Epithelial Progenitor Cells in the Rat Trachea Neil F. Johnson and Ann F. Hubbs Inhalation Toxicology Research Institute, Albuquerque, New Mexico
Highly pure populations of basal and secretory cells from the rat trachea have been inoculated into denuded tracheal grafts to determine the differentiation pathways of these two cell types. The basal cell inoculum resulted in an .epithelium comprised of only basal and ciliated cells, while the secretory cell inoculum gave rise to an epithelium comprised of secretory, basal, and ciliated cells. These results show that, in the rat trachea, the secretory cell is the major progenitorial cell type and the basal cell has only limited progenitorial capacity.
The normal tracheobronchial lining is a well-differentiated epithelium typified by the presence of basal, secretory, and ciliated cells. Under normal circumstances, few cells in this tissue undergo division at anyone time; however, marked proliferation and phenotypic modulation can occur following injury. Malignant transformation in the tracheobronchial lining in humans gives rise to approximately 90% of all primary lung tumors, even though this tissue covers less than I % of the lung surface exposed to the atmosphere (1). The neoplasms that arise express a range and mixture of phenotypes not seen in normal, mature epithelium. However, the differentiation pathways of both normal and aberrant epithelial cells are poorly understood. Of the three major cell types present in the tracheobronchial epithelium, only basal and secretory cells have thecapacity to divide and differentiate. The ciliated cells are considered to be terminally differentiated and incapable of division (2). In fact, it is only under exceptional circumstances that the ciliated cells of isolated hamster trachea are capable of synthesizing DNA, as evidenced by tritiated thymidine incorporation (3). There is conflicting evidence concerning the roles of secretory and basal cells in repair and maintenance of the ,normal epithelium, the nature of their differentiation pathways, and their relationship to the development of bronchogenic tumors. The basal cell is a small cell lying adjacent to the basement membrane of the airway. Identifying basal cells by conventionallight microscopy is difficult in tissue sections and in mixed cell populations where they can form only a small percentage of the total cell population. Electron micrQscopy shows the basal cell is very diverse in shape, size, and distribution within the upper airways (30); however, the basal cell has scant cytoplasm, containing few mitochondria, little rough endoplasmic reticulum, and .numerous keratin fila-
(Received in original form March 6, 1990 and in revised form June 5, 1990) Address correspondence to: Dr. N. F. Johnson, Inhalation Toxicology Research Institute, P.O.. Box 5890, Albuquerque, NM 87185. Abbreviations: fetal calf serum,
pes; minimum essential medium, MEM.
Am. J. Respir. CeU Mol. BioI. Vol. 3. pp. 579-585, 1990
ments. The ultrastructural appearance of basal cells suggests that they are moderately differentiated cells involved in cell adhesion (4). However, this cell is sometimes considered analogous to an undifferentiated basal progenitor cell seen in other epithelia such as skin, cornea, gut, and bladder. The results of light microscopy studies in which tritiated thymidine pulse labeling was used (5, 32) suggest that basal cells differentiate to both secretory and ciliated cells. It has been reported (6) that basal cells undergo an asymmetric division, in which one of the progeny is an undifferentiated cell capable of further division and differentiation into superficially located secretory cells, while the other cell loses its ability to divide. A further cell renewal model has been proposed (7)in which there are three compartments: the first consists of self-renewing basal cells that give rise to the second compartment, a mucous cell compartment, some of which can divide, and a third compartment consisting of terminally differentiated cells with finite life spans. The use of light mi,croscopic techniques makes interpretation of these studies difficult. However, based on the results of recent studies in which isolated populations of rabbit basal cells were used to inoculate denuded tracheal grafts, it is suggested that basal cells from this species can divide and give rise to a complete epithelial lining compriseld of basal, ~cretory, and ciliated cells (8, 9). In the intact, mature tracheobronchial epithelium, the secretory cell is a well-differentiated, tall, columnar cell with numerous secretory granules, a prominent cytoplasmic synthetic apparatus, numerous mitochondria, and a high cytoplasm/nucleus ratio. In the hamster trachea, the. major proliferative response following denuding injuries arIses in the secretory cells at the wound edge. This response is followed by re-establishment of the epithelium and the appearance of preciliated cells (10). The latter cells are large and contain apical fibrogranular bodies (evidence of ciliogenesis), which themselves frequently contain secretory granules that appear to be carried over from the parent secretory cell. However, the preciliated cells lack the well-developed synthetic apparatus seen in the mature secretory cell. These observations imply that the secretory cell, rather than the basal cell, is the major proliferative cell in the tracheobronchial
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epithelium and that the secretory cell can give rise to ciliated cells. This implication is strengthened by results from studies on the effects of vitamin A on cell differentiation. Vitamin A deficiency in rodents leads to the formation of localized areas of squamous metaplasia (II). Squamous metaplasia is frequently considered to be an expression of aberrant basal cell division, which, under certain conditions, can progress to carcinoma in situ and to invasive carcinoma (2). However, detailed light and electron microscopy studies have shown that vitamin A-deficiency lesions arise from keratinization of secretory cells (11). The implication of these results is that the secretory cell is not terminally differentiated and thus, because of its proliferative capacity, may play a major role in repair and maintenance of the tracheobronchial lining. The aim of the research reported here is to define the progenitorial capacity of tracheal basal and secretory cells by inoculating highly pure populations of each cell type into denuded tracheas and then determining the morphologic characteristics of the re-established epithelium. The rat was used as a source of tracheal cells because the cells lining the rat trachea have a similar morphologic appearance to those found in th~ major conducting airways of the human.
Materials and Methods We used female F344/N rats (10 to 12 wk old) from the breeding colony at the Inhalation Toxicology Research Institute (ITRI) as tracheal graft donors. Male F344/N rats (6 to 8 wk old) were used as epithelial cell donors, and male F344/N rats (10 to 12 wk old) were used as graft recipients. Animals used for epithelial cell and tracheal donors were killed by CO2 irthalation, after which their tracheas were removed aseptically. The epithelial cells were harvested according to a previously published method (15). Briefly, the laryngeal end of the trachea was cannulated with polyethylene tubing (ID, 1 to 1.4 mm; aD, 1.57 mm) fitted with an 18-gauge hypodermic needle and the tracheal lumen Was flushed with medium (RPMI-1640 with 25 mM Hepes buffer, 20 mg/liter DNase, and 20 ml/liter antibiotic solution; , Sigma Chemical Co., St. Louis, MO). The distal end of the trachea was occluded with an artery clamp, and the lumen was filled to distension with a solution of hyaluronidase (1.6 mg/ml) and cytochalasin B (l ~g/ml) in minimal essential medium (MEM; Sigma), then incubated for 60 min at 37° C. After the incubation period, the lumen was flushed with 10 ml medium (as above), which was collected and kept at 4° C. The lumen was reinflated with MEM containing 3.3 mg/ml pronase (protease type XIV; Sigma) and incubated for 30 min at 37° C, then flushed with 10 ml medium (as above, but containing 10 ml fetal calf serum [FCS]). This medium was pooled with the previous sample, and the pooled sample was then used to flush the tracheal lumen an additional 5 times. The resultant cell suspension was centrifuged (150 x g for 15 min at 10 C), and the supernatant was carefully removed and discarded. The cell pellet was gently resuspended in 4 ml of fresh RPMI medium (without FCS). Cell numbers were counted on a hemacytometer, and cell Viability was assessed by using the trypan blue dye exclusion technique. The cell suspensions were adjusted to a concentration of approximately 0.5 x 1()6 cells/ml and were analyzed in an EPICS V flow cytometer (Coulter Electronics, Hialeah, 0
FL). Populations of basal an,d secretory cells were sorted by using the cells' inherent forward-angle and side-scatter signals. Two-parameter, contour diagrams were used to delineate discrete cell populations. The details of this method of separating cells and the criteria for distinguishing the populations as basal and secretory cells have recently been described (21). The criteria for distinguishing these cell populations are based on flow cytometric phenotype, electron microscopy, and lectin histochemistry (21). The purities of the sorted populations were assessed by light microscopy on a cytology preparation made by sorting 25,000 cells into glass tubes, centrifuging, and resuspending the cell pellet in 200 ~l of phosphate-buffered saline. Cytospin preparations were then made from the cell suspension by using a Shandon Cytospin 2 (Shandon, Pittsburgh, PA), and staining with Diff-Quik® stain (American Scientific Products, McGraw Park, IL). Basal cells were small (approximately 9 ~m in diameter), with scant cytoplasm and darkly staining nuclei; secretory cells were approximately 14 ~m in diameter with prominent cytoplasm (21). Sorted populations of basal cells with purities in excess of 94 % and of secretory cells with purities greater than 92 % were used as. inocula for the tracheal graft repopulation studies. For each tracheal repopulation, 0.7 to 1.0 x 105 cells were sorted into 5 ml Ha'm's Fl2 medium containing 10% FCS and 1% penicillin/streptomycin. Sorted and unsorted cells (the original cell suspension) and medium alone were inoculated into tracheas denuded by repetitive freezing (-70 0 C) and thawing (37 0 C) (22). Denuded tracheas inoculated with medium without cells served as negative controls for these studies. The inoculated tracheas were transplanted into the subcutaneous tissue in the suprascapular region of the dorsal thorax of syngeneic recipients; two inoculated tracheas were implanted into each host animal. After 5 wk, the host animal was killed and both the grafted tracheas and the 'fat's own trachea were removed. Each trachea was cut in half longitudinally; one half was fixed in buffered formaldehyde for conventional light microscopy, and the other half was fixed in buffered glutaraldehyde for electron microscopy. Paraffin-embedded tissue was sectioned, and 5-~m thick sections were stained with hematoxylin and eosin, combined with alcian blue or periodic acid Schiff. These sections were also used to determine the number of cells and the proportions of the various cell types. Cells were counted in a x-IOO field by·oil immersion light microscopy; the field was calibrated by using a reticle (Nikken; Tokyo, Japan). The numbers of the various epithelial cell types· were recorded for a specific number of fields; 'for each individual trachea, the cells lining at least 2 mm of basement membrane were counted for each individual trachea.' Cells with obvious cilia were recorded as ciliated cells; cells adjacent to the basement membrane that possessed an efongated triangular profile were recorded as basal cells; all other epithelial cells were recorded as secretory cells, In addition, the height of the epithelium of each individual trachea was measured at five locations at least 1 mm apart, using a calibrated eyepiece reticle (Nikken). For electron microscopy, 70-nm sections of resin-embedded tissue were stained with lead citrate and uranyl acetate and viewed with a Hitachi HUlk electron microscope.
Johnson and Hubbs: Tracheal Progenitor Cells
Figure 1. Light micrographs of tracheal sections stained with hematoxylin and eosin combined with alcian blue. Magnification: xl,150. (a) Host trachea showing the normal epithelium comprised of basal, secretory, and ciliated tells. The secretory cells do not stain with alcian blue. (b) Tracheal graft repopulated with unsorted tracheal cells. The re-established epithelium is comprised of secretory, basal, and ciliated cells. A proportion of the secretory cells contain alcian blue-stained material. (c) Tracheal graft repopulated with sorted secretory cells. The hypertrophic, re-established epithelium contains prominent secretory/mucous, basal, and ciliated cells. The mucous cells stain prominently with alcian blue. (d) Tracheal graft repopulated with sorted basal cells. The re-established, low cuboidal epithelium is comprised only of basal and ciliated cells. Alcian blue-stained cells are absent from the epithelium.
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Cell distribution Secretory
Alcian Bluepositive Cells
Medium control Unsorted tracheal cells
% Cells/mrn Sorted secretory cells
% Cells/mrn Sorted basal cells
% Cells/mrn Host trachea
± 2.3 ± 4.3
± 1.7 ± 2.9
± 2.1 ± 5.4
± 1.5 ± 3.5
± 1.7 ± 3.8
± 2.6 ± 4.3
± 2.9 ± 2.9
± 0.1 ± 0.1
± 6.9 ± 6.0
± 1.0 ± 1.8
± 2.0 ± 5.6
± 2.5 ± 6.3
Results Host Trachea The epithelial lining of the host's own untreated trachea was composed primarily of basal cells, alcian blue-negative secretory cells (0.3 positive cells/mm basement membrane), and ciliated cells (Figure la). The epithelium was composed of 22.2 % basal cells, 46.2 % secretory cells, and 31.7% ciliated cells; the density of these cells was 224 cells/mm basement membrane (Table 1). The ultrastructure of the various cell types was similar to that previously described (23). The basal cells were typified by a low cytoplasm/nucleus ratio. The cytoplasm contained scant mitochondria and elements of rough and smooth endoplasmic reticulum, but possessed prominent cytokeratin bundles and junctional complexes, with adjacent basal, secretory, and ciliated cells. These cells, unlike the secretory and ciliated cells, possessed hemidesmosomes. The secretory cells were larger than the basal cells and possessed a higher cytoplasm/nucleus ratio. The cytoplasm of the secretory cells contained prominent secretory granules, numerous mitochondria, elements of rough and smooth endoplasmic reticulum, and a well-developed Golgi apparatus. The ciliated cells were characterized by the presence of apical cilia.
12.5 ± 3.2
contained prominent secretory granules, while the ciliated cells had fully developed cilia without overt evidence of ciliogenesis. Tracheal Graft Inoculated with Sorted Secretory Cells Inoculating denuded tracheas with a purified population of secretory cells resulted in a re-established epithelial lining in eight of 10 tracheas (Figure Ie). The lining was hypertrophic and comprised of basal (17%), ciliated (38%), and secretory cells (44%) (Table 1). The secretory cells commonly contained large amounts of alcian blue-positive material. Electron microscopy showed that, apart from their greater electron translucency, the ciliated cells were similar to those seen in the intact epithelium; the basal cells were, however, smaller (Figure 2).
Tracheal Graft Inoculated with Medium Alone All tracheas inoculated with medium alone had their lumena occluded by loose stromal elements containing macrophages, fibroblasts, and patent capillaries. These tracheas showed no evidence of re-epithelialization (Table 1). Tracheal Graft Inoculated with Unsorted Tracheal Cells Nine of ten denuded tracheas inoculated with unsorted tracheal cells re-established an epithelium (Figure Ib). The epithelial lining contained 21.0 % basal, 28.8 % secretory, and 50.2 % ciliated cells; and 7.0 secretory cells per millimeter of basement membrane were found to contain alcian bluepositive material (Table 1). The numeric density of the cells was 184 cells/mm basement membrane. The cytoplasm of both the secretory and ciliated cells was more electron translucent in these repopulated tracheas than in intact tracheas; however, the cells appeared to be mature. The secretory cells
Figure 2. Electron micrograph of the epithelium resulting from repopulation of a tracheal graft with sorted secretory cells. Unlike in the host trachea, the secretory cells of the repopulated trachea are predominantly of the mucous variety, and the basal cells are frequently small. (Bar represents 1 J,Lm.)
Johnson and Hubbs: Tracheal Progenitor Cells
Figure 3. Electron micrograph of the epithelium resulting from repopulation of a tracheal graft with sorted basal cells. The epithelium contains mature basal and ciliated cells. (Bar represents 1 /Lm.)
Figure 4. Electron micrograph of the apical region of adjacent ciliated cells from an epithelium arising from sorted basal cells. The cilia are mature, and there is in the cytoplasm no evidence of secretory cell differentiation. (Bar represents 1 /Lm.)
Tracheal Graft Inoculated with Sorted Basal Cells Inoculating denuded tracheas with a purified population of basal cells resulted in are-established epithelial lining in five of 10 tracheas (Figure Id). The lining was composed of low cuboidal ciliated cells (76 %) and prominent basal cells (24%) (Table 1). Less than 1% of the'cells were secretory cells. The numeric density of the cells was 109 cells/mm basement membrane. Electron microscopy of these epithelia revealed that the ciliated and basal cells had ultrastructural appearances similar to those of ciliated and basal cells in the intact, normal epithelium (Figure 3). The apical surface of the ciliated cells contained mature cilial structures, and there was no evidence of secretory cell differentiation in the cytoplasm (Figure 4). The basal cells frequently possessed numerous cell junctions and associated keratin filaments (Figure 5).
Basal cells in normal rat tracheas exhibit a lower proliferative potential than do secretory cells. Cell-cycle analysis showed that up to 10% of the total tracheal cell population was in S, G2, or M phases of the cell cycle; of this latter group of cells, 84% were secretory cells and 16% were basal cells (21). The fact that fewer basal cells are in G2/S/M phases of the cell cycle may be taken to support the concept of the basal cell as a stem cell and that they are recruited extensively after injury to the epithelium. However, it has been shown that after damage to the tracheal epithelium, it is the secretory cell that contributes most of the repair process (25-27). This low proliferative response combined with the lower numeric density of basal cells in the trachea compared to the secretory cells suggests that the basal cells do not rep-
Discussion The results of this study show that secretory cells form the major progenitorial compartment within the rat trachea. In denuded tracheal grafts, the secretory cell is capable of reestablishing a new epithelium composed of basal, secretory, and ciliated cells. In contrast, the basal cells were capable of only basal and ciliated cell differentiation. Our results are in marked contrast with those of two recent studies (8, 24), which showed that basal cells from the rabbit trachea were capable of re-establishing an epithelium containing basal, ciliated, and secretory cells. In the previous studies, the basal cells were obtained by centrifugal elutriation techniques. In one of the two studies, the cells were further enriched by an in vitro cloning step. The purity of the elutriation fraction was determined by electron microscopy; there was approximately 11 % contamination with other epithelial cells. This impurity may have influenced the outcome of the direct repopulation study (8). As discussed below, these investigators' selection of the largest clones during the in vitro cloning (24) may also have influenced the results of repopulations with cloned basal cells.
Figure 5. Electron micrograph of basal cells from an epithelium arising from sorted basal cells. The cells possess prominent cell junctions and associated cytokeratin filaments. The cytoplasm of the basal cell contains few organelles. (Bar represents I /Lm.)
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resent the major cell compartment involved in repair and maintenance of the tracheobronchial lining. In the lower airways, repair of the airway epithelial lining occurs in the absence of basal cells (13, 28). Contamination of slowly growing basal cells with a small fraction of more rapidly growing secretory cells could account for the differences in the results of the rat (basal cell purity = 95% ) and rabbit (basal cell purity = 83% ) studies. Similarly, differences in the proliferative potential of basal and secretory cells in culture could account for the results obtained when clones of cells isolated from cultured basal cells were used to repopulate denuded tracheas (24). Basal cells isolated from the rat trachea by either flow cytometry or centrifugal elutriation have been shown to have a colony-forming efficiency ofO.l to 1.0% (16,21). In contrast, secretory cells obtained by flow cytometry had a colonyforming efficiency of 3.6 % (21). Because the basal cell population used for in vitro cloning prior to tracheal grafting had a purity of only 82 % (24) and since secretory cells form colonies in culture with an efficiency 10 to 100 times that of basal cells (21, 24), the large, rapidly growing colonies selected fot repopulation (24) may have been derived primarily from secretory cells contained in the 18% of contaminating cells. The difference in results between our study and those in which the rabbit trachea was used as a source of basal cells (8, 24) may also have been related to a species difference. The tracheal lining of the rabbit contains Clara cells, which are found only in the lower conducting airways of the rat. Differences may also have arisen because the host and donor species were different in the earlier experiments (8, 24); rabbit cells were inoculated into rat tracheas, which were subsequently transplanted into nude mice. For the studies reported here, syngeneic rats were used as both donor and host. In addition, appropriate medium controls were also used in our studies. The use of medium controls was not reported for the repopulation studies in which cloned basal cells were used (24). It is unlikely that any of the cells of the re-established epithelium were derived from the original tracheal graft; however, this possibility cannot be ruled out based on the methods and results of the earlier studies. In our study, repopulating tracheas with unsorted tracheal cells·or sorted secretory cells resulted in hypertrophic epithelia. This hypertrophy was also associated with the appearance of alcian blue-positive cells, which was most striking in the epithelium derived from secretory cells. Electron microscopy revealed the presence of mucous cells within these epithelia, particularly the epithelial arising from inoculation of pure populations of serous cells. This observation confirms previous reports that the serous and mucous cell phenotypes are expressions of a common cell type (20). The significance of these changes will be determined by future studies in which we will follow the time course of epithelialization in tracheas repopulated with sorted secretory cells. The lower numeric density and hypertrophy of cells in the tracheas repopulated with unsorted cells, compared to host tracheas, suggest that the repopulated tracheal epithelium may have still been in the process of being re-established. In all but the secretory cell-repopulated tracheas, the ciliated cells were overexpressed numerically, while the basal and secretory cells were underexpressed numerically. The differ-
ences in number and proportion of the various cell types may indicate that all three suspensions of cells can re-establish a complete ciliated cell epithelium within the time frame of this experiment, but not a basal or secretory cell epithelium. It could also indicate that the composition of the initial inoculum may influence the outcome of the re-established epithelium; this is particularly true for basal and secretory cell repopulations. In the case of the unsorted trachea, the number of ciliated cells in the inoculum is less than those found in the intact epithelium (15). In the epithelium established from the unsorted cell inoculum, the number and proportion of ciliated cells is greater than the intact epithelium while the number and proportion of secretory cells is depressed and the proportion of basal cells is similar, but are numerically less than in the intact epithelium. The early appearance of numerous ciliated cells is not generally seen during the early phases of regeneration in the rodent trachea (31). The implications of the results of our study are that the secretory cell is represented as the major progenitorial cell type in the normal rat trachea. This same conclusion has been drawn from the results of studies in which repair of denuding injuries in the hamster trachea (10, 25-27) and N02 damage to rat airways (13, 28) was investigated. Until recently, basal cells of the tracheobronchial lining have been assumed to have a role analogous to the basal keratinocytes of skin. As a result of this assumption, basal cells generally have been considered the cell at risk from the carcinogenic and toxic effects of xenobiotics. Many calculated dose-effect relationships, especially for radioactive materials, such as inhaled radon progeny are based on this premise (29). The secretory cells, however, appear to be the critical cells at risk because they have a greater progenitorial capacity, a greater potential metabolic capability (21), a greater proliferative capacity (21), and are more numerous than the basal cells (15). In addition, the secretory cells receive a higher dose from inhaled material because they are closer to the luminal surface. The risk for a specific exposure condition does not change if the secretory cells are considered the critical cells; the effective dose changes. Acknowledgments: This research was supported by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract No. DEAC04-76EVO1013 in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. The technical help of Ms. June Hogan, Pat Lawson, and Joanne Millisa, and Mr. Bob Smith is gratefully acknowledged.
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