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1Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton; 2Department of Biology and Center for Cancer Research, Howard Hughes Medical Institute, Massachusetts Institute of Technology and 3Brigham and Woman's Hospital, Harvard Medical School, Boston, Massachusetts; and 4Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Philadelphia, Pennsylvania
Submitted 30 July 2007 ; accepted in final form 20 March 2008
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ABSTRACT |
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regeneration; pneumonectomy; proliferation; stem cells; alveolar
Several candidate endogenous stem or progenitor cell populations have been identified in the lung, including Hoechst dye-effluxing side population cells (17, 21, 26, 27) as well as variant Clara cells and bronchioalveolar stem cells (BASCs), which are located at the bronchioalveolar duct junction (12) and show similar properties of naphthalene resistance and proliferation during lung injury. BASCs in particular may be pivotal for regeneration of epithelium since they have been shown to proliferate in response to airway or alveolar injury in vivo (e.g., naphthalene, bleomycin), and cultured BASCs isolated by flow cytometry differentiate into Clara cells, AECI, and AECII, which defines these cells as multipotent (12). The present study was undertaken to determine if BASCs exhibit a proliferative response during compensatory lung regrowth, and to observe the kinetics of BASC compared with AECII in order in order to understand their relative quantitative contributions to alveolar regrowth following pneumonectomy. Physiology and cell kinetics were related using a three-compartment (BASC, AECII, and type 1 alveolar epithelial cell) systems biology model to assess whether BASC proliferation and differentiation could be sufficient to account for 100% of alveolar epithelial cell regrowth.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Animals.
Mice used for the pulmonary function, morphometry, and cell kinetics experiments were 10–12 wk old, 
20 g, C57BL/6 females, and for the cell proliferation study, we used 8-wk-old C57BL/6 females obtained from Jackson Laboratories. All experiments were performed in accordance with National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee at Tufts University. Mice were deemed free of infectious diseases by surveillance using sentinel animals, gross necropsy, and lung histology. Mice were anaesthetized by intraperitoneal injection of ketamine (50–75 mg/kg) and xylazine (5 mg/kg) and then received 2 ml of warmed normal saline and 100 mg/kg sodium ampicillin subcutaneously. Orotracheal intubation was performed under direct visualization using a 19-gauge steel-tipped endotracheal tube over a flexible stylette. Mice were secured in supine position and mechanically ventilated (AUT6110; Buxco Electronics, Wilmington, NC) at 150–200 tidal breaths of 0.3 ml of room air per minute, at positive end-expiratory pressure of 5 cmH2O during surgery and recovery.
Pneumonectomy procedure.
After assessing adequate anesthetic depth via absence of response to toe pinch, the left thoracic wall was clipped and disinfected. The skin, chest wall, and pleura were incised at the fifth intercostal space, and the left lung was gently lifted through an 7-mm incision and ligated at the hilum with 4-0 silk. The lungs were then inflated to 30 cmH2O, and the chest wall closed during this inflation with a single interrupted suture. The skin was closed with 5-0 polydioxanone in a simple interrupted pattern. Mice were extubated at the onset of vigorous spontaneous breathing. The mice recovered from surgery in a warmed cage, and postoperative pain was managed with buprenorphine subcutaneously (0.05 mg/kg) as soon as mice showed conscious motor control, and every 12 h thereafter as needed (<3 days). Chow, nutrient gel (on the cage floor), and water were provided ad libitum. Sham pneumonectomy animals underwent an identical procedure, except that after the thoracotomy, the chest was left open for 5 min to simulate the conditions of the pneumonectomy group and then closed as described.
Measurement of vital capacity and quasistatic lung compliance. On the final day of the experiment (1, 3, 7, 14, 28, or 45 days after PNX or no surgery as indicated by day 0 per protocol), vital capacity and respiratory system compliance were measured following ketamine/xylazine anesthesia. Pulmonary function testing was carried out using a whole body flow-type plethysmograph (PLY3111, Buxco Electronics). Three volume history breaths were administered at 25 cmH2O, and the lung reinflated to 30 cmH2O airway pressure. The breath was held for 0.4 s and then permitted to deflate passively to relaxation volume, when negative pressure was introduced to deflate the lung to residual volume (–30 cmH2O; Forced Maneuvers System, AUT6110, Buxco Electronics). Tests were rejected if there was any spontaneous respiratory effort during the maneuver, and results from a minimum of three maneuvers were averaged for each data point. Vital capacity was measured as the volume of deflation from +30 cmH2O to –30 cmH2O. Respiratory system compliance (i.e., chord compliance) was determined as the linear slope of the quasistatic deflation pressure-volume between 10 and 0 cmH2O airway pressure.
Tissue preparation and morphometry. After pulmonary function testing, the mice were euthanized via cervical dislocation. Following median sternotomy, right ventricular perfusion was performed with normal saline to clear the pulmonary vasculature of erythrocytes. The trachea was cannulated and the lungs removed en bloc. Tissue fixation was achieved with intratracheal 10% buffered formalin at 25 cmH2O overnight. The trachea was then ligated, and the lung was stored in 10% buffered formalin. In control mice, the left lung was removed before embedding to avoid bias during cell counting on tissue sections. Mean linear intercept (Lm) values were calculated using an automated software system (SigmaScan Pro; Systat Software, San Jose, CA) using standard methods (28). Five measurements were made per mouse, and the results were averaged.
Tissue immunofluorescence for enumeration of BASC. Immunofluorescent staining (IF) was performed on formalin-fixed, paraffin-embedded sections (5 µm). As primary antibodies, the polyclonal goat antibody anti-CC10 (Santa Cruz, dilution 1:200), the polyclonal rabbit antibody anti-proSPC (Chemicon AB3786, dilution 1:1,000), and the monoclonal mouse antibody anti-BrdU (Santa Cruz, dilution 1:200) were used. Tissue sections were deparaffinized and hydrated using standard methods, and antigen retrieval was performed using a citrate buffer (pH 6.0) and microwave heating (10 min). Tissues were washed (PBS with 0.1% Triton X-100) three times after antigen retrieval. BASCs were double-stained with CC10 and proSPC. Detection was performed as follows: when triple staining for colocalization of proSPC, CC10, and BrdU, donkey anti-rabbit Alexa Fluor 488 (green), donkey anti-goat Alexa Fluor 350 (blue), and donkey anti-mouse Alexa Fluor 594 (red); when staining for proSPC and CC10, donkey anti-rabbit Alexa Fluor 488 and donkey anti-goat Alexa Fluor 594, respectively. The appropriate single-antibody and secondary-only control assays were performed, and no dual staining or relevant background staining was observed.
Between 16–30 bronchioalveolar duct junctions (BADJ) per mouse were photographed digitally (Nikon Eclipse E600, Spot cooled CCD camera and software), and the images merged in Adobe Photoshop 6.0. BASCs were identified as those with a single nucleus (DAPI) and clear double staining for CC10 (rhodamine) and proSPC (FITC), and were counted as number of BASC per BADJ. To ensure correct identification of BASCs, deconvolution microscopy was utilized where necessary to demonstrate that the CC10 and proSPC were associated with the cell cytoplasm in question. (See Supplementary Figure 1 for deconvolution images. Supplemental data for this article is available online at the AJP-Lung web site.) AECII were identified as those with punctate staining with proSPC. Total nucleated cells per HPF (i.e., DAPI-stained nuclei) were counted, and the mean percentage of AECII cells/nucleated cells in a minimum of 15 HPF was obtained. The BASC/BADJ and AECII/nuclei were used to monitor cell densities over time.
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BASC compartment (B = BASC numbers)
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AECII cell compartment (A2 = AECII numbers)
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AECI cell compartment (A1 = AECI numbers)
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The time domain solution was derived using LaPlace transforms (see Supplemental data), and explicit expressions for B(t), A2(t), and A1(t) as functions of time and rate constants were obtained. Optimal values for the independent model parameter were derived by minimizing the root mean square model error equal to the difference between the sum of: 1) the numbers of cells predicted by the model and the actual numbers measured by immunohistochemistry at the various time points, and 2) the measured lung surface area and calculated surface area, estimated from the number of AECII cells at various time points. For simulation purposes, the study period was divided into seven intervals: days 1–3, 4–6, 7–9, 10–12, 13–15, 16–18, and >18 days, each with distinct kinetic parameters. The overall response was generated by solving the equations in a piecewise continuous fashion. Initial values for cell type were derived from published morphometry: the percentage of total lung cells that are BASC = 0.34% (12), AECI = 8%, and AECII = 16% (18). Normalized total surface area of the lung was determined as 0.9 x AECI(t) + 0.1 x AECII(t). AECI(t = 0) and AECII(t = 0) were both set equal 1, and values at all subsequent time points are expressed as a fraction of baseline (18).
To assess the potential contribution of BASC differentiation to alveolar epithelial cell regrowth following pneumonectomy, the percent differentiation of BASCs into AECII cells was systematically increased from 0 to 50%, and population doubling times were then determined by iteration to minimize modeling error. Model error is reported as the ratio of the root mean square error {which is equal to 
[(measured value – predicted value)2]1/2, where the summation is performed for BASC numbers, AECII cell numbers, and lung surface area at each time} to the root mean square signal {which is equal to [(measured value)2]1/2, where the summation is performed for BASC numbers, AECII cell numbers, and lung surface area at each time}. Initial values for BASC doubling time used in the minimization analysis were 8–24 h based on Kim et al. (12) and a doubling time of 85–270 h for AECII (29). Minimum error values on the order of 10% were achieved using realistic ranges for population doubling times of BASC (19–23 h) and AECII cells (38–51 h). Only the initial values used in the iterative calculations for error minimization were selected from the literature; the actual final values were determined from the error minimization process.
Statistics. Measures of lung function and mean linear intercept were analyzed by repeated measures ANOVA with a two-way Dunnett's post hoc test. Changes in BASC and AECII numbers and proliferation were analyzed using a mixed effect model, admitting mouse as a random effect. The ratio of AECII to total parenchymal nuclei was determined, and the distribution tested for normality using the Shapiro Wilks test (25). The ratio was examined using mixed effects modeling with intersubject variability as a random effect, to see if there was an association between ratio and observation point (day). Mixed effects analysis under these situations is routinely used to admit the subject-specific aspect of variation into the analysis. Failure to do this leads to excessive type 1 errors (i.e., smaller than appropriate estimates of errors in the regression coefficients). To explore observation point (day) association with the BASC values, we used Cuzick's trend test (5) as there were discrete, as opposed to continuous, values (or levels) for BASC. A P value of 0.05 was considered significant.
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RESULTS |
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Model predictions closely matched measured cell numbers and lung volumes for simulations in which BASCs contributed between 0 and 25% of ACEII during lung regrowth (Supplementary Fig. 2, A–D). Simulations indicate that as percentage differentiation of BASCs into AECII cells increased above this level, the BASC population doubling times required to approximate the results exceeded physiological limits, i.e., <5 h (Fig. 6A). The model cannot match the data using similar population doubling times for BASCs and AECIIs, indicating that it is very unlikely that population doubling times for BASCs and AECII cells in vivo are in fact the same. Furthermore, simulation errors were stable (10%) between 0 and 12.5% contribution of BASC to AECII and then increased abruptly at 
25% contribution of BASC to AECII, suggesting that model fit deteriorated above this level (Fig. 6B). These simulations indicate that the model cannot quantitatively match the morphometry and physiology of post-PNX lung regrowth if 25% of BASCs are required to differentiate into AECII cells during growth.
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DISCUSSION |
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Pulmonary function testing was performed in concert with Lm measurements to define the pattern of regrowth, discriminate regrowth from alveolar enlargement, and determine how changes in morphology correspond to changes in pulmonary function (4, 20, 22, 30). In this experiment, there was no change in Lm at any point after surgery, confirming previous studies in mice at days 4, 8, 10, 12, or 21 post-PNX that changes in lung volumes are associated with an increase in alveolar septation rather than simple alveolar distension (7, 22, 30). According to Fehrenbach et al. (7), compensatory lung regrowth in the mouse is characterized stereologically by an increase in alveolar numbers (neoalveolarization), the bulk of which is established by 6 days after pneumonectomy. Thus regrowth in the mouse can be reasonably defined as a form of tissue "regeneration" akin to the return of functional mass after excision of liver or pancreatic tissue. VC was chosen as a robust physiological measurement with stringently standardized starting conditions, namely the deflation from a standard airway pressure with careful attention to volume history. In measurements taken the day following PNX, VC decreased by 34%, in agreement with past studies showing the loss of 31–34% lung volume from excision of the left lung in the mouse (4, 30). By day 7, values for VC returned to control values. These data are consistent with previous descriptions of murine compensatory lung growth (4, 22, 30). The mechanism that stimulates compensatory lung regrowth and BASC proliferation was not established in this study, but prior investigations have focused on mechanical stress as the principal factor that initiates lung regeneration (2, 10, 11, 19, 24, 31). The measurements of lung physiology are useful for comparison with cell kinetics measurements and represent a novel approach to integrate stem cell numbers with functional outcome in vivo.
Compensatory lung regrowth is traditionally thought to be achieved by differentiation of AECII to AECI, and supported further by AECII proliferation. Our model simulations support the potential role of AECII to restore lung surface area through differentiation into AECI. AECII are known to proliferate after lung injury, and it has been hypothesized that this response modulates lung regrowth and repletion of the AECI population (6). We noted an increase in BrdU uptake in AECII during regrowth and increased lung volume. Our quantitative modeling analysis confirms that the observed AECII density was sufficient to account for either all or a large part of alveolar regrowth. For example, an AECII population doubling time of 38 h was adequate to ensure matching of both the cell numbers and lung volume changes. Based on our previous in vitro studies showing that BASCs are multipotent for AECII and Clara cells, BASCs may play an important role in repopulation of the lung during compensatory lung regrowth. As the contribution of BASCs to AECII repopulation is increased from 0 to 12.5% in the model, AECII doubling times that matched the experimental data increase, but remain physiological. Together, these results are consistent with prior studies demonstrating a critical role for the AECII cell in alveolar compartment reexpansion and healing, and suggest that AECII cells (or a subpopulation of AECII) exist that have reparative progenitor cell function.
The present analysis precludes the definition of the precise contribution of BASCs to postnatal lung regrowth following pneumonectomy. BASCs may be an important modulator of lung regrowth and repair, since they proliferate at appropriate times in relation to lung regrowth. Our observed BASC proliferation, which we propose occurred de novo, could potentially be confused with cells (e.g., Clara cells or AECII) that gain expression of CC10 and/or proSPC at the BADJ as a consequence of pneumonectomy; this study does not formally rule out the possibility that Clara cells or AECII give rise to new BASCs in this setting. Arguing against this possibility, BASCs do not derive from AECII cells in our previous culture studies. In addition, our data do not rule out the possibility that an undefined precursor cell gives rise to BASCs.
Importantly, the fact that BASCs are the first cells to significantly expand in number, are the most highly proliferative subpopulation based on the fold change in BrdU incorporation compared with sham-treated animals, and continue to proliferate for an extended period suggests that BASCs likely play an important role in lung regrowth. Potential roles for the BASCs that are consistent with our data set include the following: 1) a stem cell that gives rise to a subset of AECII cells that subsequently undergo rapid expansion and/or differentiation to produce AECI cells for regeneration, 2) a stem cell for Clara cells that contributes to regrowth of the small airways during expansion of the alveolar compartment following PNX, and thereby contributing only to local bronchiolar growth rather than distal AECI expansion, 3) a critical modulatory cell that regulates AECII and Clara cell regrowth through paracrine mechanisms, and 4) a synthetic cell that secretes extracellular matrix components to ensure structural integrity of the BADJ. Importantly, these roles for BASCs are not necessarily mutually exclusive, and large numbers of BASCs are not required to assume these roles. Given the combination of our cell kinetic measurements and model simulations, our data support a dual compartment model in which both BASCs and AECII proliferate in response to pneumonectomy, and AECII act as the major progenitor cell for AECI cells. Future analysis using lineage tagging will better define the quantitative role of BASC vs. AECII in cell regrowth. As additional cell kinetic data becomes available, the current model can be used to study these data.
Our current hypothesis places BASCs at the top of a hierarchy (Fig. 1) and AECII cells as transit amplifying cells between BASCs and AECI. While there are no data to support the role of Clara cells or AECII to produce BASC, this theory cannot be excluded. Several additional limitations of the model exist that may affect our interpretation of the role of BASCs in lung regeneration. The assumption that BASCs represent an independent self-renewing pool, rather than a cell derived from an as yet unidentified precursor may be incorrect, although there is no data to the contrary. Given our earlier findings that BASC cultures give rise to Clara, AECII, and AECI cells in culture (12), the assumption in our model that AECI arise only from AECII may underestimate the contribution of BASCs to lung regeneration. Furthermore, differences in how BASC vs. AECII density were quantified for entry into the three-compartment model introduce a potential impediment to comparing the kinetics of both groups. Specifically, BASC were measured within an anatomically restricted zone (the BADJ), and the cell density of non-BASC in this region were not considered in the denominator. Hence, BASC per BADJ unlike AECII per nuclei did not represent the relative density of BASC compared with other cells at the BADJ that may have proliferated. However, quantifying the cell density of non-BASC, which proliferate along the axis of the airways, was beyond the scope of this study. While the measurement of "BASC per BADJ" differed qualitatively from "AECII per nuclei" in this respect, the number of BASC per BADJ should accurately reflect proliferation of this cell at this anatomically restricted site. The measurement of AECII per nuclei on the other hand may underestimate the proliferative potential of AECII since multiple cell types in that compartment were likely proliferating concomitantly (i.e., contributing to a larger denominator). Hence, the studies employing BrdU incorporation were used to complement the interpretations of these data. Finally, in the current model, the death rate of lung cells during rapid regrowth is assumed to be negligible. This assumption, supported by previous data (13), may be incorrect, since previous studies show significant apoptosis of endothelial and epithelial cells after the initial surge of compensatory lung regrowth in mice (23). Further investigation will need to be performed to demonstrate if the return of BASC from a state of proliferation back to quiescence is mediated by differentiation or cell death.
In summary, we found that pneumonectomy caused a transition from quiescence to proliferation that is temporally regulated in lung epithelia. Three cell types were observed to proliferate: 1) the Clara cell, 2) the AECII, and 3) the BASC. Cell cycle kinetics for these three cells differed substantially. BASCs expanded in number soon after PNX, had the highest fold proliferative index, and remained in increased numbers out to 14 days. Proliferation of all three cell types was transient, returning to baseline after the completion of lung regrowth. Kinetic modeling and quantification of BASCs and AECII suggests that BASCs and AECII may both contribute to lung regrowth following pneumonectomy in the mouse, with AECII proliferation dominating the proliferative landscape. Specific lineage tracing will be necessary to identify the exact contribution of BASCs and AECII cells to alveoli and airways during lung regeneration.
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ACKNOWLEDGMENTS |
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Present address of R. D. Nolen-Walston: Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square, PA. Present address of C. F. Kim: Stem Cell Program, Children's Hospital Boston, Department of Genetics, Harvard Medical School and the Harvard Stem Cell Institute, Boston, MA.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* R. D. Nolen-Walston and C. F. Kim contributed equally to this work. 
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