Establishing cell-specific markers that identify the cellular populations of the steady-state tracheal epithelium
We examined a panel of markers identified from published gene expression array databases and previously described markers of complicated epithelia and glands in order to confirm previously identified markers and identify novel markers for each epithelial cellular subtype in tracheal SE and SMG. This was performed to allow us to examine each of the cell populations as they after hypoxic-ischemic injury. We performed immunofluorescence staining on longitudinal tracheal sections from naïve wild-type mice to confirm the morphology and distribution of each cell subtype of the SE and the SMG. demonstrates a typical longitudinal section of the upper mouse trachea with the location of the SMGs and ducts.
Figure 1 Location and expression of markers of the different cell types of the mouse tracheal SE and SMG and ducts. (a) Longitudinal section of the mouse trachea demonstrating the normal location of SMGs, with the largest group being between the larynx and C1 (more ...)
EpCAM and E-cadherin expression were present on all epithelial cells in the SE and SMGs (). We identified that TROP-2, which is a marker of prostate basal cells,10
was expressed on all SE and SMG duct cells, but not on SMG tubules ().7
SE basal cells were identified by their morphology (low pyramidal to cuboidal cells that reside on the basement membrane (BM), and their positive staining for cytokeratin (K)5,11
and nerve growth factor receptor 12
(). They also expressed Integrin-β4 on their basolateral surface (). Secretory cells (Clara and serous cells) were identified by their columnar shape and expression of Clara cell secretory protein and polymeric immunoglobulin receptor, respectively (). Ciliated cells in the SE and the proximal part of SMG ducts were identified by their characteristic columnar shape and cilia that stained positively for acetylated β-tubulin ().
SMGs consist of serous and mucus tubules, which are lined with polyhedral serous and mucus cells with rounded basal nuclei. Serous cells express lactoferrin, lysozyme and the polymeric immunoglobulin receptor, while mucus cells stain for MUC5AC, MUC1 and DMBT1, and mucins can also be visualized with Alcian Blue-Periodic Acid Schiff staining (). The SMG tubules are surrounded by elongated myoepithelial cells (MEC), which expressed K5, K14 and α-SMA (). SMG tubules converge into the SMG ducts that are lined with a thin layer of stratified epithelium. The basal cells of the SMG duct cells express K5, K14, Integrin-α6 and nerve growth factor receptor in addition to TROP-2 ().
Reproducibility and consistency of the hypoxic-ischemic injury in the tracheal transplant model
The severity of injury and stages of repair were consistently the same at day 1 and day 3 post-transplant in all tracheas examined. At the later time points post-transplant, the repair stages were consistent among tracheas, except for one to two tracheas per group, which showed a failure of re-epithelialization of the trachea. In these tracheas, no living epithelial cells were seen on the BM or in the SMG bed.
Repair of the SE, SMGs and SMG ducts occurs in a reproducible spatio-temporal pattern
To assess the time course of the in vivo
regeneration of the mouse trachea SE, SMG and SMG ducts after severe hypoxic-ischemic injury, we performed syngeneic heterotopic murine tracheal transplants. This model3-5,13
allowed us to examine the surviving cell populations after injury and to study the temporal and spatial repair of the SE, SMG and SMG ducts.
At day 1, many SE cells expressed annexin v and sloughed into the lumen of trachea or SMG duct. Cells that remained attached to BM were 94.5 ± 2.1% K5+ basal cells of which only 4.7 ± 4.8% were also K14+, 4.9 ± 1.2% were CC10+ Clara cells and there were no ciliated cells. 55.5 ± 3.2% of the cells that remained attached to the BM were also annexin v+. In SMGs, 7.1 ± 7.2% of duct cells and 80.5 ± 7% of tubular cells were annexin v+ ().
Figure 2 Repair and regeneration of the mouse trachea at days 1 and 3 post-tracheal transplant. At day 1 post-transplantation (i–iii), initial injury and necrosis of all epithelial tissues were seen by haematoxylin and eosin (i). Most surface epithelial (more ...)
At day 3 post-transplantation, wide areas of the BM were denuded and all cells that remained attached to BM were K5+ basal cells with no CC10+ Clara cells or ciliated cells. About 12.9 ± 4.2% of the K5+ basal cells also expressed K14. Some of these K5+ cells still had rounded or low pyramidal nuclei, while other cells were found to have elongated nuclei with elongated cytoplasmic processes. Only 8.1 ± 1.8% of the cellsthat remained attached to BM were annexin v+. In the SMGs, there were no annexin v+ duct cells. Most of the SMG beds located deeper in the submucosa than the ducts showed no nuclei, with only halos of dead cells. Of the few remaining nuclei, 95.1 ± 6.8% of cells were annexin v+ ().
On day 5 post-transplantation, apart from the sloughed cells in the lumen of the trachea or SMG ducts, no annexin v+ cells were detectable. The BM was completely covered with 90.3 ± 3.1% of SE cells being K5+, 8.5 ± 2.7% were CC10+, 8.2 ± 1.6% were lysozyme+ and there were no tubulin positive cells. 88 ± 3.5% of the K5+ cells also expressed K14. 92.7 ± 3.4% of SMG cells were K5+, 16.5 ± 5.9% were α-SMA+ and 14 ± 5.6% expressed lysozyme (). Scattered areas of the tracheal SE were two to three cell layers thick with round to oval-shaped cells. TROP-2,10
a previously described marker of prostate basal cells, was detected in all the SE cells and was also detectable in all the SMG-like structures that were forming in the SMG area (). This is in contrast to steady-state tracheas, where TROP-2 is present in all SE cells but only in SMG duct cells and not in SMG tubules (). 16.5 ± 5.9% of the TROP-2+ SMG-like structures developed two to three layers of epithelial cells in which the outer cells expressed α-SMA, in addition to K5 and K14 ().
Figure 3 Repair and regeneration of the mouse trachea at days 5 and 7 post-tracheal transplant. On day 5 post-transplantation (i–iv), the denuded basement membrane was now completely covered with TROP-2+K14+ cells (ii, iii). Most areas of the tracheal (more ...)
14 ± 5.6% of the inner cells of the SMG-like structures expressed lysozyme, lactoferrin and polymeric immunoglobulin receptor (all markers of serous cells) that were now also detectable in secretions within the lumen of the SMG-like structures ( and data not shown). Although mucus secretions as determined by DMBT1 expression were not present (), AB-PAS staining detected neutral mucus secretions in the lumen of the healing SMG ducts, indicating at least initial differentiation towards mucus cells ().
The repair continued through day 7, where the BM was completely covered with hyperplastic SE consisting of three to five cell layers of stratified to pseudostratified epithelium (). TROP-2 was still present in all the SMG-like structures in the submucosa (). About 85 ± 4.6% of the cells of the SE were K5+ and of these 79.1 ± 9% also expressed K14 (). Of the remainder of the cells on the SE, 14.4 ± 1.9% were CC10+, 8.3 ± 2.3% were lysozyme+ and no tubulin positive cells were seen. Many of the cells that expressed these differentiation markers in SE and SMG were also positive for K5.
The epithelial hyperplasia extended into the SMG ducts too. 82.1 ± 6.9% of all the cells of the SMG ducts still expressed K5 and K14, while other ducts expressed K5/K14 in cells in the basal layer only, as is seen in steady-state ducts (data not shown). In the SMGs, 43.8 ± 5.5% were α-SMA+/K5+/K14+ MECs but were still rounded in morphology and not elongated, as in the fully repaired state (). About 20.2 ± 2.7% of the SMG cells expressed lysozyme, but the mucus cell marker DMBT1 was still undetectable (). Similar to the day 5 post-transplant time point, neutral mucus was detected by AB-PAS staining, indicating the presence of some functional mucus cells ().
By day 10 post-transplantation, 89 ± 3% of the cells of the SE were K5+ and of these 51.3 ± 3.5% still expressed K14. 18.6 ± 2.2% of SE cells were CC10+, 8.5 ± 2.4% were lysozyme+ and 13.3 ± 4.1% were tubulin positive ciliated cells. Many of the differentiation markers in SE and SMGs were also positive for K5 (). The SMGs now had distinct duct-like and tubule-like structures. TROP-2 was detected in all the SE, SMG ducts and in all the tubule-like structures, including the MECs (). The α-SMA+/K5+/K14+ MECs represented 14.9 ± 8.8% of all SMG cells and were now thinner and longer (). 10 ± 3.4% of the differentiated cells of the SE were serous secretory cells (expressing lysozyme). No DMBT1 expression was seen yet (), although both neutral and acidic mucus were detectable with AB-PAS staining, indicating the development of differentiated mucus cells ().
Figure 4 Repair and regeneration of the mouse trachea at days 10 and 14 post-tracheal transplant. By day 10 post-transplantation, there were more areas of pseudostratified SE (i–iv). Many cells were still K14+ (ii). The SMGs now had distinct duct-like (more ...)
By 14 days post-transplantation, there was near complete return of SE to its normal pseudostratified appearance with 37.8 ± 6% of SE cells expressing K5, which were mostly basal cells and K14+ cells represented only 15.4 ± 4.3% of all cells of the SE (). About 31.4 ± 5.4% of SE cells were CC10+, 14.8 ± 1.8% were lysozyme+ and 20.4 ± 3.4% were tubulin positive. Clara, serous and ciliated cells were therefore present in the SE in numbers similar to the steady state.
71.5 ± 5.5% of SMG cells were K5+ and 11.5 ± 2.7% were lysozyme+, and DMBT1 was detectable and stained 6.3 ± 3.5% of the SMG cells (). Many of the cells that expressed differentiated markers in the SE and SMG also expressed K5. Neutral and acidic mucus were now detectable in the SMGs and in SE, indicating the development of mucus tubules and goblet cells (). TROP-2 was still detected in the SE, SMG ducts and in all the tubule-like structures, including the MECs (). The α-SMA+/K5+/K14+ MECs represented 20.8 ± 7.1% of cells and were elongated in morphology (). We found that repair of the SE and SMG was almost complete by 14 days post-transplantation and that tracheas collected at 21 days post-transplantation were similar to the 14-day tracheas but their lumens, SMG ducts and tubules were distended with secretions, which probably reflects the lack of proper drainage of secretions in the grafts placed in a heterotopic position (data not shown). A graphical representation of the quantitative immunostaining data is shown in and demonstrates the temporal change in cell populations during injury and repair.
A graphical representation of the quantitative immunostaining data., annexin;, K5;, K14; , secretory cells;, tubulin.
Identification of the proliferating cell type and distribution
We performed pulsing studies with BrdU to delineate the proliferating cell populations. All recipient mice with the heterotopic tracheal grafts received two doses of BrdU 6 and 3 h prior to euthanasia. At day 1, no BrdU+ cells were seen in the SE or SMG (). At day 3, 53.4 ± 9.1% of cells on the SE were BrdU+ and K5+ basal cells. 7.7 ± 4.9% of cells in SMG ducts were BrdU+. No BrdU+ cells were seen in SMG tubules (). At day 5, there was a marked increase in the number of BrdU+ cells both on the SE and in the SMGs. In the SE, 22 ± 9.8% of cells were BrdU+ and K5+. No cells were double positive for BrdU and the differentiation markers CC10, lysozyme or tubulin. In the SMGs, 29.4 ± 8.1% of cells in the repairing ducts were BrdU+ and K5+ with 5 ± 2.2% also staining for α-SMA. No BrdU+ cells were seen in the deeper portions of the SMG bed. Some unidentified parenchymal cells were also BrdU positive (). At day 7, 24 ± 17.3% of cells on the SE were BrdU+ and K5+ with 4.2 ± 2% also staining for CC10 or lysozyme but not tubulin. In the SMGs, 39.9 ± 14.6% of cells in SMG ducts were BrdU+ and K5+ with 12.3 ± 4.4% also staining for α-SMA. BrdU+ cells were seen throughout the SMG tubules (). At day 10, 2.9 ± 1.4% of cells on the SE were BrdU+ and K5+ with no cells being double positive for BrdU with CC10, lysozyme or tubulin. In the SMGs, 8.9 ± 4.2% of cells were BrdU+ and K5+ with 4.6 ± 3.5% also staining for α-SMA (). At day 14, only 0.5 ± 0.3% of cells on the SE were BrdU+ and K5+ with no cells being double positive for BrdU with CC10, lysozyme or tubulin. In the SMGs, 5.8 ± 2.6% of cells were BrdU+ and K5+ with 2.7 ± 1.5% also staining for α-SMA (). These data collectively indicate that the surviving K5+ basal and duct cells proliferate and then begin to differentiate at days 3–5. α-SMA+ differentiating MEC cells in the SMGs also start to proliferate from day 5 onward, while CC10+ differentiating secretory cells in the SE briefly participate in proliferation only at around day 7 post-transplantation.
Figure 6 BrdU expression at time points post-tracheal transplantation. All recipient mice with the heterotopic tracheas from all time points received 2 mg of BrdU 6 and 3 h prior to sacrifice. At day 1, no BrdU+ cells were seen in the SE or SMGs (i). At day 3, (more ...)
SMG duct cells that survived the severe hypoxic-ischemic injury can regenerate the SE
The hypoxic-ischemic injury leaves behind some SE basal cells that are the presumed source of the regenerated SE after tracheal transplantation. To examine whether SMG duct cells have the ability to regenerate the SE when basal cells are absent, we incubated dissected tracheas in pronase for 4 h, an enzymatic digestion that we showed previously removes all basal cells, leaving SMG duct and SMG cells in place.7
Then we performed heterotopic syngeneic transplants with the pronase-digested tracheas and compared them with heterotopic syngeneic tracheal transplants, which were not pronase digested and had basal cells. We found that the remnants of the SMGs and their ducts could regenerate pronase-denuded SE (). However, notably, the SE of the pronase-digested tracheas repaired more slowly than the non-pronase-digested tracheas, with a delay in repair of the SE of about 7 days. The BM was still denuded at 7 days post-transplant in the pronase-digested tracheal grafts compared with the non-pronase-digested grafts that were lined by several layers of K5+K14+ epithelial cells (). K5+K14+ epithelial cells were seen lining the BM at 14 days post-transplant in the pronase-digested tracheal grafts () but were delayed in repair as compared with the epithelium of non-pronase-digested tracheal grafts (). The repair of the SMGs was not delayed after pronase digestion and the return of the different SMG and SMG duct cell types remained the same (). These data suggest that in the absence of basal cells, SMG duct cells may have the ability to regenerate the SE.
Figure 7 Repair and regeneration of the mouse SE, SMGs and SMG ducts after severe hypoxic-ischemic injury after removal of basal cells. (a) Delayed repair and regeneration of the SE after pronase digestion of tracheal grafts. The basement membrane was still denuded (more ...)