Our data show that SMG duct cells are a population of multi-potent stem/progenitor cells that survive a severe hypoxic-ischemic injury in the tracheal epithelium and play a role in repair of the SMGs, SMG ducts and SE analogous to the identification of stem cell populations in the ducts of other tissues [10
]. We developed a novel method to isolate SMG duct cells by FACS and used in vitro and in vivo models of stem/progenitor cell self-renewal and differentiation to compare the regenerative potential of known stem/progenitor cells in the airway SE, namely the BCs, to the SMG duct cells. We found that unlike BCs, SMG duct cells were capable of repairing the SMG serous and mucus tubules and MECs and that they were also able to regenerate SE. We speculate that previous reports of surface airway epithelial cell and BC self-renewal and differentiation ability may have been affected by enzymatic digestion methods that also allowed duct cells to be included with BC preparations [2
]. This likely explains why air-liquid interface cultures from the proximal half of the mouse trachea are more efficient at making airway epithelium than the distal half, which has no SMGs and ducts, but both halves are capable of generating differentiated airway epithelium in culture [15
]. We believe that our data demonstrate that BCs and duct cells both contain stem/progenitor cells, but that within duct cells there is a more multipotent stem/progenitor cell.
We found important similarities and differences between the SMG duct stem/progenitor cell population and the BC stem/progenitor population. The SMG duct cells are distinct from the BCs in that (a) they give rise mostly to dense clonal spheres in culture, (b) they are capable of generating SMG-like structures in an in vivo model of regeneration, (c) lineage tracing shows that this population forms SMGs, SMG ducts, and SE adjacent to the SMG ducts, and (d) microarray analysis demonstrates key differences in the nature of the epithelium of the duct cells as compared with BCs. However, there are also similarities between the SMG duct and BC stem/progenitor populations: (a) they both express the surface markers ITGA6 and NGFR and (b) in the sphere-forming and air-liquid interface assays, they have similar differentiation capacity.
SMG duct cells express K14 and we showed previously that under steady-state conditions, only 10% of mouse BCs and just 1% of human BCs of the SE express K14 [14
]. However, premalignant lesions of the SE all express K14 and the presence of K14 in primary non-small cell lung cancer (NSCLC) tissue was associated with a poor prognosis [14
]. The presence of K14 in the airway epithelium therefore appears to correlate with response to injury and the presence of stratified rather than pseudostratified epithelium. The microarray gene expression profile comparison of SMG duct and BCs demonstrates that the duct cells are programmed toward stratified squamous epithelium and express genes that are associated with stratified squamous epithelial cancers, such as squamous skin cancer. This leads us to speculate that SMG duct cells may be a stem cell of origin for NSCLC. We further hypothesize that because duct cells can give rise to glandular structures (the SMGs), as well as SE, that it is possible that they could give rise to NSCLCs with both squamous and adenocarcinoma histologies.
In mice, the SMGs and SMG ducts are only present in the upper third of the trachea [8
] and the SMG duct cells, therefore, likely do not play the major role in repair of the airway epithelium. However, in other mammals such as pigs and humans, SMGs and SMG ducts are found throughout the cartilaginous airways and it is therefore possible that SMG ducts play a larger role in repair of the airway SE, in addition to repair of the SMGs and SMG ducts after injury in humans. There are a number of clinical scenarios where injury results in sloughing of the SE, for example in respiratory syncytial virus infection or acute smoke inhalation. We envision that the SMG duct cells will play an important role in repair of the airway epithelium in these situations.
A normal human bronchial epithelial cell line (Clonetics Cell Systems, Lonza, Walkersville, MD) has been shown to generate spheres and possess self-renewal and differentiation potential in matrigel cultures [27
]. These spheres were able to differentiate to mucus and serous cell types and produced luminal spheres in culture. However, it is not clear whether SMG duct cells might be included in the culture system, as the nature of the human samples used to generate these cell lines is proprietary information. On the basis of our studies, it seems likely that SMG duct cells as well as BCs are included in these cell preparations.
A similar strategy of lineage tracing K14-expressing cells in the airway has been recently published [28
]. Although the investigators did not focus on SMGs or SMG ducts, they did comment that they detected cells derived from K14+ cells in the SMG tubules and ducts but not in the SE adjacent to the glands. Two aspects of this previous study hampered the investigators’ ability to assess the role of SMG ducts in repair and regeneration of the airway epithelium. Firstly, the authors found that endogenous B-galactosidase activity was present in peritracheal glands and they could therefore not perform lineage tracing in these areas. Secondly, the naphthalene model of airway epithelial injury used in the study is not as severe an injury as the hypoxic-ischemic tracheal transplant model and may not therefore require the recruitment of SMG duct cells for repair of the SE.
We found that SMG duct cells have approximately a fourfold lower colony generating capacity than BCs. There are a number of possible explanations for this. One major reason is that SMG duct cells are comprised of at least three distinctive cell populations, namely, BCs of the duct (ITGA6+/NGFR+/tubulin−), ciliated cells of the duct (ITGA6−/NGFR−/tubulin+), and nonciliated, non-BCs of the duct (ITGA6-/NGFR−/tubulin−). It is possible that only one of these cell populations is the genuine stem/progenitor cell and thus with our current method of sorting all TROP-2+ duct cells, we are only obtaining a population that is enriched for the true duct stem/progenitor cell. Another possible explanation is that SMG duct cells are in a more protected location and not exposed to constant environmental injury, as the BCs are. They, therefore, only need to be recruited for repair in the setting of a severe injury and so may cycle more slowly.
Recently, a stem cell was identified in the lung that appears to have the potential to differentiate into a number of different cell types of the proximal and distal airway epithelium as well as into the lung vasculature [29
]. If this work can be replicated, then it suggests that there is a lineage hierarchy in the lungs and that duct cells are considerably further down the lineage than these lung stem cells.