DISCUSSION

In this study, we found that neonatal SSEA-1⁺ pulmonary cells were stem/progenitor cells capable of self-renewal and differentiating into pneumocytes and tracheal epithelial cells. The SSEA-1⁺ PSCs highly expressed CCSP and inhibited LPS-induced TSLP and IL-4-induced eotaxin production in primary lung epithelial cells. Adoptive transfer of SSEA-1⁺ PSCs reduced AHR and suppressed airway damage in acute OVA-induced asthmatic mice by preserving of the level of CCSP; decreasing the infiltration of eosinophils; inhibiting the production of IL-4, IL-5, IL-13, eotaxin, and TSLP; and increasing Foxp3⁺ Treg cells. We have demonstrated that the SSEA-1⁺ PSCs play an immunomodulatory role in the progression of asthma by inhibiting allergen-induced acute inflammatory responses and lung damage.

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 disease⁷². We found that neonatal SSEA-1⁺ PSCs were located in the bronchioles, terminal bronchioles, and BADJ in neonatal lung tissues. Moreover, neonatal SSEA-1⁺ PSCs were able to self-renew, proliferate, and differentiate into pneumocytes, 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-1⁸⁸. However, 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 exploration. Moreover, SSEA-1⁺ PSCs were found in the bronchioles, terminal bronchioles, and BADJ in neonatal lung tissues, but were located in the deep parenchyma after transplantation into asthmatic mice. In this model,

suggested that SSEA-1⁺ PSCs should be localized 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, respectively⁹⁹, mouse Oct-4-expressing stem/progenitor cells comprise 0.0016-0.0022% of the neonatal lung cells⁹³, and a population termed bronchioalveolar stem cells represents 0.4% of the total lung cell preparations from adult mice⁸⁸. 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 were used in our study. The reasons underlying the discrepancy of the size of stem/progenitor cell population between laboratories may be differences in the species and age of the mice, the surface markers used, and the methodologies employed for sampling and analysis. In this study, the SSEA-1⁺ PSCs in the neonatal airways were found to represent approximately 2.5% of the pulmonary cell suspensions. These data indicated that the enzyme-based digestion method produced a high yield of stem/progenitor cells.

Phenotypic characterization of the SSEA-1⁺ PSCs is pivotal to clarify their biological functions in vivo. We found that CD9, CD24, CD26, CD29, CD47, CD54, CD98, CD147, and CD324 are positive, but CD24 and CD54, and CD324 are distributed heterogeneously within the SSEA-1⁺ cells. In our preliminary results, we found that enriched SSEA-1⁺ CD133⁺ cells simultaneously expressed CD24 and CD35, suggested that these SSEA-1⁺ cells comprise two distinct cell populations that can be distinguished

CD133⁺ and CD133^- populations and might with different lineage stem/progenitors or a different maturation status. Therefore, further studies are needed to explore whether distinct sub-groups of cells within the SSEA-1⁺ PSCs are critical for the anti-inflammatory effects.

MSCs have been shown to improve inflammation in a variety of disease models including protection airway from allergen-induced pathology^105-107. Although MSCs could likely modulate an inflammatory microenvironment, various studies also indicated that MSCs promote fibrogenesis by directly differentiating to myofibroblasts and contribute to disease progression^{108, 109}. Most basic biological studies indicate that MSCs function as bone marrow stromal cells, modulating the marrow microenvironment and serving as precursors to differentiated skeletal lineages.

Therefore, injection of MSCs into lung tissue is more likely an artificial therapy rather than an augmentation of naturally occurring mechanisms for lung repair⁷². In this study, we identified that SSEA-1⁺ PSCs existed in lung and specifically differentiated into lung epithelial lineage; hence, it might have advantages over MSCs.

IL-5, eotaxin, and TSLP, secreted by airway epithelial cells, are critical for maintaining asthmatic inflammation and stimulating TH2 polarization¹¹⁰. TSLPR-deficient mice fail to develop an inflammatory lung response to inhaled antigen indicted that TSLP plays a critical role in the development of inflammatory and/or allergic responses in the asthma model⁵³. Lung-specific overexpression of TSLP induces hyperreactivity and TH2-related inflammation⁵⁰. TSLP induces the proliferation of bronchial epithelial cells and bronchial repair through regulating the production of IL-13⁴⁸. We found that the

SSEA-1⁺ PSCs inhibited TSLP and eotaxin production in activated airway epithelial cells in vitro. These results suggested that SSEA-1⁺ PSCs might decrease inflammation-induced epithelial damage and stabilize epithelium through interaction with lung structural cells to inhibit TSLP and eotaxin production, and suppress the infiltration of inflammatory cells. Moreover, SSEA-1⁺ PSCs inhibited the production of eotaxin and IL-5 in the airway epithelium and reduced the infiltration and activation of eosinophils.

These data might explain the suppression of AHR in the SSEA-1⁺ PSCs recipient group.

Both naturally occurring Treg (nTreg) and inducible Treg (iTreg) cells are thought to effectively attenuate airway inflammation and improve airway function^{111, 112}. Foxp3⁺ Treg population in thoracic LN was increased in SSEA-1⁺ PSCs-treated asthmatic mice which raises the possibility that engrafted SSEA-1⁺ PSCs might increase the recruitment of Treg into inflamed site or promote the generation of iTreg in local tissue. Transgenic mice overexpressing IL-4 in the airway have decreased expression of CCSP^{42, 113}, suggested that IL-4 regulate CCSP expression. SSEA-1⁺ PSCs inhibited IL-4 and increased airway CCSP production in our asthmatic mice, the possible molecular mechanism is that engrafted SSEA-1⁺ PSCs ameliorate allergen-induced airway inflammation might associated with increase of Treg population or regulate original CCSP-positive epithelial cell through paracrine manner in the inflamed lung tissue, and subsequently inhibit TH2 cytokines and preserve airway CCSP expression and sustain its anti-inflammation effects.

Previous study indicated that CCSP suppressed allergen-induced inflammatory responses in vivo⁸⁷. We found that anti-CCSP antibody restored SSEA-1⁺ PSCs-induced

for eotaxin inhibition. Therefore, SSEA-1⁺ PSCs might inhibit the airway epithelial cells to produce inflammatory cytokines/chemokines through at least two different mechanisms. In this study, we reported that SSEA-1 was used as a marker for pulmonary stem cells and benefit for airway inflammation inhibition in asthmatic mice.

Therefore, we suggested that lung SSEA-1⁺ cells might used to be a potentially useful airway inflammatory biomarker in diagnosis and management of asthma in the future.