Stem Cells Dev. 2014 Nov 15;23(22):2744-57.

Behavioral heterogeneity of adult mouse lung epithelial progenitor cells.

Chernaya O, Shinin V, Liu Y, Minshall RD.

Department of Anesthesiology, University of Illinois at Chicago , Chicago, Illinois.



The existence and identity of multi-potent stem cells in the adult lung is currently highly debated. At present, it remains unclear whether candidate stem/progenitor cells are located in the airways, alveoli, or throughout the epithelial lining of the lung. Here, we introduce a method of airway micro-dissection which enabled us to study the progenitor behavior of pulmonary epithelial cells in region-specific contexts. The progenitor characteristics of epithelial cells isolated from the trachea, proximal and distal airways, and lung parenchyme were evaluated in vitro and in vivo. We identified a population of airway-derived basal-like epithelial cells with the potential to self-renew and differentiate into airway and alveolar lineages in culture and in vivo after sub-cutaneous transplantation. The multi-potent candidate progenitors originated from a minor fraction of the airway epithelial cell population characterized by high expression of α6 integrin. Results of the current study provide new insights into the regenerative potential of region-specific α6 integrin positive pulmonary epithelial cells.

PMID: 24950291



The major functional characteristic of adult stem cells is their potential to self-renew over the lifetime of an organism. In the adult lung, due to complexity of its architecture and limited number of available functional in vivo stem cell assays, existence and location of stem cells remain uncertain.

The goal of the current study was to evaluate growth and self-renewal potential of region-specific adult mouse lung epithelial cells. Proximal and distal airways were separated (micro-dissected) from the rest of lung tissue following protease treatment (Chernaya et al., 2014). The remaining parenchyme contained alveoli, bronchoalveolar duct junctions (BADJ), and terminal airways. The micro-dissection method can be used across various mouse strains as it does not rely on transgene expression to discriminate between cells from different regions of the lung. Clonogenic potential of airway- and parenchyme-derived epithelial cells was evaluated in vivo in subcutaneous Matrigel implants. To study self-renewal of region-specific lung epithelial cells, heterotopic injections were extended into serial transplantation assays (Fig. 1). In subcutaneous grafts, airway and parenchymal cells generated morphologically distinct cysts (Fig. 2) lined by region-specific epithelium. When introduced into secondary recipient mice as single cell suspensions, a subset of airway and parenchymal cells gave rise to secondary epithelial cysts, thus demonstrating self-renewal in vivo under these conditions (Fig. 1). Interestingly, a fraction of airway cell-derived cysts contained p63+, CCSP+ and pro-SPC/ABCA+ cells indicative of differentiation into airway as well as alveolar-specific cell phenotypes. The p63+, CCSP+ and pro-SPC/ABCA+ cysts were not observed in grafts of parenchymal cells. Thus, under the assay conditions described, only progenitors located in the airway epithelium demonstrated potential to generate basal-, secretory- and alveolar-like cell types.



Previous studies suggested that in the adult mouse lung, elevated expression of integrin α6 and commonly co-expressed subunit β4, marked multi-potent stem/progenitor cell populations (McQualter et al., 2010; Chapman et al., 2011). Whether α6β4-expressing candidate stem cells were located in the air-conducting or gas-exchange compartment was not shown conclusively (McQualter et al., 2010; Chapman et al., 2011). Moreover, stem cell status of the α6β4-positive cells was questioned based on low self-renewal of these cells in vivo (Oeztuerk-Winder et al., 2012). To re-evaluate the stem/progenitor potential of this population, we compared growth and self-renewal of integrin α6high epithelial cells isolated from the airways and parenchymal regions. Within the airway-derived epithelium, high expression of α6 integrin strongly correlated with high clonogenic potential in vitro and in heterotopic transplantation assays. A fraction of airway integrin α6high epithelial cells demonstrated self-renewal and multi-potentiality in sub-cutaneous Matrigel implants (Fig. 3). Candidate α6high stem/progenitor subsets demonstrated lower expression of secretory markers, as compared to the major (non-clonogenic) population of mature airway epithelium. In contrast to airway-derived epithelial cells, parenchymal α6high epithelial cells had low clonogenic potential regardless of assay conditions. Together, our results suggested that adult mouse airways contain rare integrin α6high epithelial cells with primitive immune-phenotype, and the potential to self-renew and differentiate into basal-, airway- and alveolar-like phenotypes. Although a subset of candidate progenitors expressed p63, we did not observe p63-expressing cells in lower airways in situ or in freshly sorted airway α6high epithelial cells. Thus, it is possible that the described subset of cells underwent conversion from p63-negative to p63-posivite phenotype when plated in culture or transplanted subcutaneously.



The major limitation of our study is that it did not address the origin (lineage) of candidate progenitors or establish linear relationships between p63, CCSP and pro-SPC expressing cells. It has been traditionally accepted that adult endogenous stem/progenitor cells are primarily defined by their functional properties, rather than by immune-phenotype (Bertoncello, McQualter, 2013). In this context, p63 expression may reflect an “active state” of the described stem/progenitor cell subset. Two recently published studies provided evidence for the role of p63-expressing stem cells in distal lung regeneration using an in vivo injury model and keratin 5 (Krt5, transcriptional target of p63) lineage labeling. It was shown that undifferentiated Krt5-expressing cells migrate from the airways in response to influenza injury and contribute to lung repair (Zuo et al., 2015, Vaughan et al., 2015). However, similar to the airway α6high subset, the population of Krt5-expressing cells appeared heterogeneous, and differentiation potencies of Krt5+ subsets were dependent upon the extent of injury and other model characteristics (Rawlins, 2015). Considering potential future applications of multi-potent adult lung stem/progenitor cells in tissue engineering for airway/lung replacement and drug testing studies, further characterization of the candidate multipotent airway stem cell populations will be critical.




Chernaya et al., Stem Cells Dev. 2014; 23(22):2744-57 McQualter et al., Proc Natl Acad Sci U S A. 2010;107(4):1414-9

Chapman et al., J Clin Invest. 2011;121(7):2855-62

Oeztuerk-Winder et al., EMBO J. 2012; 31(16):3431-41

Bertoncello and McQualter, Respirology. 2013;18(4):587-95

Zuo et al., Nature. 2015; 517(7536):616-20

Vaughan et al., Nature. 2015; 517(7536):621-5

Rawlins, Nature. 2015; 517(7536):556-7




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