Basal Cells As Stem Cells of the Mouse Trachea and Human Airway Epithelium

Basal cells as stem cells of the mouse trachea and human airway epithelium Jason R. Rocka, Mark W. Onaitisb, Emma L. Rawlinsa, Yun Lua, Cheryl P. Clarka, Yan Xuea, Scott H. Randellc, and Brigid L. M. Hogana,1 Departments of aCell Biology and bSurgery, Duke University Medical Center, Durham, NC 27710; and cCystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27559 Contributed by Brigid L. M. Hogan, June 18, 2009 (sent for review May 16, 2009) The pseudostratified epithelium of the mouse trachea and human Results and Discussion airways contains a population of basal cells expressing Trp-63 (p63) In Vivo Lineage Tracing of Mouse Tracheal BCs. Previous lineage T2 and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreER trans- tracing of mouse tracheal BCs after injury used a KRT14-CreER genic mouse line for lineage tracing, we show that basal cells transgene (8). However, this allele is inefficient for labeling BCs generate differentiated cells during postnatal growth and in the in steady state. We therefore made a new line in which a 6-kb adult during both steady state and epithelial repair. We have human keratin 5 (KRT5) promoter drives CreERT2 (Fig. 1A). We fractionated mouse basal cells by FACS and identified 627 genes used this allele, in combination with the Rosa26R-lacZ reporter, preferentially expressed in a basal subpopulation vs. non-BCs. to lineage trace BCs in adult mice for up to 14 weeks (Fig. 1B). Analysis reveals potential mechanisms regulating basal cells and Soon after the last tamoxifen (Tmx) injection, most of the allows comparison with other epithelial stem cells. To study basal labeled cells (Ϸ98%) were scored as BCs (Fig. 1C). Over time, cell behaviors, we describe a simple in vitro clonal sphere-forming the percentage of labeled cells scored as BCs declined, whereas assay in which mouse basal cells self-renew and generate luminal that of labeled secretory and ciliated cells increased (Fig. 1 D–F cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, and Table 1). Lineage-labeled BCs give rise to more Clara than ITGA6 and NGFR, which can be used in combination to purify ciliated cells over the chase period. There are 2 possible expla- human lung basal cells by FACS. Like those from the mouse trachea, nations for this result. First, BCs may give rise to Clara cells more CELL BIOLOGY human airway basal cells both self-renew and generate luminal frequently than ciliated cells—possibly because the latter have a daughters in the sphere-forming assay. longer half-life than Clara cells and need to be replaced less often. Alternatively, BCs first give rise to Clara cells, which then epithelial basal cells ͉ lung ͉ NGFR ͉ stem/progenitor ͉ sphere-forming assay slowly transition to ciliated cells. We currently favor the second model because it is supported by tritiated thymidine pulse-chase experiments in the rat (1) and our own pulse-chase lineage asal cells (BCs) make up approximately 30% of the labeling studies with Scgb1a1-CreERϩ (Clara) cells in the mouse Bpseudostratified mucociliary epithelium of the lung. They are relatively undifferentiated and characteristically express the trachea (13). transcription factor Trp-63 (p63) and cytokeratins 5 and 14 During postnatal growth there is an increase in the length and (Krt5/14) (1–3). In the rodent, BCs are confined to the trachea, diameter of the rodent trachea and in the number of epithelial where they are interspersed among the ciliated, secretory, and cells, including BCs (1). In previous studies we showed BrdU neuroendocrine cells. By contrast, in the human lung, BCs are labeling of tracheal epithelial cells at postnatal (P)6 days, P3 weeks, and P6 months (13). Further analysis of the material present throughout the airways, including small bronchioles (1, ϩ 4, 5). demonstrated that 55% of p63 BCs and 10% of non-BCs are Cell turnover in the adult trachea is normally very low (6). BrdU labeled at P6 days, whereas the corresponding values for However, epithelial injury elicits rapid proliferation of surviving P3 weeks were 9% of BCs and 2% of non-BCs (Fig. S1). To cells, except ciliated cells (7), and the tissue is soon repaired. follow the behavior of tracheal BCs during postnatal growth a Several lines of evidence suggest that tracheal BCs function as cohort of KRT5-CreERT2;Rosa26R-eYFP mice was given a pulse stem cells for this repair. First, lineage tracing of KRT14-CreER– of Tmx at P10 days or P12 days (Fig. 1G). At P3 weeks, the expressing cells after naphthalene injury, which depletes secre- majority (96%) of lineage labeled cells were BCs (Fig. 1H and tory cells, suggested that some BCs can both self-renew and give Table S1). These represented 7% Ϯ 3% of all BCs in the trachea rise to ciliated and secretory cells (8). Second, a subset of BCs (Table S2). This percentage remained approximately the same retains BrdU label over the long term after epithelial damage by when members of the cohort were examined at P6 weeks and at SO2 inhalation (9). Third, BCs isolated on the basis of high P15 weeks, suggesting that labeled BCs self-renew and are not expression of a KRT5-GFP transgene have a greater capacity to diluted by descendants of unlabeled cells. As before, there was proliferate and give rise to large colonies in vitro than KRT5- an increase over time in the percentage of labeled cells scored GFPϪ cells (3). Finally, fractionated rat tracheal BCs can restore as Clara or ciliated cells (Fig. 1I and Table S1). Thus, during the entire epithelium of a denuded trachea in a xenograft model (10). Similar xenograft assays have been performed with BCs from human nasal polyps and fetal trachea (11, 12). Author contributions: J.R.R., M.W.O., E.L.R., S.H.R., and B.L.H. designed research; J.R.R., M.W.O., E.L.R., Y.L., C.P.C., and Y.X. performed research; J.R.R., M.W.O., E.L.R., S.H.R., and Despite the evidence that BCs are stem cells, relatively little B.L.H. analyzed data; and J.R.R., M.W.O., E.L.R., S.H.R., and B.L.H. wrote the paper. is known about their biology and the mechanisms regulating The authors declare no conflict of interest. their behavior. To address these problems, we have generated a Freely available online through the PNAS open access option. transgenic mouse line to more efficiently lineage trace and Data deposition: The data reported in this paper have been deposited in the Gene genetically manipulate BCs. We have also isolated mouse BCs Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession number and obtained a transcriptional profile. Finally, we have devel- GSE15724). oped a clonal sphere-forming culture system that will greatly 1To whom correspondence should be addressed. E-mail: b.hogan@cellbio.duke.edu. facilitate studies on the self-renewal and differentiation of This article contains supporting information online at www.pnas.org/cgi/content/full/ mouse and human BCs. 0906850106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906850106 PNAS Early Edition ͉ 1of5 Downloaded by guest on September 23, 2021 A B C D E G H I F Fig. 1. Lineage tracing of tracheal BCs. (A) KRT5-CreERT2 transgene con- struct. (B) Timeline for steady-state experiments with KRT5-CreERT2;Rosa26R- Fig. 2. Characterization of mouse tracheal epithelial populations. Tracheal LacZ adult mice. (C–E) Paraffin sections of X-gal–stained (blue) trachea. Anti- epithelial cells were sorted on the basis of labeling with GSI-A3B isolectin and acetylated tubulin (brown, cilia) (C) 6 days, (D) 3 weeks, and (E) 12 weeks after expression of KRT5-GFP.(A) KRT5-GFP tracheal epithelium not labeled with final Tmx injection. (F) Bar graph showing percentage of labeled cells scored GSI-A3B. (B) Transgenic cells labeled with GSI-A3B. p63ϩ cells make up 90.5% as basal, Clara, or ciliated after Tmx exposure and chase. Data shown are of cells in P4, 40.4% of cells in P6, and 1.1% of cells in P5. Averaged over 7 mean Ϯ SEM. (G) Timeline for postnatal growth experiments with Rosa26R- experiments, P4 contained 6% of total sorted cells, P5 67%, and P6 12%. (C) eYFP reporter. (H and I) Cryosections stained for GFP (green, lineage label), Heatmap showing the 100 most differentially expressed genes by Affymetrix Scgb1a1 (red, Clara cells), and T1␣ (blue, BCs). (H) P3 weeks. (I) P15 weeks. microarray of P4, P5, and P6. (D) RT-PCR confirmed the upregulation of 11 Ϫ [Scale bar in C (for C–E), 20 ␮m; in H (for H and I), 25 ␮m.] genes in KRT5-GFPhi BCs vs. KRT5-GFP cells. postnatal growth BCs both self-renew and give rise to Clara and transgenic mouse line in which GFP is expressed at high levels ciliated cells. in approximately 60% of p63ϩ tracheal BCs (3). The Finally, we followed the fates of labeled BCs in a repair model. lectinϩ;KRT5-GFPϩ cells (P4) were an essentially pure BC Ͼ Inhalation of SO2 leads to extensive damage of the tracheal subpopulation because 90% express p63. By contrast, the epithelium, followed by proliferation of surviving cells and lectinϪ;KRT5-GFPϪ (hereafter, non-BC) population (P5) con- restoration of normal histology by 2 weeks (14). During the tained very few p63ϩ cells. The lectinϩ;KRT5-GFPϪ cells (P6) repair process, labeled BCs proliferate and give rise to relatively contained both basal and dendritic cells (Fig. 2 A and B).

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