O R I G I N A L A R T I C L E E X P E R I M E N T A L A L L E R G Y A N D I M M U N O L O G Y
Lung-derived SSEA-1
+stem/progenitor cells inhibit allergic airway inflammation in mice
C.-J. Chiu1, T.-Y. Ling2& B.-L. Chiang1,3
1Graduate Institute of Immunology, College of Medicine, National Taiwan University;2Department of Pharmacology, College of Medicine, National Taiwan University;3Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
To cite this article: Chiu C-J, Ling T-Y, Chiang B-L. Lung-derived SSEA-1+stem/progenitor cells inhibit allergic airway inflammation in mice. Allergy 2015;
DOI: 10.1111/all.12567.
Background: Asthma is characterized by chronic airway inflammation and airway hyperresponsiveness (AHR). Little is known about the role of pulmonary stem/
progenitor cells (PSCs) in allergic airway inflammation.
Methods: To identify and investigate the role of PSCs in the bronchial epithelium of neonatal mice, we developed an enzyme-based digestion method to obtain sin-gle-cell suspension from lung tissues. Characterization of PSCs was performed using flow cytometry, real-time PCR, immunofluorescence staining, confocal microscopy, and scanning electron microscopy. The effects of SSEA-1+ (stage-specific embryonic antigen-1) PSCs was studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of cell-based regulation using flow cytometry, real-time PCR, and immune-blotting.
Results: Single-cell suspensions derived from neonatal lung tissue included popu-lations that expressed either SSEA-1+ or Sca-1+ (stem cell antigen-1). The SSEA-1+ PSCs were highly prevalent in neonatal mice, and they were rare in adult mice. Enriched neonatal SSEA-1+PSCs had the ability of self-renewal and differentiated into pneumocytes and tracheal epithelial cells. SSEA-1+ PSCs reduced AHR and airway damage in asthmatic mice by decreasing eosinophil infiltration, inhibiting chemokines/cytokines production, and preserving the level of CCSP.
Conclusions: Here, we demonstrated that neonatal SSEA-1+PSCs play an immu-nomodulatory role in the progression of asthma by reducing lung damage and inhibiting inflammatory responses. Further understanding the molecular mecha-nisms of neonatal SSEA-1+PSCs might shed light on exploring the novel thera-peutic approaches for allergic airway inflammation.
Asthma is a heterogeneous inflammatory disorder that is characterized by chronic airway inflammation, airway hyper-responsiveness (AHR), and excessive airway mucous produc-tion. After exposure to allergens, various inflammatory cells infiltrate the airway (1). Eosinophils have been found to be the predominant effector cells for tissue damage and pulmo-nary dysfunction. The intensity of the pulmopulmo-nary recruitment of eosinophils is strongly correlated with the severity of AHR (2).
In addition to function as a physical barrier, the airway epithelium plays a critical role in the process of T-helper type
which attracts and activates the dendritic cells (DCs) to prime naive CD4+T cells to differentiate into TH2 cells (3, 4). Eotaxin secreted by airway epithelial cells is an eosino-phil-specific chemokine that plays a critical role in eosinophil recruitment in asthma (5, 6). Clara cell secretory protein (CCSP), which is produced by nonciliated bronchiolar Club cells (also known Clara cells), is critical in inhibiting the dif-ferentiation of naive T cells into TH2 cells via the DCs (7).
In addition, CCSP not only inhibits the infiltration of inflam-matory cells but also directly reduces allergic inflammation in the lung (8). A recent study showed that the LPS and house Allergy
development of TH2 immune response and allergic inflamma-tion (9). These observainflamma-tions suggest that lung structural cells not only function as a barrier but also modulate the allergic immune response.
Asthma in most patients is only symptomatically con-trolled by the available medications (10). Therefore, the pos-sibility that stem/progenitor cells could modulate the immune system has led to an increasing interest in using stem/progen-itor cells as a potential therapeutic modality for severe refrac-tory asthma (11). Airway stem cells were first identified in a lung injury model used to demonstrate that bronchiolar stem cells are pollutant-resistant, CCSP-expressing stem cells that contribute to the repair of injured airways (12). Ling et al.
reported a serum-free culture system that can support the in vitrocolony growth of primary neonatal pulmonary epithelial cells expressing octamer-binding transcription factor-4 (Oct-4). These cells express not only Oct-4 but also other markers, such as stage-specific embryonic antigen-1 (SSEA-1) and stem cell antigen-1 (Sca-1) (13). SSEA-1 and Sca-1 are cell markers for induced pluripotent stem cell and hematopoietic stem cell, respectively (14, 15). Previous study indicated that the pro-genitor epithelial colony cells from the lungs expressed stem cell markers such as Oct-4 and SSEA-1. The colony cells had extensive self-renewal and differentiation potential (16).
These studies raised the possibility that pulmonary stem/pro-genitor cells (PSCs) could regulate immune responses and imply their therapeutic potential for treating asthma.
The aim of this study was to identify the PSCs in the bron-chial epithelium of neonatal mice and to investigate their stem cell capacity in vitro. We also explored whether neonatal SSEA-1+ PSCs could exert a therapeutic benefits in the progression of allergic airway inflammation.
Results
Identification of potential stem/progenitor cells from neonatal mouse lungs
The development of the lungs occurs as a continuous process from embryogenesis to early adolescence in humans as well as mice (17). A stem cell pool present in the neonatal lung contributes to both the bronchiolar and alveolar lineages during lung development (18), whereas these stem cell popu-lations are a rare and quiescent population in the adult lung (19, 20). Previous study showed that Oct-4-, SSEA-1-, Sca-1-, or CCSP-expressing pulmonary stem/progenitor cells undergo terminal differentiation to alveolar pneumocytes (13). There-fore, we speculated that mouse PSCs might reside among the Sca-1+, SSEA-1+, Oct-4+, and CCSP+ cells. To test this hypothesis, single-cell suspensions from lung tissues of neona-tal and adult mice were prepared. CCSP and Oct-4 are expressed in cytosol and nucleus, respectively. Therefore, we used fluorescence-activated cell sorting (FACS) to analyze the expression pattern of cell surface Sca-1 and SSEA-1. Sin-gle cells were identified by forward scatter, and immune cells (CD45+) were excluded. FACS analysis showed that Sca-1+
-evaluate the putative stem/progenitor cell population, we analyzed the total cell number of SSEA-1+and Sca-1+cells derived from the lungs of mice of different ages. We found that the number of SSEA-1+cells significantly decreased in an age-dependent manner (Fig. 1B,C). The numbers of SSEA-1+ cells at postnatal day 1 and day 7 were 1.7 0.4 9 105 and 6.0 0.8 9 105, respectively. In con-trast, the Sca-1+cell population significantly increased with age. Compare with neonatal lung section, adult SSEA-1+ cells were difficult to detect in whole-mount view (Fig. S1).
The frequency of lung SSEA-1+cells presented in the adult mice was far fewer than the frequency seen in neonatal mice by whole-mount staining. These data were consistent with the results of Fig. 1. The enlarged immunofluorescence image showed that adult lung SSEA-1+cells were localized in the bronchoalveolar duct junction (BADJ), which was almost the same as neonatal mice (Fig. S2).
Phenotypic characteristics of neonatal SSEA-1+pulmonary cells
To characterize this potential stem/progenitor cell popula-tion, we performed an unbiased FACS-based screen of the SSEA-1+pulmonary cells using a collection of monoclonal antibodies directed against cell surface markers. Fluores-cence-activated cell sorting analysis showed that the neonatal SSEA-1+pulmonary cells expressed epithelial lineages mar-ker E-cadherin (CD324), while negative for CD31 (endothe-lial marker), CD34 (hematopoietic stem cell marker), CD90.2, CD73, and CD105 (mesenchymal stem cell; MSC markers) by FACS analysis. In addition, neonatal SSEA-1+ pulmonary cells expressed CD9, CD24, CD26, CD29, CD47, CD54, CD98, and CD147 (Fig. 2A). Lung is a complex organ that requires the specification of various epithelial cell types for proper homeostasis. To verify the cell lineage of SSEA-1+cells, we checked the expression of p63 (a basal cell marker), T1a (a type I pneumocyte marker), surfactant pro-tein C (SPC, a type II pneumocyte marker), and CCSP (a Club cell marker) by FACS analysis, real-time quantitative PCR (RTQ-PCR), and immunoblotting. Interestingly, SSEA-1+ pulmonary cells were negative for p63 and T1a, but positive for SPC (Fig. 2B) and CCSP (Fig. 2C,D). Immuno-fluorescence staining of whole airway tissue mounts revealed that SSEA-1+cells resided in the bronchioles, terminal bron-chioles, and the BADJ in the lungs of neonatal mice (Figs S1 and S2). The bronchoalveolar stem cells (BASCs) were defined as CCSP/SPC dual-positive population at the BADJ as described previously by Kim et al. (21). Therefore, these results raised the possibility that SSEA-1+ cells might be multifunctional and comprise the regenerative cell popula-tions within the airway microenvironment.
Neonatal lung SSEA-1+cells possess self-renewal, clonogenicity, and multipotency ability
To test whether pulmonary SSEA-1+cells fulfill the criteria
Pulmonary stem/progenitor cell and asthma Chiu et al.
cells were resuspended in Matrigel-based three-dimensional culture. We found that primary sphere colonies were observed 10–15 days after cell culture (Fig. 3A left). To fur-ther clarify the self-renewal capacity of pulmonary SSEA-1+ cells, primary spheres were subsequently dissociated to single cell and then resuspended to Matrigel-based three-dimen-sional culture. The formation of secondary spheres was observed after culturing for 7–10 days (Fig. 3A right). Sphere colony assay showed that SSEA-1+cells exert higher sphere-forming ability than SSEA-1-cells. The sphere formation effi-ciency of SSEA-1+ cells was 1–2 spheres/2500–5000 total cells, as determined by limiting dilution assay (Fig. 3B).
These results indicated that SSEA-1+pulmonary cells might expand through self-renewal. Although SSEA-1+pulmonary cells only expressed SPC (type II pneumocyte marker) when initially isolated from neonatal mice (Fig. 2B), after culturing on Matrigel-coated plate for 15–20 days, the SSEA-1+ pul-monary cells differentiated into pro-surfactant protein C+
pulmonary cells have the capacity to differentiate into tra-cheal epithelial cell (TEC) because SSEA-1+ cells were located at the BADJ in the lungs of neonatal mice (Figs S1 and S2). Immunofluorescence staining of tight junction mar-ker ZO-1 and centrosome marmar-ker c-tubulin showed that SSEA-1+ pulmonary cells differentiated into both ciliated and nonciliated cells 15 days after grown in air–liquid inter-face (ALI) cultures (Fig. 3D). Scanning electron microscopy (SEM) also showed the similar results (Fig. 3E). These obser-vations suggested that neonatal SSEA-1+ pulmonary cells had the capability to differentiate into both pneumocytic and TEC lineages. Based on these in vitro studies, we suggested that neonatal SSEA-1+pulmonary cells are stem/progenitor cells with self-renewing, clonogenic, and multipotent proper-ties.
SSEA-1+PSCs reduced TSLP and eotaxin production
A C
B
Figure 1 Identification of neonatal pulmonary stem/progenitor cells. (A) Neonatal pulmonary cells were labeled with Sca-1 and SSEA-1 antibodies and analyzed using flow cytometry. The data are representative of five independent experiments. (B) Representative flow cytometry histograms of SSEA-1 and Sca-1-stained cell
popu-lation in the lung single-cell suspension. (C) Cell number of SSEA-1+- and Sca-1+-enriched pulmonary cell extracted from whole lung/
mouse (n= 3–5 mice per group). Data are means SD and repre-sentative of three independent experiments.**P < 0.01.
Chiu et al. Pulmonary stem/progenitor cell and asthma
protecting role in lung damage. However, whether PSCs play a critical role in the process of inflammation is not well understood. To explore the biological functions of neonatal SSEA-1+ PSCs, we developed an adult lung epithelial cells and neonatal SSEA-1+ PSCs coculture system in the pres-ence of stimulators. As TLR4 ligation on airway epithelial cells induces the release of innate cytokines including TSLP, which promote the development of pathogenic TH2 cells and asthmatic inflammation (9). In addition, IL-4 plays a critical role in the differentiation of TH2 cells and induces inflamma-tion through stimulating the expression of eotaxin from lung
and eotaxin, respectively. ELISA measurements of cell cul-ture supernatant indicated that the primary lung epithelial cells produced high levels of TSLP and eotaxin upon LPS and IL-4 stimulation, respectively. However, the neonatal SSEA-1+ PSCs inhibited LPS-induced TSLP and IL-4-induced eotaxin production (Fig. 4A). To clarify whether the neonatal SSEA-1+ PSC-mediated inhibitory effect was dependent on soluble or cell–cell contact-dependent factors, coculture of neonatal SSEA-1+PSCs and adult lung epithe-lial cells was physically separated by a Transwell insert, and it found that neonatal SSEA-1+ PSCs suppressed LPS- and A
C D
B
Figure 2 Characterization of neonatal SSEA-1+ pulmonary cells.
(A) Flow cytometric analysis of surface marker expression in neonatal SSEA-1+ pulmonary cells. The gray areas represent matched isotype controls. Data are representative of three inde-pendent experiments. (B) Fluorescence-activated cell sorting analysis of intracellular surfactant protein C (SPC) and surface T1a expression in SSEA-1+ pulmonary cells. SSEA-1+pulmonary cells expressed SPC (a type II pneumocyte marker) but were negative for T1a (a type I pneumocyte marker). Data are repre-sentative of two independent experiments. (C) Enriched SSEA-1+
pulmonary cells were purified from neonatal mice, and the Clara cell secretory protein (CCSP) expression levels were determined using RTQ-PCR with specific primers. The mRNA levels were normalized using the housekeeping gene GAPDH. Bars indicate mean SD of three independent experiments. **P < 0.01. (D) Lysates of enriched neonatal SSEA-1+pulmonary cells were pre-pared, and the CCSP protein levels were determined from immu-noblots stained with a specific antibody against CCSP. b-actin was used as the internal control. Data are representative of two independent experiments.
Pulmonary stem/progenitor cell and asthma Chiu et al.
effect was mainly dependent on soluble factors. To clarify the mechanism of inhibition of inflammation and airway damage by SSEA-1+ PSCs, we tested whether SSEA-1+ PSCs could inhibit TSLP and eotaxin production in the pres-ence of CCSP neutralization antibody. We found that anti-CCSP antibody restored SSEA-1+ PSC-induced TSLP but not eotaxin, suggesting that CCSP might not to be the pre-dominant pathway for eotaxin inhibition (Fig. S3).
Transplantation of SSEA-1+PSCs preserved the epithelium and alleviated the severity of asthmatic features
neonatal SSEA-1+ PSCs into asthmatic mice might have therapeutic potential. Both SSEA-1-positive and SSEA-1-neg-ative fractions isolated from neonatal mice were collected and used for further adoptive transfer studies. To provide a precise area of their niche that would permit the stem/pro-genitor cells to survive and to investigate the anti-inflamma-tory effects of this stem/progenitor cell population, SSEA-1+ PSCs were intravenously delivered into mice after the second ovalbumin (OVA) aerosol exposure (Fig. 5A). We used pul-monary cells isolated from enhanced GFP transgenic (EGFP-tg) mice to monitor the localization of the SSEA-1+PSCs in the recipient animals. Six days after transfer,
anti-GFP-A C
D
E B
Figure 3 Neonatal SSEA-1+pulmonary cells are self-renewal and multipotent. (A) Representative photographs of primary and sec-ondary sphere colonies. Bars, 100lm. (B) Efficiency of second-ary spheres formation. The results are expressed as means SD (n = 3). (C) SSEA-1+ pulmonary cells cultured on Matrigel-coated plate for 15–20 days were stained with pro-sur-factant protein C (proSPC; green; left) and AQP5 (green; right)
antibody to identify the type I pneumocyte; the nuclei were counterstained with DAPI (blue). Bars, 20lm. (D) SSEA-1+ pul-monary cells seeded in an ALI culture for 15 days were labeled for ZO-1 (proSPC; green), c-tubulin (red), and DAPI (blue). Bars, 20lm. (E) Scanning electron microscopic images of in vitro-cul-tured SSEA-1+ pulmonary cells at ALI for 15 days, showing cili-ated and noncilicili-ated cells. Bar, 10lm.
Chiu et al. Pulmonary stem/progenitor cell and asthma
the anti-OVA IgE titer (Fig. S4C). Notably, SSEA-1+PSCs significantly suppressed the invasive AHR to methacholine (Fig. 5B) and decreased the infiltration of inflammatory cells into peribronchovascular areas in the OVA-induced asth-matic mice (Fig. 5C and Fig. S5). The total cell counts and the proportion of eosinophils in BAL fluid were significantly decreased in the SSEA-1+PSC-treated group compared with untreated group (Fig. 6A,B). ELISA showed that neonatal SSEA-1+PSCs treatment significantly inhibited the secretion of eotaxin, TSLP, IL-4, IL-5, and IL-13 in the BAL fluid of OVA-induced asthmatic mice (Fig. 6C). The levels of CCSP in both the BAL fluid and blood serum were lower in patients with asthma compared with healthy controls (24, 25). Reduction of CCSP expression in bronchiolar epithelium is associated with epithelial cell damage (26). Immunofluores-cence staining showed that CCSP was weakly expressed in asthmatic lung tissue compared with healthy lung tissue (Fig. 6D). In the asthmatic model, CCSP was strongly expressed in the SSEA-1+PSCs recipients (Fig. 6D). There-fore, transplantation of neonatal SSEA-1+PSCs might
pre-Discussion
In this study, we found that mouse neonatal SSEA-1+ pul-monary cells were stem/progenitor cells capable of self-renewal and differentiating into pneumocytes and tracheal epithelial cells. Neonatal SSEA-1+ PSCs highly expressed CCSP and inhibited LPS-induced TSLP and IL-4-induced eotaxin production in primary lung epithelial cells. Trans-plantation of neonatal SSEA-1+ PSCs reduced AHR and suppressed airway damage in OVA-induced asthmatic mice, which might be associated by preserving of the level of CCSP; decreasing the infiltration of eosinophils; and inhibiting the production of IL-4, IL-5, IL-13, eotaxin, and TSLP. We have demonstrated that neonatal SSEA-1+ PSCs play an immunomodulatory role in the progression of asthma by inhibiting allergen-induced inflammatory responses.
It is suggested that lung tissue comprises multiple spatially and temporally restricted stem or progenitor cell lineages that have varying abilities to respond to injury and disease (27).
A B
Figure 4 SSEA-1+ pulmonary stem/progenitor cells (PSCs) sup-press thymic stromal lymphopoietin (TSLP) and eotaxin production.
(A) Lung epithelial cells derived from adult mice were cocultured with neonatal SSEA-1+PSCs in the presence of LPS or IL-4 for 24 h. TSLP and eotaxin contents in LPS- and IL-4-stimulated culture
supernatants were determined using ELISA, respectively. (B) Co-culture system of adult lung epithelial cells and neonatal SSEA-1+ pulmonary cells was separated with a Transwell. Data are means SD and representative of three independent experiments.
*P < 0.05, **P < 0.01.
Pulmonary stem/progenitor cell and asthma Chiu et al.
lung tissues. Moreover, neonatal SSEA-1+ PSCs were able to self-renew, proliferate, and differentiate into pneumocytes, and ciliated and nonciliated TECs. Based on these findings, it is probable that SSEA-1+ cells might serve as progenitor/
stem cells for all differentiated airway epithelial cells. In adult mice, BASCs were identified at the BADJ within the terminal bronchioles by expressing CCSP, SPC, and Sca-1(21). How-ever, neonatal SSEA-1+PSCs expressed CCSP and SPC, but not Sca-1; therefore, whether these two cell populations share similar functional therapeutic benefit remains further
explora-but were located in the deep parenchyma after transplanta-tion into asthmatic mice. In this model, we transplanted SSEA-1+ PSCs into asthmatic mice by intravenously injec-tion, which suggested that SSEA-1+ PSCs should be local-ized inside lung parenchyma. However, whether SSEA-1+ PSCs migrate and localize in the parenchyma and execute their anti-inflammation or repair ability is still unknown.
Recent studies demonstrated that c-kit+human lung stem cells constitute an average of 0.0042% and 0.0244% of the lung cells of the adult and fetus, respectively (22), mouse A
C
B
Figure 5 Transplantation of SSEA-1+ pulmonary stem/progenitor cells (PSCs) improves airway hyper-responsiveness in the asth-matic mice. (A) Flowchart of the method used to produce the oval-bumin-induced asthmatic murine model. (B) Airway function was measured by invasive body plethysmography. The results are expressed as means SEM of the lung resistance (RL) in the ratio
of RL after PBS nebulization. Data are representative of two inde-pendent experiments (n= 4–7 mice per group). **P < 0.01. (C) H&E staining of lung sections in untreated, SSEA-1+PSC-, SSEA-1 -pulmonary cells-treated, and healthy group. Bars, 200lm. Arrows denote infiltrated leukocytes; br, bronchus; v, vessel. Data are rep-resentative of two independent experiments.
Chiu et al. Pulmonary stem/progenitor cell and asthma
represent 0.4% of the total lung cell preparations from adult mice (21). Morphometric analysis of total lung sections showed that SSEA-1+ PSCs constituted an average of 0.138 0.036% of the lung cells in the neonatal mice that
represent 0.4% of the total lung cell preparations from adult mice (21). Morphometric analysis of total lung sections showed that SSEA-1+ PSCs constituted an average of 0.138 0.036% of the lung cells in the neonatal mice that