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 ofaCell Biology andbSurgery, Duke University Medical Center, Durham, NC 27710; andcCystic 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
and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreERT2trans-
genic mouse line for lineage tracing, we show that basal cells
generate differentiated cells during postnatal growth and in the
adult during both steady state and epithelial repair. We have
fractionated mouse basal cells by FACS and identified 627 genes
preferentially expressed in a basal subpopulation vs. non-BCs.
Analysis reveals potential mechanisms regulating basal cells and
allows comparison with other epithelial stem cells. To study basal
cell behaviors, we describe a simple in vitro clonal sphere-forming
assay in which mouse basal cells self-renew and generate luminal
cells, including differentiated ciliated cells, in the absence of
ITGA6 and NGFR, which can be used in combination to purify
human airway basal cells both self-renew and generate luminal
daughters in the sphere-forming assay.
epithelial basal cells ? lung ? NGFR ? stem/progenitor ? sphere-forming assay
are relatively undifferentiated and characteristically express the
transcription factor Trp-63 (p63) and cytokeratins 5 and 14
(Krt5/14) (1–3). In the rodent, BCs are confined to the trachea,
where they are interspersed among the ciliated, secretory, and
neuroendocrine cells. By contrast, in the human lung, BCs are
present throughout the airways, including small bronchioles (1,
Cell turnover in the adult trachea is normally very low (6).
However, epithelial injury elicits rapid proliferation of surviving
cells, except ciliated cells (7), and the tissue is soon repaired.
Several lines of evidence suggest that tracheal BCs function as
stem cells for this repair. First, lineage tracing of KRT14-CreER–
expressing cells after naphthalene injury, which depletes secre-
tory cells, suggested that some BCs can both self-renew and give
rise to ciliated and secretory cells (8). Second, a subset of BCs
retains BrdU label over the long term after epithelial damage by
SO2 inhalation (9). Third, BCs isolated on the basis of high
expression of a KRT5-GFP transgene have a greater capacity to
proliferate and give rise to large colonies in vitro than KRT5-
GFP?cells (3). Finally, fractionated rat tracheal BCs can restore
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).
Despite the evidence that BCs are stem cells, relatively little
is known about their biology and the mechanisms regulating
their behavior. To address these problems, we have generated a
transgenic mouse line to more efficiently lineage trace and
genetically manipulate BCs. We have also isolated mouse BCs
and obtained a transcriptional profile. Finally, we have devel-
oped a clonal sphere-forming culture system that will greatly
facilitate studies on the self-renewal and differentiation of
mouse and human BCs.
asal cells (BCs) make up approximately 30% of the
pseudostratified mucociliary epithelium of the lung. They
Results and Discussion
In Vivo Lineage Tracing of Mouse Tracheal BCs. Previous lineage
tracing of mouse tracheal BCs after injury used a KRT14-CreER
transgene (8). However, this allele is inefficient for labeling BCs
in steady state. We therefore made a new line in which a 6-kb
human keratin 5 (KRT5) promoter drives CreERT2(Fig. 1A). We
used this allele, in combination with the Rosa26R-lacZ reporter,
to lineage trace BCs in adult mice for up to 14 weeks (Fig. 1B).
Soon after the last tamoxifen (Tmx) injection, most of the
labeled cells (?98%) were scored as BCs (Fig. 1C). Over time,
the percentage of labeled cells scored as BCs declined, whereas
that of labeled secretory and ciliated cells increased (Fig. 1 D–F
and Table 1). Lineage-labeled BCs give rise to more Clara than
ciliated cells over the chase period. There are 2 possible expla-
nations for this result. First, BCs may give rise to Clara cells more
frequently than ciliated cells—possibly because the latter have a
longer half-life than Clara cells and need to be replaced less
often. Alternatively, BCs first give rise to Clara cells, which then
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
labeling studies with Scgb1a1-CreER?(Clara) cells in the mouse
During postnatal growth there is an increase in the length and
diameter of the rodent trachea and in the number of epithelial
cells, including BCs (1). In previous studies we showed BrdU
labeling of tracheal epithelial cells at postnatal (P)6 days, P3
weeks, and P6 months (13). Further analysis of the material
demonstrated that 55% of p63?BCs and 10% of non-BCs are
BrdU labeled at P6 days, whereas the corresponding values for
P3 weeks were 9% of BCs and 2% of non-BCs (Fig. S1). To
follow the behavior of tracheal BCs during postnatal growth a
cohort of KRT5-CreERT2;Rosa26R-eYFP mice was given a pulse
of Tmx at P10 days or P12 days (Fig. 1G). At P3 weeks, the
majority (96%) of lineage labeled cells were BCs (Fig. 1H and
Table S1). These represented 7% ? 3% of all BCs in the trachea
(Table S2). This percentage remained approximately the same
when members of the cohort were examined at P6 weeks and at
P15 weeks, suggesting that labeled BCs self-renew and are not
diluted by descendants of unlabeled cells. As before, there was
an increase over time in the percentage of labeled cells scored
as Clara or ciliated cells (Fig. 1I and Table S1). Thus, during
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
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.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: The data reported in this paper have been deposited in the Gene
Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession number
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
August 4, 2009 ?
vol. 106 ?
no. 31 ?
postnatal growth BCs both self-renew and give rise to Clara and
Finally, we followed the fates of labeled BCs in a repair model.
Inhalation of SO2 leads to extensive damage of the tracheal
epithelium, followed by proliferation of surviving cells and
restoration of normal histology by 2 weeks (14). During the
repair process, labeled BCs proliferate and give rise to relatively
large patches of descendants that include Clara and ciliated cells
(Fig. S2). By contrast to the findings at steady state, BCs
responding to injury give rise to more ciliated than Clara cells
over the same period (Table 1). This suggests that the fates of BC
daughters can vary in response to local conditions.
Trancriptional Profile of Tracheal BCs. To separate BCs from co-
lumnar cells for transcriptional profiling, we first sorted cells on
the basis of binding of fluorescently labeled Griffonia simplici-
folia isolectin A3B (GSI-A3B), which binds all BCs (3, 10, 15).
It also binds a population of dendritic cells present within the
epithelium (16). We further purified the lectin?cells on the basis
of expression of GFP using a previously described KRT5-GFP
transgenic mouse line in which GFP is expressed at high levels
in approximately 60% of p63?tracheal BCs (3). The
lectin?;KRT5-GFP?cells (P4) were an essentially pure BC
subpopulation because ?90% express p63. By contrast, the
lectin?;KRT5-GFP?(hereafter, non-BC) population (P5) con-
tained very few p63?cells. The lectin?;KRT5-GFP?cells (P6)
contained both basal and dendritic cells (Fig. 2 A and B).
RNA from all 3 fractions was analyzed by Affymetrix microar-
ray. Clustering of the 100 most differentially regulated genes
between the fractions showed good correlation between repli-
cates (Fig. 2C). The genes most highly expressed in the non-BC
population included those associated with ciliated and secretory
cells (Table S3). As expected, some of the genes upregulated in
the lectin?;KRT5-GFP?population are associated with den-
dritic cells. In the future, new strategies will be used to remove
the dendritic cells from this BC fraction. We found 627 genes
upregulated at least 21.5-fold in the lectin?;KRT5-GFP?BCs
compared with non-BCs (P ? 0.001) (Table S4 and Table S5).
The validity of the screen was confirmed in several ways. First,
RT-PCR confirmed the enrichment of transcripts in KRT5-
struct. (B) Timeline for steady-state experiments with KRT5-CreERT2;Rosa26R-
LacZ adult mice. (C–E) Paraffin sections of X-gal–stained (blue) trachea. Anti-
final Tmx injection. (F) Bar graph showing percentage of labeled cells scored
as basal, Clara, or ciliated after Tmx exposure and chase. Data shown are
mean ? SEM. (G) Timeline for postnatal growth experiments with Rosa26R-
eYFP reporter. (H and I) Cryosections stained for GFP (green, lineage label),
Scgb1a1 (red, Clara cells), and T1? (blue, BCs). (H) P3 weeks. (I) P15 weeks.
[Scale bar in C (for C–E), 20 ?m; in H (for H and I), 25 ?m.]
Lineage tracing of tracheal BCs. (A) KRT5-CreERT2transgene con-
Table 1. BCs of the trachea give rise to ciliated and Clara cells at steady state and in response
to epithelial injury
Time of chaseBasal (%) Clara (%)Ciliated (%)
Total no. of
Total no. of
Tmx ? 6 d
Tmx ? 3 wk
Tmx ? 6 wk
Tmx ? 12–16 wk
Tmx ? SO2? 2 wk
97.8 ? 0.9
53.1 ? 3.8
38.5 ? 1
1.6 ? 0.8
6.6 ? 1.5
14.7 ? 0.6
28.6 ? 1.4
21.4 ? 2.6
0.5 ? 0.3
1.3 ? 0.9
6.2 ? 1.9
18.4 ? 2.6
40.1 ? 2.7
Percentage of lineage labeled cells, ? SEM, scored as basal, Clara, or ciliated after Tmx exposure and varying
times of chase, with or without SO2injury.
epithelial cells were sorted on the basis of labeling with GSI-A3B isolectin and
expression of KRT5-GFP. (A) KRT5-GFP tracheal epithelium not labeled with
GSI-A3B. (B) Transgenic cells labeled with GSI-A3B. p63? cells make up 90.5%
of cells in P4, 40.4% of cells in P6, and 1.1% of cells in P5. Averaged over 7
experiments, P4 contained 6% of total sorted cells, P5 67%, and P6 12%. (C)
Heatmap showing the 100 most differentially expressed genes by Affymetrix
microarray of P4, P5, and P6. (D) RT-PCR confirmed the upregulation of 11
genes in KRT5-GFPhiBCs vs. KRT5-GFP?cells.
Characterization of mouse tracheal epithelial populations. Tracheal
www.pnas.org?cgi?doi?10.1073?pnas.0906850106Rock et al.
GFPhiBCs (Fig. 2D). Second, published reports confirmed
expression of several enriched genes in BCs of murine trachea
and/or human airways. Examples include genes encoding Trp-63,
which is required for BC development in the mouse trachea (2),
Snai2, Cd109, claudin 1, Icam1, aquaporin 3, and keratins 5, 14,
and 17 (3, 17–21). Finally, immunohistochemistry on sections of
mouse trachea and normal human bronchus confirmed BC
expression of several genes, including Ngfr (see below).
The genes upregulated in lectin?;KRT5-GFPhiBCs were
sorted into functional categories (Table S4). Those associated
with cell adhesion include genes encoding components of
hemidesmosomes and anchoring fibrils—integrins ?6 and ?4,
laminins ?3 and ?3, dystonin (BPAG1), and collagen 17a1
(BPAG2) (22). This reflects the abundant hemidesmosomal
attachments of BCs to the underlying basal lamina (1). Our
results also suggest that BCs produce components of the ECM,
including fibulin1, fibrillin2, Tnc, collagen18a1, Sparc, and
TGF?-induced. They are also enriched for cytoskeletal compo-
nents that influence membrane organization, shape, and motil-
ity. Transcription factors that are upregulated in KRT5-GFP?
BCs include Snai2 (Slug) and its corepressor, ajuba (Jub),
basonuclin, Lmo1, Tbx2, Barx2, Etv4, Hlf, AP-2 epsilon, Myc, and
Tracheal BCs are normally relatively quiescent but respond
rapidly to injury. Both quiescence and activation are likely
regulated by reciprocal signaling between BCs and their niche.
Transcripts for various signaling ligands (Bmp7, delta-like 1 and
2, jagged2, Ccl20, PdgfC, pleiotrophin, Tgfb1, Ntf3, Wnt3a,
Wnt5b, and Wnt9a) and receptors (Notch1, Gfra1, Ngfr, Ptch2,
Egfr, Tgfbr3, ephrin receptor B4, and several G protein-coupled
receptors) were enriched in this BC subpopulation. In addition,
antagonists of intercellular signaling pathways (Socs3, sprouty1,
TGF? binding protein 4) were also upregulated.
The rapid proliferative response of BCs to epithelial injury
suggests that, although they are normally relatively quiescent,
they are poised to respond to activating stimuli. This character-
istic is not unique to respiratory BCs. For instance, stem cells in
systems that undergo cycles of growth, destruction, rest, and
regrowth (e.g., the mammary gland and hair follicle) undergo
similar phenotypic switches from quiescence to activation. It is
therefore of great interest that a number of genes expressed in
stem cells of the hair follicle bulge and mammary gland, includ-
ing transcription factors, components of the ECM, signaling
ligands, receptors, and, importantly, negative regulators of in-
tercellular signaling, are also expressed in tracheal BCs (23, 24).
and, potentially, the ability of BCs to respond to activating
The fact that BCs generate different proportions of ciliated
and Clara cells at steady state vs. repair after injury (Table 1)
suggests that progeny cell fates are influenced by local tissue
conditions. This concept has been proposed for the Drosophila
midgut, where varying levels of Notch ligand in the intestinal
stem cells regulates the fates of their progeny (25, 26). Signifi-
cantly, BCs are enriched for transcripts of the Notch pathway
(e.g., Notch1, Dll1, and Jag2). Future studies will determine their
function in BCs.
Novel Assay for Self-Renewal and Differentiation of BCs. Previous
assays for the potential of tracheal BCs to proliferate and
differentiate include repopulation of denuded tracheal xeno-
grafts and air–liquid interface (ALI) culture. Neither is ideal for
quantitative analysis because xenograft models are low-
throughput, and we found it difficult to reproducibly differen-
tiate BCs at the ALI in the absence of non-BCs. We therefore
adapted 3-dimensional sphere-forming assays from other stem
cell populations [e.g., prostate (27)] for the expansion and
differentiation of BCs. When single, viable KRT5-GFP?tra-
cheal BCs were seeded in this assay, ‘‘tracheospheres’’ with a
visible lumen formed within 1 week, even in the absence of
stroma or non-BCs (Fig. 3 A and B). The sphere-forming
efficiency was ?3% of plated KRT5-GFP?cells. In comparison,
the colony-forming efficiency of KRT5-GFP?cells in 2-dimen-
sional ALI culture in combination with a 500-fold excess of
KRT5-GFP?cells was 5% (3). Approximately 0.5% of KRT5-
GFP?cells, likely KRT5-GFP?BCs, formed spheres histolog-
ically indistinguishable from the KRT5-GFP?BC-derived
spheres (Fig. 3C and Table S6).
By 9 days of culture, we observed tracheospheres with diam-
eters ranging from ?50 ?m to ?300 ?m, suggesting that
heterogeneity exists within the sphere-forming population (Fig.
S3 and Table S7). Rarely, lobed colonies were observed, but
these colonies did not survive over the long term and so have
limited capacity for self-renewal in this assay. By day 9, tracheo-
spheres consisted of a pseudostratified epithelium with p63?,
KRT14?BCs peripheral to luminal KRT8?cells (Fig. 3 D and
E). By day 20, surviving spheres had undergone luminal expan-
sion and a thinning of the pseudostratified epithelium, and
beating cilia were observed. This was confirmed by antibody
staining for acetylated tubulin (Fig. 3G). In 26-day spheres the
percentage of KRT8?columnar cells that were ciliated (?50%)
was approximately the same as in adult wild-type mouse tra-
cheas. Nonciliated KRT8?cells persisted in spheres but did not
express markers of Clara (Scgb1a1), neuroendocrine (CGRP),
or mucus-producing (Muc5AC) cells at levels detectable by
immunohistochemistry at 9 or 20 days of culture. In addition,
very few cells expressed claudin 10. Further experiments are
needed to identify this columnar cell type and to optimize
conditions for the differentiation of mature secretory cells from
BC progenitors. BCs in spheres maintained expression of KRT5-
GFP at least up to P21 days. To further demonstrate the
self-renewal potential of BCs, we have serially subcultured
KRT5-GFP?cells from spheres twice at the time of submission.
Tracheospheres formed within 9 days of seeding. (C) Approximately 3% of
spheres. (D and E) 9 days. Tracheospheres stained for (D) p63 (red) and DAPI
(blue, nuclei). (E) Basal KRT14 (red) and luminal KRT8 (green). (F and G)
Twenty-day differentiated tracheospheres. (F) Hematoxylin and eosin stain.
Arrows indicate ciliated cells. (G) Acetylated tubulin (red, cilia) and NGFR
(green, BCs). (Scale bars, 1 mm in B; 50 ?m in D–G.)
A novel sphere-forming assay for BC behavior. (A) Single viable
Rock et al.PNAS ?
August 4, 2009 ?
vol. 106 ?
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For wide application of the tracheosphere assay, it is impor-
tant to be able to isolate BCs independently of any transgene.
Our transcriptome analysis revealed that Ngfr (p75, Tnfrsf16), a
member of the TNF receptor superfamily, is enriched in tracheal
BCs (Table S4). Significantly, this gene is also expressed in
basal/basal-like cells of other epithelia, including esophagus,
corneal limbus, and mammary gland (28–30). Ntf3, encoding a
ligand for NGFR, is also enriched in mouse tracheal BCs (Table
S5), raising the possibility of autocrine signaling within this
population. Immunohistochemistry showed that NGFR is spe-
cifically localized to 98% of p63?mouse BCs (Fig. 4A). We
into basal and nonbasal populations (Fig. 4B). After FACS, 86%
of NGFR?cells were p63?, whereas ?1% of the NGFR?cells
express p63. The sphere-forming efficiency of NGFR?BCs was
?4%, whereas ?0.05% NGFR?cells gave rise to spheres (Table
S6). This finding suggests that although a subset of BCs is able
of BC-depleted populations to do so is significantly reduced. In
fact, because 4% of BCs generate spheres and ?1% of NGFR?
cells are p63?BCs, the 0.05% sphere-forming efficiency in the
NGFR?population is potentially attributable to BCs. Spheres
derived from NGFR?cells were histologically the same as those
derived from KRT5-GFP?cells. Furthermore, when NGFR?
were mixed after sorting, all spheres fluoresced either red or
green (Fig. S3D). This finding suggests that spheres are derived
from single NGFR?BCs.
of BCs are found throughout the lung, although numbers decline
distally (1, 4, 5). These cells express a number of genes enriched
in mouse BCs, including NGFR (Fig. 4C). To apply our in vitro
assay to human BCs, we isolated total epithelial cells from
human bronchi and cultured them overnight to enrich for p63?
is preferentially expressed in tracheal BCs. We therefore sorted
human lung epithelial cells by FACS using NGFR and ITGA6
antibodies (Fig. 4D). Under these conditions, ?96% of the
ITGA6?;NGFR?fraction was p63?, whereas only ?15% of the
ITGA6?;NGFR?cells expressed p63. When seeded in the assay,
ITGA6?;NGFR?BCs gave rise to ‘‘bronchospheres’’ by day 10,
with a single lumen that underwent expansion over 20 days of
the ITGA6?;NGFR?cells. Spheres contained KRT14?, p63?
BCs peripheral to KRT8?luminal cells, and ciliated cells were
observed within 25 days of culture. These findings suggest that
human BCs are capable of both self-renewal and the generation
of differentiated daughters.
In conclusion, we have demonstrated in vivo that BCs of the
mouse trachea function as progenitor cells both during postnatal
growth and in the adult at steady state and in a repair model.
Using a clonal assay, we have shown that BCs of both mouse and
human airways can self-renew and differentiate in the absence of
stroma or columnar epithelial cells. This assay will facilitate the
study of mechanisms regulating the development, maintenance,
and repair of the airway epithelium. Finally, we have derived a
transcriptional profile of mouse BCs that will inform future
investigations on the phenotypic control of this important stem
cell population in both normal development and disease.
Materials and Methods
(Gt(Rosa)26Sortm1Sor), Rosa26R-eYFP (Gt(Rosa)26Sortm1(eYFP)Cos), C57BL/6-
Tg(CAG-EGFP)131Osb/LeySopJ, and Gt(ROSA)26Sortm4(ACTB?tdTomato,?EGFP)Luo
mice have been described before. The Krt5-GFP transgenic mouse line is
maintained by interbreeding on the C57BL/6xC3H background. Rosa26R-LacZ
(Gt(Rosa)26Sortm1Sor) and Rosa26R-eYFP (Gt(Rosa)26Sortm1(eYFP)Cos) reporter
mice were used at the N3 C57BL/6 backcross generation. To establish the
KRT5-CreERT2line, DNA was injected into fertilized zygotes. The construct is
identical to that used to generate the KRT5-GFP line, except that a CreERT2
cassette was inserted in place of GFP. Offspring of 11 founders were screened
to obtain 1 that gave robust and inducible recombination in tracheal BCs in
response to Tmx. The line is presently on the N3 backcross to C57BL/6.
For adult lineage tracing, 8–10-week-old male and female mice hemizy-
gous for KRT5-CreERT2and homozygous for Rosa26R-LacZ were injected i.p. 4
Food Companies). Postnatal mice hemizygous for both KRT5-CreERT2and
on P10 or P12. Mice injected with corn oil only were included in all experi-
ments. For SO2injury, adult male mice were exposed to 500 ppm SO2in air for
3 h 1 week after the final Tmx injection (7).
Immunohistochemistry. For adult lineage tracing, X-gal–stained paraffin sec-
were scored as ciliated (tubulin?), Clara (columnar, tubulin?), or basal (low,
For lineage tracing during postnatal growth, cryosections were stained
with chick anti-GFP (lineage label, 1:500 GFP1020; Aves Labs), goat anti-
Scgb1a1 (Clara cells, 1:10,000; kindly provided by Barry Stripp), mouse anti-
(BCs, 1:1000 clone 8.1.1; DSHB). Alexa-Fluor coupled secondary antibodies
(Invitrogen) were used at 1:500. Z-stacks of optical sections were captured on
scored manually to distinguish cell boundaries.
For NGFR staining, after citrate buffer antigen retrieval paraffin sections
were stained with mouse antihuman NGFR (1:100 05–446; Millipore) and
Vectastain ABC Kit (Vector Laboratories) or rabbit antimouse NGFR (Ab8875;
Abcam) and Alexa-Fluor coupled secondaries. For staining spheres, paraffin
sections were incubated with mouse anti-p63 (BCs, 1:200 sc-8431; Santa Cruz
to p63?(red) BCs in the mouse trachea. (B) FACS using an anti-NGFR antibody
to isolate BCs from primary mouse tracheal epithelium. p63? cells make up
?86% of cells in P6. (C) NGFR expression is confined to the BCs of normal
human bronchus. (D) FACS using anti-NGFR and anti-ITGA6 antibodies to
enrich for BCs from primary human bronchial epithelial cultures. p63? cells
Human spheres at 18 days of culture. (E) Light micrograph. (F) Hematoxylin
KRT14?basal layer appeared somewhat discontinuous. (H and I) Human
spheres at 25 days stained for (H) p63 (green, BCs) and (I) acetylated tubulin
(red, cilia). (Scale bars, 50 ?m in A and C; 25 ?m in E–I.)
www.pnas.org?cgi?doi?10.1073?pnas.0906850106 Rock et al.
Biotechnology), mouse anti-KRT14 (BCs, 1:500 MS-115-P1; Lab Vision), rat
anti-KRT8 (luminal, 1:100 TROMA-I; DSHB), and NGFR and acetylated tubulin
Mouse Tracheal Cell Isolation and FACS. Mouse tracheas were cut into pieces
and incubated in Dispase (BD Biosciences, 16 U/mL) in PBS (30 min) at room
temperature. Digestion was stopped by addition of DMEM with 5% FBS.
EDTA 20 min at 37 °C, followed by gentle pipetting and passage through a
40-?m cell strainer. GSI-A3B isolectin (a kind gift from Dr. Irwin Goldstein,
University of Michigan) was fluorescently labeled using Lightning-Link R-
Phycoerythrin Conjugation Kit (Innova Biosciences) and made functionally
monovalent by incubation with 5 mM N-acetyl-D-galactosamine (Sigma) (10)
before labeling of cells for 40 min on ice. For FACS with NGFR antibody, cells
were diluted to 1 ? 106cells/mL in 2% FBS, 2% BSA in PBS and incubated in 18
?g/mL rabbit anti-NGFR (Ab8875; Abcam) or IgG isotype control followed by
washing and incubation in allophycocyanin-conjugated or Alexa Fluor 488
(BD Biosciences). Cells were collected in DMEM with 2% BSA and cultured
immediately or frozen for RNA extraction.
Mouse Tracheosphere Culture. Sorted cells were resuspended in MTEC/Plus
?L/cm2pipetted into a 12-well 0.4-?m Transwell insert (Falcon). MTEC/Plus (1
mL) was added to the lower chamber and changed every other day. Cultures
were maintained at 37 °C, 5% CO2. On day 7, MTEC/SF (31) was placed in the
was counted on days 7–9 using an inverted microscope. Samples were fixed in
4% paraformaldehyde in PBS, embedded in 3% agarose, then paraffin, and
Human BC Isolation and Culture. Bronchi approximately 1 cm in diameter were
processed as described previously (32). Briefly, under institutional review
board–approved protocols, excess unaffected airways at the time of tumor
resection or donor airways unacceptable for transplantation were obtained
and cells dissociated using Protease XIV (Sigma). Cells were plated onto type
I/III collagen-coated tissue culture dishes in bronchial epithelial growth me-
dium. After 24 h, day 1 passage 1 cells were harvested using trypsin/EDTA and
labeled for FACS using mouse anti-NGFR (5 ?g/mL Ab8875; Abcam), Alexa
Fluor 488 donkey antimouse, and rat anti-ITGA6 (3 ?g/mL Ab19765; Abcam).
medium and fed with ALI medium every other day.
into 3 populations: lectin?;K5-GFPhi, lectin?;KRT5-GFP?, and lectin?;KRT5-
to generate triplicates. RNA was extracted from frozen cells using RNeasy
Micro kit (Qiagen) and quality checked with a 2100 Bioanalyzer (Agilent
Technologies). RNA (20–30 ng per sample) was amplified and labeled using
the Ovation RNA Amplification Kit V2 and FL-Ovation Biotin V2 (NuGEN) by
the Duke Microarray Facility. Standard Affymetrix protocols and mouse
genomic 430 2.0 chips were used to generate .cel files deposited in the Gene
Expression Omnibus of the National Center for Biotechnology Information
(accession number GSE15724). These were imported into Bioconductor in the
R software environment and preprocessed using robust multichip averaging.
Calculation of fold change difference and t tests was performed for each
probe set between the nonbasal and lectin?;K5-GFPhigroups. Probe sets with
a P value ?0.001 and fold change ?2.8 were annotated. The functions lmFit,
the robust multichip average–normalized data for the 3 groups of cells. The
functions decideTests and order were used to order significantly different
probes, and the top 100 were used to create the heatmap using the function
KRT5-GFP?populations from 8 adult KRT5-GFP hemizygous mice and cDNA
synthesized from 120 ng RNA using SuperScript III reverse transcriptase. PCR
primers are listed in Table S9.
ACKNOWLEDGMENTS. We thank Xiaoyan Luo and Tim Oliver for assistance
with FACS and imaging, respectively, and members of the Hogan laboratory
for critical reading of the manuscript. This work was supported by National
Institutes of Health Grants HL071303 (to B.L.M.H.) and K12 CA100639–03 (to
J.R. is supported by training grant 2T32HL007538–26. E.R. is a recipient of a
Parker B. Francis Fellowship.
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no. 31 ?