymes and proteinases, including neutrophil elastase and matrix metalloproteinase-9, which destroy the alveolar structure . Apoptosis of lung epithelial and endothelial cells is critical for the pathogenesis of emphysema caused by cigarette smoke exposure . Cigarette smoke exposure can progressively reduce antioxidant and autophagic defensive abilities in lung epithelial and endothelial cells, thereby driving cells towards apoptosis . Persistent oxidative stress could deplete the balance between self-renewal and cell differentiation in stem cells and progenitors, which results in impaired alveolar regeneration in lung tissue with emphysema .
Current treatments for COPD include reduction of dyspnea, prevention of exacerbation and disease progression, and improvement of quality of life to reduce mortality . After decades of efforts to identify therapeutic strategies to treat COPD, smoking cessation and anti-inflammatory pharmacologic agents, such as antagonists of cytokines such as tumor necrosis factor-α and interleukin-8, are the mainstays of treatment . However, under these therapeutic approaches, while COPD patients do improve their life quality and reduce both symptoms and acute exacerbations, there is no prevention of disease progression and therefore no reduction in mortality . Stem and progenitor cell therapies have been widely considered as the best potential candidates for the treatment of respiratory diseases and destructive disorders such as COPD/emphysema [9, 10]. Their capability of differentiating into various cell types , immunomodulatory effects , paracrine effects , and anti-apoptosis  can help to repair and regenerate lung tissue after injury.
Various stem cells and delivery routes have been used to treat experimental models of elastase-induced emphysema . Transplantation of embryonic stem cells (ESCs) that can be differentiated to type II alveolar epithelial cells have been shown to improve symptoms in the mouse and patient with emphysema [14, 15]. Other sources of MSCs harvested from various tissues (including bone marrow, adipose, umbilical cord blood, amniotic fluid and lung tissue) revealed some beneficial effects on the reversal of lung structure destruction, tissue regeneration, neutrophil infiltration, and collagen deposition [9, 16].
Although transplanted stem or progenitor cells contribute only marginally to lung regeneration, a variety of microenvironmental mediators in the damaged lung can affect precisely differentiation of engrafted cells. This may influence the therapeutic efficacy for the repair of the damaged lung. Thus, in other disease models, such as neurological disease and myocardial infarction, predifferentiating stem cells into specific cell types prior to transplant might be a better approach [17, 18]. Direct transplantation of type II alveolar epithelial cells, which are known progenitor cells for alveolar epithelium, also modulates the inflammatory response and alveolar edema fluid clearance. This is accomplished by secretion of soluble paracrine factors, and alveolar epithelial barrier restoration occurs via differentiation into type I alveolar epithelial cells in acute lung injury [19, 20] and fibrotic animal models [21, 22]. Similarly, established stable hESCs or induced pluripotent stem cells (iPSCs) that can be differentiated and enriched into a pure population of type II alveolar epithelial cells may be suitable for transplantation to treat lung injury [23, 24].
AFMSCs are particularly interesting because of their potential benefits when considering them for medical use. Amniotic fluid is a novel stem cell source that is regarded as medical waste. AFMSCs have the potential to differentiate into each of the embryonic germ layer cells, remarkable self-renewal ability, and a lack of ethical concern and are easy to isolate and abundantly available, and they possess privileged immunological characteristics that make them an ideal and reliable candidate for cell therapy . Several papers have reported that engrafted AFMSCs can differentiate into lung lineage cells and integrate into damage sites or induce local regeneration in lung injury animal models [16, 26, 27].
In this work, we aim to identify if predifferentiation of AFMSCs to LEPLCs before transplantation would result in a better outcome in terms of cell survival, anti-inflammatory response, tissue regeneration, and histopathological improvement in a lung emphysema animal model.
Isolation and characterization of murine AFMSCs
AFMSCs were derived from the amniotic fluid of pregnant eGFP-transgenic mice (days 11.5). First, the yolk sac was ruptured with a 27-gauge needle. AFMSCs were recovered and cultured in α-minimal essential medium (α-MEM) (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin and were incubated at 37 °C in a 5% CO2 incubator as described in Wen et al.  (Additional file 2: Figures S1A and S1B).
Flow cytometry analysis
Flow cytometry was used to examine the purity of isolated AFMSCs by detection CD44, stem cell antigen 1 (Sca-1), CD105 (eBioscience), CD34, CD90 (BD Biosciences), CD29, CD11b, CD73, CD106, and CD45 (R&D Systems) expression on the cell surface as described in Peng et al.  (Additional file 2: Figure S1E). To confirm the percentage of differentiated AFMSCs towards LEPLCs, we detect the presence of TTF-1, SP-C, AQP-5, and CCSP in cells according to the manufacturer’s instructions. Briefly, detached cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 for 15 min, blocked with 4% BSA in PBS for 1 h, and then incubated overnight at 4 °C with the following intracellular marker antibodies at appropriate dilutions: TTF-1 (Abcam, 1:250 dilution), SP-C, AQP-5, and CCSP (Millipore, all at 1:250 dilutions). The cells were then incubated with an appropriate Alexa Fluor® 546 dye-conjugated secondary antibody (1:200) at room temperature for 2 h. After rinsing the cells twice, fluorescence was detected and analyzed using flow cytometry (FACSCalibur).
In vitro differentiation
To induce the isolated AFMSCs towards differentiation into adipogenic and osteogenic cell lineages, cells were cultured to 100% confluency and then cultured in either an adipogenic medium (α-MEM containing 10% FBS, 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 10 μg/mL insulin, and 100 μM indomethacin), or an osteogenic medium (α-MEM containing 10% FBS, 1 μM dexamethasone, 10 mM glycerol 2-phosphate, and 50 μM ascorbic acid 2-phosphate). The medium was changed twice a week. After 3 weeks of induction, Oil Red O staining was performed to evaluate the adipogenesis efficiency by examining the intracellular accumulation of lipid droplets (Additional file 2: Figure S1C). Additionally, alizarin red staining was performed to evaluate the osteogenesis efficiency by detecting calcium mineralization (Additional file 2: Figure S1D).
To induce the isolated AFMSCs towards differentiation into lung epithelial progenitor-like cells, we use optimal media formulation described previously, with slight modifications . AFMSCs were plated on 0.1% gelatin-coated culture dishes for 24 h and cultured to confluency. Then, the culture medium was changed to, which comprises a small airway basal medium (SABM) supplemented with 0.5 mg/mL BSA, 5 mg/mL insulin, 10 mg/mL transferrin, 30 mg/mL bovine pituitary extract, 0.5 mg/mL epinephrine, 0.5 mg/mL hydrocortisone, 0.5 ng/mL human EGF, 1% penicillin-streptomycin, and 50 ng/mL FGF-10 . The medium was changed every 2 days. After 7 days of induction, flow cytometry analysis was performed to detect lung epithelial cell lineage markers.
Intratracheal transplantation of stem cells into PPE-induced lung emphysema murine model
Pulmonary emphysema was induced by intratracheal instillation, as previously described [12, 31]. Eight-week-old male ICR mice were purchased from the Lasco (Taipei, Taiwan). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Chang Gung University (Taoyuan, Taiwan; IACUC No. CGU15-156), and the experiments were performed in accordance with the guidelines. Mice were randomly picked to different groups, and there were at least 5 or more mice in each group. Each mouse was intratracheally administered 1.5 mg/kg porcine pancreatic elastase (PPE) dissolved in 50 μL sterile PBS on day 0. After 2-week period of PPE treatment, mice were randomly selected for intratracheal injection (1 × 105 cells in 50 μl PBS) of AFMSCs, LEPLCs, or PBS. After 4-week period of stem cells/PBS treatment, the mice were sacrificed by an overdose of 2.5% avertin and the therapeutic effects were examined.
Histology and immunofluorescence (IF)
Left lung tissues were fixed with 10% formalin overnight and paraffin embedded. Tissues were sectioned and stained with hematoxylin and eosin (H&E) according to standard protocols . The severity of lung emphysema was assessed by measuring the mean linear intercept (Lm) . The distance in airspace size between the alveolar walls was calculated by drawing equally distributed horizontal lines across each alveolus from wall to wall and recording the length for each measurement. Five random microscopic fields within each lung section were observed . For IF analysis, the paraffin-embedded sections were deparaffinized and then blocked with 4% fetal bovine serum in PBS with 0.2% Triton X-100 for 2 h at room temperature. Sections were then incubated overnight at 4 °C with primary antibody as follows: anti-pro-SPC (Millipore, 1:4000), anti-AQP-5 (Millipore,1:800), and anti-GFP (Millipore, 1:750). Sections were incubated with secondary antibody (Alexa Fluor® 488 conjugated goat anti-mouse IgG or Alexa Fluor® 647 conjugated goat anti-rabbit IgG) for 1 h at RT. After washing with PBS, the slides were stained with DAPI for nuclear counterstaining and mounted with FluoreGuard Mounting Medium (Biosystems).
Masson trichrome and Sirius red stain for lung fibrosis analysis
To determine collagen content, the lung tissue sections were subjected to Masson’s Trichrome staining (TRM-2, SCYTEK Laboratories) according to the manufacturer’s protocol. For Picrosirius red staining, slides were deparaffinized and rehydrated and then stained with hematoxylin for 1 min. After washing, the slide was immersed in Picrosirius red solution for 30 min. Then, the slides were washed in 4% Glacial acetic acid solution, dehydrated, and mounted. Stained slides were examined using bright field microscopy.
Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining (TUNEL) assay
To detect the apoptotic cells in the damaged lung, lung tissue sections were subjected to TUNEL staining using the In Situ Cell Death Detection Kit, Fluorescein (Roche) according to the manufacturer’s protocol. The stained slides were then stained with DAPI for nuclear counterstaining and mounted with FluoreGuard Mounting Medium.
RNA isolation and quantitative real-time RT-PCR
Total RNA was prepared from lung tissue using the EasyPrep Total RNA Kit (TOOLS). RNAs were reverse transcribed into cDNAs using an MMLV Reverse Transcription kit (Protech). Quantitative real-time RT-PCR was performed using LightCycler 480 SyberGreen I Master Mix and the LightCycler® 480 Instrument (Roche) as previously described . Sequences of the gene-specific primers used are listed in Additional file 1: Table S1. Relative gene expression was determined by the ∆∆Ct method, where Ct is the threshold cycle. The relative mRNA expression levels were normalized to the mRNA level of the reference Gapdh gene.
Western blot analysis
Western blot analysis to examine the indicated proteins was performed as described previously . Brief, 50 μg of total proteins from cell lysates was loaded onto each lane and the proteins were separated in sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE; Bio-Rad Laboratories). After electrophoresis, the resolved proteins were transferred to PVDF membrane (Millipore). The membranes were blocked with 5% skimmed milk powder (Anchor) in phosphate-buffered saline-Tween (PBS-T): phosphate-buffered saline (PBS, Sigma-Aldrich) containing 0.1% Tween-20 in (Sigma-Aldrich) for 2 h and probed overnight with the following antisera at appropriate dilutions: 1:500 dilution of the anti-proSPC and anti-AQP-5 (Millipore) and a 1:10,000 dilution of the anti-β-actin (Novus Biologicals) antisera in PBS-T. Identification of each protein was achieved with the Western Lightning ECL Plus (Millipore) using an appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories). Protein levels in the western blot analysis were detected and quantified by the Amersham Imager 600 imaging system (GE Healthcare Life Sciences). To adjust for loading differences, the optical density of each protein was normalized to that of the β-actin band.
Data are presented in bar graphs as the mean ± SD. Differences between groups were analyzed using one-way analysis of variance analysis (ANOVA), followed by the Dunnett’s post hoc test. When results were not normally distributed, a Kruskal-Wallis test followed by Dunn’s tests between groups was performed. All data were plotted and analyzed using GraphPad Prism. For all analyses, a p value < 0.05 was considered statistically significant.
Optimization of lung cell lineage differentiation in AFMSCs
Downregulated expression of emphysema factors in LEPLC-transplanted PPE-induced pulmonary emphysema mice
We next investigated the lung histopathologic sections from each experimental group at 4 weeks post stem cell transplantation (Fig. 2a). H&E staining showed that alveolar space increased in the lungs injected with PPE but was significantly attenuated by transplantation of AFMSCs and LEPLCs (Fig. 2a). Quantification of alveolar destruction was measured using the mean linear intercept length (Lm). The Lm was markedly increased in the PPE group when compared to the PBS control group, and the Lm was decreased in the AFMSC-transplanted mice and was further decreased in the LEPLC-transplanted mice (Fig. 2b).
Downregulated expression of inflammatory and fibrotic factors in LEPLC-transplanted PPE-induced pulmonary emphysema mice
Potential roles of transplanted LEPLCs in PPE-induced pulmonary emphysema mice
Taken together, our data indicated that transplanted LEPLCs engrafted, attenuated inflammatory and fibrotic effects, increased alveolar regeneration, and reduced structural alveolar cell apoptosis in PPE-induced pulmonary emphysema mice.
This is the first report using predifferentiated AFMSCs to treat mouse lung emphysema. Because of the low efficiency of accurate differentiation at lung damaged site and the heterogeneity of undifferentiated MSCs, these are unsuitable for clinical application. Thus, the predifferentiated strategies not only could enrich the lung progenitors to accelerate and reconstruct the damaged area, but also be great help in developing minimally self-originating regenerative therapeutic.
The use of PPE to induce experimental emphysema, where lung injury develops rapidly, easily, inexpensively, and severely than cigarette smoke . In the present study, we used a model of a single intratracheal instillation of PPE, which causes histological and morphological characteristics, lung inflammation, and collagen deposition closely resembling human emphysema . MSCs were administered 2 weeks after the PPE administrate, when alveolar structure was destructed, and mild emphysema were already established .
Following the promising results of MSC-based cell therapy in an experimental model of pulmonary emphysema in 2006 , a variety of sources of MSCs (bone marrow, adipose tissue, lung, amniotic fluid) have been transplanted into proteolytic enzyme- or cigarette smoking-induced COPD-like animal models. These studies have shown a therapeutic potential for transplanted MSCs on improving lung architecture, lung tissue remodeling, modulating inflammatory response, and decreasing apoptosis and collagen accumulation in pulmonary emphysema mouse model [43, 44, 45]. In this study, we used amniotic fluid mesenchymal stem cells, which possess self-renewal capacity, clonal properties , and multilineage differentiation ability (Additional file 2: Figure S1), and lack teratoma formation potential and ethical concerns . In comparison with that of Li et al. in which AFMSC-based therapy can alleviate the lung emphysema , our results further observed that transplanted AFMSCs had yielded beneficial effects on reducing inflammation and fibrosis, enhancing lung alveolar epithelium regeneration and reversing emphysema.
The low efficiency of accurate and precise differentiation of engrafted stem cells in the damaged lung reinforces the notion that progenitor cell populations are considered as better therapeutic agents for accelerate healing and reconstruct the damaged area . In addition, CCSP-positive Clara cells are thought as a progenitor cell population for the distal conducting airways and could theoretically contribute to alveolar epithelial repair in naphthalene-injured rodent model . Other evidence observed that β4 integrin-expressing AEC-progenitor or p63/keratin 5-expressing basal epithelial-progenitor populations may play a role in alveolar epithelial regeneration in fibrotic lung . However, a lung progenitor cell showed benefit on directly differentiating to specific cellular lineage, but studies mentioned that progenitor cell cannot be maintained in a proliferative state over multiple passage. Therefore, generating a pure and abundant population of lung progenitor cells is a key problem . Taken together, transplantation of undifferentiated cells and progenitors had both therapeutic benefit and limitation; we therefore see value in using predifferentiated MSCs into alveolar epithelial lineage prior to transplant in the emphysema model.
Based on this idea, Wang et al. established a stable genetic modified hESCs that can be differentiated and enriched into a pure population of type II alveolar epithelial cells may be suitable for transplantation to treat lung injury . Other reports have established predifferentiating protocol for ESCs  or iPSCs [24, 52] into alveolar epithelial cells, and transplantation of these predifferentiated cells exerts a better therapeutic approach in lung disease models.
Mounting evidence has shown that transplanted MSCs exert their beneficial effects in part via paracrine factors and/or transdifferentiation to epithelial cells that contribute to restoring the destructed lung architecture [26, 44]. In addition, evidence showed that MSC-based tissue repair partly relies on secreting paracrine factors to establish a regenerative microenvironment and repair the damaged sites . The potential mechanism of transplanted AFMSCs may act by modulating inflammation, apoptosis, fibrosis, and cell proliferation via the paracrine effectors [54, 55]. We further evaluated the therapeutic potential of these predifferentiated AFMSCs in a PPE-induced lung emphysema mouse model. Reports showed that both type I and II alveolar epithelial cells are constantly contributing to airway defense. Specifically, type II alveolar epithelial cells may secrete a variety of mediators to modulate the inflammatory response in lung injury [56, 57]. In this study, we observed downregulation of the expression of inflammatory factors, pro-IL-β, IL-6, INOS, and MCP-1 upon transplantation of either AFMSCs or LEPLCs. Both transplantation groups showed improvement in terms in the presence of fibrosis, and both groups had increased SPC+ (type II alveolar epithelial cell) and AQP-5+ (type I alveolar epithelial cell) cell regeneration.
Previous studies have shown that transplanted stem cells via intratracheal injection may distribute around the bronchi [32, 35] and may be found in alveolar capillaries or larger blood vessels after an intravenous injection . Our results also observed that transplanted AFMSCs or LEPLCs were engrafted surrounding the bronchi. Thus, we propose that transplantation of either AFMSCs or LEPLCs may restore damaged type II alveolar epithelial cell around the bronchi and repair the alveoli structure collapse which heavily relies on paracrine effects to establish a regenerative microenvironment eventually repair the damaged sites.
Cumulative observations found that coexistence emphysema (ECM breakdown) and fibrosis (ECM overaccumulation) should not be that unusual , which is in line with the increased and accumulated collagen as observed in airways of our PPE-induced emphysema mouse model. One possible mechanism is that elastase degradation relative abundance of collagen and elastin leads to alveolar destruction. At the same time, elastase may promote myofibroblast differentiation at the regions of alveolar wall destructive tissue to restore collagen content, eventually causing fibrosis . In the remodeling process of emphysema, TGF-β plays a key role in stimulating myofibroblast proliferation and secretion of collagen fibers . Either AFMSCs or LEPLCs reduced collagen accumulation in airways and TGF-β1 expression level; hence, LEPLCs were able to completely restore collagen level in the lung parenchyma.
This study uses an optimal differentiation condition of mouse amniotic fluid mesenchymal stem cells towards LEPLCs in vitro under modified small airway growth medium (mSAGM). Transplantation of pre-differentiated LEPLCs through intratracheal injection adapted to the microenvironment, attenuated inflammatory and fibrotic effects, recovered alveolar regeneration, and reduced structural alveolar cells apoptosis in PPE-induced pulmonary emphysema mice.
We thank the Molecular Medicine Research Center at Chang Gung University for help with tissue imaging.
CMC, KYC, and HLC designed the experiments. YWL, CCY, and JCY performed the experiments. TTH, YCC, HLC, KYC, and CMC performed the data analysis. YWL, JCY, and CMC prepared the manuscript and figures. All authors read and approved the final manuscript.
This research was supported by grants MOST-104-2313-B-005-043-MY3, MOST-104-2320-B-182-020-MY3, MOST-105-2314-B-182-042-MY3, MOST-105-2811-B-182-036, MOST-106-2811-B-182-020, and MOST-107-2811-B-182-513 from the Ministry of Science and Technology of Taiwan and CMRPD-1H-0031, CMRPD-1E-0233, and CMRPD-1G-0592 from Chang Gung Memorial Hospital, and partially supported by the iEGG and Animal Biotechnology Center from Feature Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE-107-S-0023-A) in Taiwan.
Ethics approval and consent to participate
Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Chang Gung University (Taoyuan, Taiwan; Document CGU15-156), and the experiments were performed in accordance with the guidelines.
Consent for publication
The authors declare that they have no competing interests.
- 10.Black AC Jr, Williams S, Young HE. From bench top to bedside: formation of pulmonary alveolar epithelial cells by maintenance cells and healing cells. J Stem Cell Res. 2017;1:1–16.Google Scholar
- 17.Abeysinghe HC, Bokhari L, Quigley A, et al. Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke. Stem Cell Res Ther. 2015;6:186–204.CrossRefGoogle Scholar
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