Protein Cell

DOI 10.1007/s13238-018-0506-y Protein & Cell

RESEARCH ARTICLE
Regeneration of functional alveoli by adult
human SOX9+ airway basal cell transplantation
Qiwang Ma1, Yu Ma1,5, Xiaotian Dai2, Tao Ren3, Yingjie Fu4, Wenbin Liu1, Yufei Han1, Yingchuan Wu1,
Yu Cheng4, Ting Zhang5, Wei Zuo1,5,6&
1
Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai 200433, China
2
Southwest Hospital, Third Military Medical University of PLA, Chongqing 400038, China
3
Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
4
The Institute for Biomedical Engineering and Nano Science, School of Medicine, Tongji University, Shanghai 200029, China

Protein & Cell

5
Kiangnan Stem Cell Institute, Zhejiang 311300, China
6
Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University,
Guangzhou 510120, China
& Correspondence: zuow@tongji.edu.cn (W. Zuo)
Received December 8, 2017 Accepted December 24, 2017

ABSTRACT human lung structure can be reconstituted by orthotopic

transplantation of tissue-specific stem/progenitor cells,
Irreversible destruction of bronchi and alveoli can lead
which could be translated into a mature regenerative
to multiple incurable lung diseases. Identifying lung
therapeutic strategy in near future.
stem/progenitor cells with regenerative capacity and
utilizing them to reconstruct functional tissue is one of
KEYWORDS lung, regeneration, transplantation, stem
the biggest hopes to reverse the damage and cure such
cell, bronchiectasis, alveoli
diseases. Here we showed that a rare population of
SOX9+ basal cells (BCs) located at airway epithelium
rugae can regenerate adult human lung. Human SOX9+ INTRODUCTION
BCs can be readily isolated by bronchoscopic brushing
Regeneration of human skin (Gallico et al., 1984), corneal
and indefinitely expanded in feeder-free condition.
epithelium (Rama et al., 2001; Rama et al., 2010) and
Expanded human SOX9+ BCs can give rise to alveolar
hematopoietic system (Copelan, 2006) by autologous
and bronchiolar epithelium after being transplanted into
transplantation of tissue-specific stem/progenitor cell has
injured mouse lung, with air-blood exchange system
been achieved decades ago and now has become a routine
reconstructed and recipient’s lung function improved.
therapeutic approach. However, regeneration of large inner
Manipulation of lung microenvironment with Pirfenidone
organs, such as lung, remains one of the biggest challenges
to suppress TGF-β signaling could further boost the
to modern medicine. Lung-related diseases are the third-
transplantation efficiency. Moreover, we conducted the
leading cause of human death globally. Most of the lethal
first autologous SOX9+ BCs transplantation clinical trial
lung diseases such as chronic obstructive pulmonary dis-
in two bronchiectasis patients. Lung tissue repair and
ease (COPD) (Mannino, 2002), idiopathic pulmonary fibrosis
pulmonary function enhancement was observed in
(Selman et al., 2004) and bronchiectasis (Moulton and Bar-
patients 3–12 months after cell transplantation. Alto-
ker, 2012) are characterized by irreversible, progressive
gether our current work indicated that functional adult
damage of lung tissues (alveoli and/or bronchi). Besides the
mitigating treatments available, lung transplant surgery is the
only solution for the exacerbated patients but its application
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13238-018-0506-y) contains sup-
is largely limited due to the extreme lack of donor lung as
plementary material, which is available to authorized users. well as severe side effects resulted from immune-rejection.
As a potential substitute, the transplantable artificial lung
Qiwang Ma, Yu Ma, Xiaotian Dai, and Tao Ren have contributed technique is promising but still in its infancy (Ott et al., 2010).
equally to this work.

© The Author(s) 2018. This article is an open access publication

 RESEARCH ARTICLE
Therefore, millions of patients are in urgent need of a new
strategy to cure such diseases and stem/progenitor cell-
based regenerative therapy is likely to be the biggest hope
for them. Among all cells with clinical potential, mesenchy-
mal stem cells (MSCs) or other stroma-derived cells are
easy to obtain and handle. However, it is widely recognized
that transplanted MSCs function mainly through paracrine or
immunomodulatory mechanism (Meirelles Lda et al., 2009),
with no evidence showing that they can reconstitute lung
structure for regeneration purpose. Induced pluripotent stem
cells (iPSCs) could be another source of “self” stem cells for
autologous transplantation and indeed, iPSCs have been
successfully coaxed to alveolar and airway lineage in vitro
(Huang et al., 2014). However the capability of iPSC-derived
cells to generate real lung structure and their tumorigenic
risk remains to be evaluated in vivo (Kotton and Morrisey,
2014). To this end, tissue-resident progenitor cells from an
Protein & Cell
adult’s own lung—if can be identified, isolated and expanded
—can be a new option for transplantation therapy.
In adult rodent, different populations of lung stem/pro-
genitor cells have been identified in last decade with capa-
bility to reconstruct lung epithelium. Most of the mouse lung
stem/progenitor cells are facultative and can be induced to
proliferate in response to injury as well as differentiate into
one or more lung cell types (Kotton and Morrisey, 2014; Kim
et al., 2005; Barkauskas et al., 2013; Hogan et al., 2014;
Desai et al., 2014). More recently, we and others found a
rare population of p63+/Krt5+ distal airway stem cells
(DASCs), which play essential role in murine lung repair after
influenza-induced acute injury (Zuo et al., 2015; Vaughan
et al., 2015). However in adult human, whether there are
lung cells with regenerative capacity in vivo need to be
explored. Given the huge differences between human vs.
mouse of their respiratory systems in terms of develop-
mental process, lung lobulation, branching pattern and cell
composition, the identity of human lung progenitor cells need
to be rigorously evaluated.
In the current work, we discovered the putative adult
human lung progenitor cells located at the bottom of “rugaes”
in airway epithelium, with a SOX9 marker to distinguish them
from other SOX9−/P63+/KRT5+ airway basal cells (BCs).
From a trace amount of bronchoscopic brush-off lung tis-
sues, we isolated SOX9+ BCs and expanded them in vitro
indefinitely. SOX9+ BCs transplanted into injured immune-
deficient mouse lung can regenerate functional lung
epithelium with both human bronchiolar and alveolar
epithelium reconstituted. Most importantly, for the first time
we explored the clinical feasibility of autologous SOX9+ BC
transplantation to treat two patients with chronic lung dis-
eases. The clinical trial result is highly consistent with our
observation on mouse model, and making it a solid basis for
future large-scale clinical study.
Qiwang Ma et al.
RESULTS
Bronchoscopic isolation of clonogenic airway basal
cells
In current study, we worked on the P63+/KRT5+ BCs in the
airway epithelium of human lung which could possibly be the
counterpart of mouse DASC. The workflow of BC isolation
and expansion is summarized in Fig. 1A. Approximately
20,000–30,000 cells were brushed off from the luminal sur-
face of donor’s 3rd–4th order bronchus using a 2-mm
bronchoscopic brush (Wimberley et al., 1982) (Fig. 1B). The
brushed-off cells were seeded onto embryo-derived feeder
cells with the culture medium favoring BC growth (Zuo et al.,
2015; Wang et al., 2015). After seeding 5,000 live cells onto
6-well plate, 9 (±2) cells grew up into visible tight colonies 3–
5 days later with expression of human nucleus specific
antigens, lung progenitor marker NKX2.1 and proliferation
marker KI67 (Figs. 1C and S1A). All of the P0 colonies were
confirmed epithelium origin (E-cadherin+, Fig. S1A) and
stained double positive for airway basal cell markers KRT5
and P63 (Fig. 1C and 1D). We did not observe any P63
single positive colonies (Vaughan et al., 2015). Considering
that BCs take for about 20% of total cell number in brushed
samples of 3rd–4th order bronchus, it appeared that
approximately 1% of the BCs in human airway could be
clonogenic lung epithelium progenitors.
We seeded one single BC onto feeder cells and grew them
into one single colony which was then picked up by cloning
cylinders and passaged continually. The latest passage of
BC clones had gone through 50 doublings (=1015 fold
expansion) in our lab. The single cell-derived BC clones and
their original brush-off tissue samples were analyzed by high-
throughput RNA sequencing (RNA-Seq). On average, we
detected 16,230 genes and 25,223 transcripts. Thus, more
than 60% of known human genes and transcripts were
expressed in clonogenic BCs. Gene expression value cor-
relation analysis showed that the clone transcriptome profiles
are distinct from their original brush-off tissues, but the two
clones from two independent persons share very similar
transcriptome (Fig. 1E, Pearson correlation coefficient =
0.95). Single nucleotide polymorphism (SNP) analysis
showed that BC clones have around 70% less polymorphism
comparing to the brush-off tissues, which is in consistency
with their single cell origination. High expression of BC
markers (KRT5, P63, NGFR and S100A) and another puta-
tive mouse stem cell marker integrin α6β4 (Chapman et al.,
2011) were observed in clones. In contrast, clonogenic BCs
do not express other bronchial or alveolar lineage markers as
shown by RNA-Seq and confirmed by immunostaining
(Figs. 1F and S1B). Protein-protein interaction analysis of
overexpressed genes indicated three major signal molecule
networks including Notch1/2/3, FGF10/7 and Wnt7 ligand
and their downstream components. All three signaling
© The Author(s) 2018. This article is an open access publication
Regeneration of human lung RESEARCH ARTICLE

A B

Br Brush

Brush
Bronchoscopic Basal cell clones on Pedigree clone
brushing-off cells feeder cells from a single Feeder-free expansion
cells
C D
Br

Br
Br

KRT5 P63 KRT5 P63 CC10

GO term enrichment

Protein & Cell

E F G FGFBP1 H
Pearson correlation level (clone vs. brush)
of transcriptome ITGB4
ITGA6 FGF1 FGFR3 -4 -2 0 2 4 6 8
Brush-1 JAG2 HSPG2
TP63
NGFR NOTCH3JAG1 NOTCH1 Laminin complex
Brush-2 FGFR4 FGF7
MKI67
FGF10 Basement membrane
Clone-1 KRT5 DLL1 NOTCH2
S100A2 MAML1 Epithelium development
Clone-2 TUBA1A
Wnt receptor signaling pathway
SCGB1A1 HES5 HES1 BACE1
C e-1
-2
Br h-1
C h-2

SCGB3A1 FZD8 FZD1 Organ morphogenesis

ne
n
us
us

FOXJ1
lo
lo

LRP6 LRP5
Br

Cell proliferation
MUC5AC
WNT7A WNT7B
0.8
0.6
0.4
0.2

Secretion
-2

-1
1

2
1

h-

h-

CTNNB1
ne

ne

Cilium
us

us

lo

lo
Br

Br

Figure 1. Isolation and characterization of BCs from SOX9+ human airway. (A) Diagram showing the process of clonogenic BCs
isolation and expansion. (B) Bronchoscopic image showing brushing of cells from human airway. (C) Left, BC colonies grown on
feeder cells; right, anti-KRT5 and anti-P63 immunostaining of BC colonies with nuclei counterstain. Human sample number n = 10.
Scale bar, 100 μm. (D) Left, BCs in human airway by anti-KRT5 and anti-P63 immunostaining. Inset, high magnification with club cell
(CC10+, cyan color) costaining; right, hematoxylin & eosin staining of the same section. Br, bronchus. Scale bar, 100 μm.
(E) Heatmap showing transcriptome profile correlation value of BC clones and brush-off tissues. (F) Expression heatmap of selected,
differentially expressed genes (P < 0.05) comparing BC clones and brush-off tissues. (G) Protein-protein interaction network of
selected genes with high expression level in BC clones. (H) Enriched gene ontology classes of BC clones versus brush-off tissues.

networks are previously known to play essential roles in
embryonic lung development (Bellusci et al., 1997; Rajagopal
et al., 2008; Tsao et al., 2008) (Fig. 1G). Gene ontology (GO)
term analysis demonstrated critical biological processes
enriched in BCs (Fig. 1H).
Clonal analysis of SOX9+ BCs
Importantly, RNA-Seq data also showed that clonogenic BCs
highly express SOX9 (Sex Determing Region Y- Box 9), a
transcriptional factor known to be enriched in branching tips
of developmental lung. In embryonic development, SOX9
activity is required to maintain the undifferentiated status of
distal lung progenitor and disruption of SOX9 function pre-
vents adult alveoli formation (Perl et al., 2005; Rockich et al.,
2013). Here we confirmed SOX9 expression in P63+/KRT5+
BC clones by immunostaining (Fig. 2A). Accordingly, by
histological examination of human 2nd order (Fig. S2) and
3rd–4th order airway (Fig. 2B), we observed 1.3% ± 0.3%
and 1.7% ± 0.5% SOX9-expressing P63+ BCs, respectively.
The proportion of SOX9+ cells in total BCs is very close to
our estimation in clonogenic assay as mentioned above
(∼1%), suggesting SOX9 as a marker to distinguish clono-
genic BCs vs. other non-clonogenic BCs. Interestingly, we
noticed that there are a few invaginations (rugaes) in 2–4
order human airway epithelium and the SOX9+ BCs are
exclusively located near the base of the rugaes. There are
averagely 3 (±1) SOX9+ BCs in each individual rugae. Fur-
ther immunofluorescent examination showed a very small
portion of them (<1%) are proliferative (KI67+) (Fig. 2C). Of

© The Author(s) 2018. This article is an open access publication

 RESEARCH ARTICLE Qiwang Ma et al.

A B C
Br Br
Br

P63 KRT5 SOX9 SOX9 P63 P63 CC-10 SOX9 Ki-67 P63

D Feeder-free F H

es
es
Human lung

asg
sag
Relative expression

ng
1.5 Early passages

ass
n lu
Late passasges

pas

ep
ma

rly
1.0

Lat
Hu

Ea
0.5 P63
SOX9
0.1
KRT5
H 5
PX

LA B

G 3

M 1
1
Protein & Cell

SC MP

A
C
SP
SP
AQ

B1

U
O

MUC1
E G Acet-a-Tub
200
Relative expression

150 Human lung CC-10

1 2 3 4 5 100 Early passages
Late passasges SPB
50
SPC
6 7 8 9 10 11 12
10 LAMP3
13 14 15 16 17 18 5 HOPX
0 AQP5
T5

63

X9

19 20 21 22 X Y GAPDH
TP
KR

SO

Figure 2. Feeder-free expansion of SOX9+ BCs. (A) Immunostaining of SOX9+ BCs with anti-P63, anti-KRT5 and anti-SOX9
antibodies. (B) SOX9+ BCs in rugae of 3rd order human airway by anti-SOX9, anti-P63 and anti-CC10 immunostaining. Scale bar,
100 μm. (C) SOX9+ BCs in rugae of 3rd order human airway by anti-KI67 immunostaining. (D) BC colony cultured on feeder-free
condition. (E) Karyotyping of cultured BCs. (F) qPCR showing alveolar and bronchial epithelium marker gene expression of human
lung sample and SOX9+ BCs in early (P2) and late (P8) passages. n = 3, biological replicates. Error bars, S.E.M. (G) qPCR showing
progenitor cell marker (Krt5, P63 and SOX9) gene expression of human lung sample and SOX9+ BCs in early (P2) and late (P8)
passages. n = 3, biological replicates. Error bars, S.E.M. (H) Western blotting showing marker gene expression of human lung sample
and SOX9+ BCs in early (P2) and late (P8) passages.

note, SOX9+ BCs can also be isolated and expanded from We further analyzed SOX9+ BCs at single cell resolution.
those small airway (∼1 mm diameter) samples, which is 5 single cells from one person in normal group were selected
accessible by open-chest surgery or autopsy but not by at Passage 0 and expanded to Passage 1 and Passage 2.
bronchoscopy (data not shown). Great variation of their clonogenic capacity was observed at
The whole brushing sampling and SOX9+ BCs cloning Passage 1 (coefficient variation = 59.9%) and Passage 2
procedure was carried out on 15 individuals with a recovery (coefficient variation = 75.7%). Similar clonogenicity varia-
rate of 100%. Donors are from 4 different disease categories tion was observed in individuals from other disease cate-
including 5 normal healthy volunteers, 2 bronchiectasis gories and the average coefficient variation of all clones is
patients, 3 chronic COPD patients, and 5 interstitial lung 52.2% (Fig. S3C).
disease (ILD) patients with pulmonary fibrosis. The SOX9+ SOX9+ BCs grown on feeder cells can be transferred onto
BCs from different categories of diseases showed no petri dish pre-coated with collagen fibers for feeder-free
apparent difference in colony morphology (Fig. S3A) or culture. The feeder-free cultured SOX9+ BC can also form
marker expression (Fig. S3B). Their clonogenic efficiency colonies though their cell-cell contact within one colony is
seemed similar—but still need future investigation in much less tight comparing to those on feeders (Fig. 2D). The
larger cohort to get statistically meaningful conclusion. feeder-free cultured BCs are able to be passaged for at least
30 doublings with no obvious morphology change.

© The Author(s) 2018. This article is an open access publication

Regeneration of human lung
Karyotyping indicated their stable genetic characteristics
along with passaging (Fig. 2E). Quantitative analysis of
progenitor markers (KRT5, P63 and SOX9) and lung
epithelium lineage markers at both RNA and protein level
indicated that there is no spontaneous differentiation of BCs
in the culture process (Fig. 2F–H).
+
Xeno-transplanted SOX9 BCs give rise to human lung
in vivo
Next we examined whether the SOX9+ BC could differentiate
and regenerate lung tissue by transplanting such cells into
mouse lung parenchyma. Firstly, immunodeficient NOD-
SCID mice were subjected to bleomycin intratracheal instil-
lation, which lead to rapid onset (8 days after bleomycin)
damage of centrilobular and surrounding regions as shown
by microCT-scan and immunostaining. Masson trichrome
staining for collagen and α-SMA immunostaining indicated
severe tissue fibrosis of mouse lung at later time points
(Zhang et al., 1996) (Fig. S5A–C). Scarce endogenous
mouse p63+/Krt5+ distal airway stem cell expansion was
observed in damaged lung parenchyma as reported previ-
ously (Vaughan et al., 2015) (Fig. S5D). Then we intratra-
cheally delivered (Zuo et al., 2015) 1 × 106 GFP-labeled
SOX9+ BCs into the injured mouse lung and analyzed the
lung 3 weeks after transplantation. As shown in Fig. 3A, we
observed large-scale incorporation of GFP+ human SOX9+
progenitors and their progeny into mouse lung. Direct fluo-
rescence after tissue sectioning showed distribution of GFP+
human cells in mouse distal lung, some of them are mor-
phologically indistinguishable from neighboring GFP− mouse
lung structures (Fig. 3A). The chimerism of human-mouse
lung was further confirmed by human-specific nucleus anti-
gen Lamin A+C co-staining with GFP (Fig. 3B) and qPCR
with human specific GAPDH primers (Fig. S6A). A few fully
differentiated human cells have lost SOX9 marker expres-
sion and form air-sacs of similar size to mouse alveoli with
AEC1 marker (AQP5 and HOPX) expression (Fig. 3C–E).
Some transplanted GFP+ human cells could also incorporate
into bronchiolar region of lung, where some of them gave
rise to Club cell with CC10 marker expression while a few
others became ciliated cells (acetylated-tubulin+, FOXJ1+),
respectively (Fig. S6B–D). However, we hardly observed
human SPC+ AEC2 in transplanted mouse lung. The differ-
entiation potential of SOX9+ BCs was further confirmed by
qPCR analysis of multiple marker genes with human specific
primers. Both AEC1 and bronchiolar cell marker genes were
strongly expressed in the chimera. For AEC2 marker genes,
though SPB and LAMP3 were highly expressed, we did not
detect SPC expression in the chimera, which was consistent
with the immunostaining result (Fig. 3F).
In control experiment, we found that the transplanted
SOX9+ progenitors cannot incorporate into non-injured
healthy mouse lung or porcine pancreatic elastase-injured
mouse lung (data not shown). Also, human lung-derived
© The Author(s) 2018. This article is an open access publication
RESEARCH ARTICLE
fibroblast cells (data not shown) or human cervix-derived
P63+/KRT5+/SOX9+ progenitor cells (Figs. 3G and S6E) can
barely incorporate into injured mouse lung either. This data
indicated the tissue specificity of different adult stem/pro-
genitor cells.
Regenerated lung by SOX9+ BC transplantation
contributed to mouse pulmonary function
Functional alveolar unit requires close epithelium-capillary
interaction for exchange of gas, energy and other sub-
stances. In the optically cleared mouse lung, we observed
branching major blood vessels in transplanted mouse lung
(Fig. S7A). We also found that the thin, long-shape human
AEC1 aligned together with microvascular vessels which are
positive for capillary endothelial markers CD34 and PECAM/
CD31, with approximately 1 μm-thick integrinβ-1+ basement
Protein & Cell
membrane between epithelium and capillary endothelium
(Fig. 4A–C). And engrafted GFP+ human cells form adhe-
rens junctions and tight junctions with neighboring alveolar
epithelial cells as shown by E-cadherin and ZO-1 staining on
the border (Fig. 4D and 4E), which makes a closed space to
maintain air pressure. In order to examine whether such
blood-gas exchanging units are functionally connected with
circulation, we developed a gold nanoparticle (AuNP)
(Cheng et al., 2008)-based approach to mimic gas exchange
and transport in vivo. The nanoparticles can be transported
in blood and diffuse across cells (like O2 and CO2) due to its
small size (∼5 nm), water solubility and lipophilicity, and
meanwhile can be detected by histology. One hour after
injection of AuNPs into mouse tail vein, we detected signif-
icant gold signal in healthy mouse alveoli (Fig. S7B) as well
as in GFP+ human alveoli (Fig. 4F), indicating the regener-
ated human tissues are functionally linked with circulation
system. On the other side, after intratracheally aspiration of
AuNPs, some GFP+ part of mouse lung showed significant
gold signal, indicating the regenerated human tissues are
anatomically linked with atmospheric air (Figs. 4G and S7C).
As control, no or very little AuNPs signal was observed in
damaged alveolar area by either way of particle delivery
(Fig. S7B and S7C). These evidences implicated that the
regenerated lung tissue has vascularized gas-exchange
capacity, probably through recruitment of self-organizing
capillary endothelial cells by SOX9+ BCs.
We also found that SOX9+ BC transplantation effectively
blocked the progression of mouse pulmonary fibrosis mani-
fested as fibronectin accumulation and α-SMA positive
myofibroblast expansion (Phan, 2012) in the human cell-
enriched area (Fig. 5A and 5B), suggesting that regenerated
human lung can replace damaged tissue in mouse model.
Accordingly, alveoli regeneration by SOX9+ BC transplan-
tation also improved the recipient mouse pulmonary function
as shown by the decrease of CO2 partial pressure, increase
of O2 partial pressure and O2 saturation in artery blood
(Fig. 5C–E).
 RESEARCH ARTICLE Qiwang Ma et al.

A B
- SOX9+ BCs HuLamin GFP

+ SOX9+ BCs

C D E
GFP SOX9 HuLamin AQP5 SPC

AIv AIv

AQP5 HOPX
F +
SOX9 BC Chimera G
Protein & Cell

800 SOX9+ BC
Human gene expression

600
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400 from lung

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200
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Figure 3. Transplantated SOX9+ BCs regenerate functional human lung in vivo. (A) Left, direct fluorescence image under
stereomicroscope showing NOD-SCID mouse lung without (upper panel) or with (lower panel) GFP-labeled SOX9+ BC
transplantation. Right, cryo-section and direct fluorescence imaging of transplanted GFP-labeled SOX9+ BCs in lung parenchyma.
Scale bar, 100 μm. (B) Immunofluorescence imaging of transplanted GFP-labeled SOX9+ BCs in lung parenchyma with human
specific Lamin A+C marker costaining. (C) Fully differentiated GFP+ cells lost SOX9 marker expression (arrowhead indicated). Scale
bar, 10 μm. (D) Confocal image with human specific Lamin A+C immunostaining (HuLamin) showing regenerated type I (AQP5+)
alveolar cells. No type II (SPC+) cells were observed. (E) Confocal image showing regenerated AEC1 (AQP5+ and HOPX+). AQP5 as
a membrane-bound protein distributes on surface of GFP+ cells. Arrowheads indicated the overlay of HOPX with GFP signal in
nucleus. Scale bar, 20 μm. (F) qPCR with human specific primers showing alveolar and bronchiolar epithelium marker gene
expression in SOX9+ BC transplanted chimeric lung (AEC1: AQP5 and HOPX; AEC2: SPB and LAMP3; bronchiolar cells: SCGB1A1
and MUC1). Biological replicates, n = 3. Error bars, S.E.M. (G) Left, clonogenic BCs isolated from human cervix epithelium obtained
by biopsy. Right, transplantation of equal numbers of BCs from lung and cervix indicated different incorporation efficiency.

TGF-β signaling modulates SOX9+ BC proliferation by TGF-β treatment together with mild change of some other
cell cycle-related genes (Fig. 6F). TGF-β had little effect on
To further improve the transplantation efficiency of SOX9+
the apoptosis of SOX9+ BCs (Fig. S8B). Collectively these
BCs, we screened multiple drugs and found Pirfenidone, an
experiments showed that the TGF-β/SMAD/P15 signaling
FDA approved anti-pulmonary fibrosis drug (King et al.,
axis could effectively modulate SOX9+ BC proliferation.
2014) could facilitate the SOX9+ BC transplantation effi-
Similar proliferation inhibitory effect of TGF-β/SMAD was
ciency significantly. Interestingly, transforming growth factor-
recently reported on TBC as well (Mou et al., 2016).
β (TGF-β) had the opposite effect (Figs. 6A and S8A). This
discovery prompted us to study the underlying molecular and
cellular mechanism. We found that Pirfenidone treatment Autologous SOX9+ BCs transplantation clinical trial
can abolish TGF-β-induced phosphorylation of SMAD2/ in bronchiectasis patients
SMAD3 (Fig. 6B). In turn, TGF-β treatment significantly
Bronchiectasis is a chronic lung disease radiographically
suppressed the clonogenicity and cell viability of SOX9+
characterized by permanent pathologic dilation of the small
BCs, which can be rescued by the SMAD2/SMAD3 inhibitor
and medium-sized bronchi, which may lead to respiratory
SB-431542 (Fig. 6C–E). Simutaneously, the expression of
failure and eventually to death. Patients with bronchiectasis,
p15(INK4B), a G1 cell cycle inhibitor, was strongly induced

© The Author(s) 2018. This article is an open access publication

Regeneration of human lung RESEARCH ARTICLE

A GFP CD34 B GFP CD34

Bv Bv
AIv AIv

C D E
GFP CD31 ITGB1 GFP CD31 E-Cad GFP ZO-1

AIv

AIv AIv
AIv

Bv

Protein & Cell

F GFP Au NP G GFP Au NP

Figure 4. Regenerated alveoli with functional epithelium-capillary system. (A) Transplanted SOX9+ BCs (anti-GFP) and
capillary endothelium marker (anti-CD34). Scale bar, 100 μm. (B) Confocal image of SOX9+ BCs regenerated alveoli (Alv) and the
neighboring capillary blood vessel (Bv). Left, immunofluorescence; right, bright field. Scale bar, 20 μm. (C) Confocal image showing
the basement membrane (ITGB1+, white color, arrowhead indicated) between regenerated alveoli epithelium and capillary
endothelium (CD31+). Scale bar, 10 μm. (D) Confocal image showing the cell adherens junction (E-cadherin+, white color) between
regenerated alveoli epithelial cells. Scale bar, 20 μm. (E) Confocal image showing the cell tight junction (ZO-1+) between regenerated
alveoli epithelial cells. Scale bar, 20 μm. (F) Direct fluorescence image of the transplanted GFP-labeled SOX9+ BCs (green) and
bright-field image of tail vein delivered gold nanoparticles (AuNPs) of the same region (brown). Scale bar, 100 μm. (G) Direct
fluorescence image of the transplanted GFP-labeled SOX9+ BCs (green) and bright-field image of intratracheally delivered gold
nanoparticles (AuNPs) of the same region (brown). Scale bar, 100 μm.

if left untreated, will have a continual decrease of their pul-
monary function. Current pharmacological strategies to treat
bronchiectasis such as antibiotics, mucolytics and anti-in-
flammatory agents could only control the disease exacer-
bation but not improve the pulmonary function nor repair the
damaged lung tissue (ten Hacken et al., 2007). To explore
the clinical feasibility of autologous SOX9+ BC transplanta-
tion, we conducted a pilot trial aiming to treat bronchiectasis
by regenerating functional human lung. The general trial
protocol and the cell manufacturer (Regend Therapeutics
Co.Ltd) were archived by China Food and Drug Adminis-
tration (CFDA) and National Health and Family Planning
Commission of China, and the trial was performed in national
approved stem cell clinical research institute (Southwest
Hospital) after strict ethic commission review of preclinical
data (A part of but not all preclinical data was released in the
current manuscript).
Two patients diagnosed as non-CF bronchiectasis were
firstly enrolled for autologous SOX9+ BC transplantation on
April, 2016. Both patients are men in 50s, non-smokers.
Patient 1 was diagnosed as bronchiectasis 8 years ago with
productive cough and dyspnea on exertion symptom, which
worsens continually under regular pharmacological treat-
ment. CT scan shows multiple bronchial cylinder dilation and
patchy consolidation in his lung. Patient 2 was diagnosed as
bronchiectasis and COPD decades ago, with productive
cough and dyspnea on exertion symptom, which worsens
continually under regular pharmacological treatment. CT
scan shows multiple bronchial cystic dilation, thicken bron-
chial wall and patchy consolidation in his lung.
For both patients, tissues were bronchoscopically col-
lected from random region of left upper lobe and right upper
lobe and transported to GMP (Good Manufacture Practices)
level tissue culture facility for SOX9+ BC isolation and

© The Author(s) 2018. This article is an open access publication

 RESEARCH ARTICLE Qiwang Ma et al.

A - SOX9+ BCs + SOX9+ BCs B + SOX9+ BCs

FN1 GFP α-SMA

C D E
15 pCO2 20 pO2 100 * sO2

Fraction (%)
15 *
10
80
kPa

kPa
10
5
5 60
0 0
al

al

s
BC

BC

al

s
PB

PB
m

BC
PB
m
or

or
Protein & Cell

or
+

+
X9

X9
N

X9
eo

eo

N
SO

SO

eo
Bl

Bl

SO
Bl
+

+
eo

eo

eo
Bl

Bl

Bl
Figure 5. BC transplantation rescued mouse pulmonary function. (A) Injured mouse lung without or with GFP-labeled SOX9+
BCs transplantation by anti-GFP and anti-Fibronectin co-staining. Scale bar, 200 μm. (B) Left, immunofluorescence image of injured
mouse lung transplanted with GFP-labeled SOX9+ BCs; right, immunostaining on the same section showing exclusion of α-SMA+
myofibroblasts from GFP+ area. Scale bar, 200 μm. (C) CO2 partial pressure of mouse arterial blood before and 1 month after
bleomycin-induced injury with or without SOX9+ BCs transplantation. Each dot indicates an individual mouse. (D) O2 partial pressure
of mouse arterial blood 1 month after bleomycin-induced injury with or without SOX9+ BCs transplantation. Each dot indicates an
individual mouse. (E) O2 saturation of mouse arterial blood before and 1 month after bleomycin-induced injury with or without SOX9+
BCs transplantation. Each dot indicates an individual mouse.

expansion (Fig. 7A). Isolated SOX9+ BCs were cultured on
clinical-level feeder cells and then shifted to feeder-free
culture condition. Totally 1 × 106/kg body weight of SOX9+
BCs were infused into distinct lobes of patients through
bronchoscopy (Tzouvelekis et al., 2013) (Fig. 7B). Clinical
status of patients was evaluated 1 day before and 1, 3 and
12 months after cell transplantation. Although it is almost
impossible to directly track unlabelled transplanted cells in
human, we did observe regional repair of cystic dilation after
cell transplantation by high-resolution computed tomography
(HRCT) scan for Patient 2 (Fig. 7C). The thickened bronchial
wall also became thinner after cell therapy for Patient 2.
Spirometry results indicated remarkable recovery of pul-
monary function in both patients after transplantation as
measured by FEV1, FVC and DLCO/VA (Fig. 7D). Impor-
tantly, no aberrant cell growth or other related adverse
events were observed during the whole follow-up time. In the
last follow-up (20 months after transplantation), Patient 1
described improvement of dyspnea, improvement of exer-
cise capacity, less productive cough and less times of
exacerbation after cell therapy; Patient 2 described less
productive cough and less times of exacerbation after cell
therapy. As it is well documented that bronchiectasis is a
permanent, irreversible disease that cannot resolve
spontaneously or with regular medicine, the recovery of
patients suggested high probability that transplanted SOX9+
BCs were able to regenerate functional lung in human, which
is consistent with our observation in animal models. And we
will continue life-long observation on the two patients.
DISCUSSION
In the current study, we revealed that a small population of
SOX9+ BCs in adult airway can regenerate human lung
epithelium. A few SOX9+ BCs brushed off from human air-
ways can be expanded to sufficient number in feeder-free
condition and transplanted into injured mouse lung to
regenerate human air exchanging units. In unperturbed lung,
the SOX9+ BCs are located in the base of airway epithelium
invagination, which is reminiscent of intestinal stem cells
residing in gut crypt compartments. However unlike the
intestinal stem cells which are constantly in active cell cycle
to replenish gut epithelium in a fast turn-over mode (Clevers,
2013), the SOX9+ BCs in airway are quiescent most of the
time, and could only be activated once a particular injury
signal from more distal lung was received. Like its counter-
part p63+/Krt5+ BCs in mouse, endogenous SOX9+ BCs
might migrate towards inflamed parenchymal region and

© The Author(s) 2018. This article is an open access publication

Regeneration of human lung RESEARCH ARTICLE

A B
TGF-β - + - +
0.49 ± 0.024 0.00 ± 0.00 0.69 ± 0.042
Pirfenidone - - + +
P-Smad2/3

Smad2/3
C D

Clonony formation units

30 **
25
20
15
10
5
**
0
- TGF-β + TGF-β 1 2 3
DMSO TGF-β
E F
Cell viability assay 50 0h
40
Absorbance of OD450

30 12 h

**
0.8 20

**
10 24 h
0.6 5
Relative expression

**
48 h

Protein & Cell

0.4 *
*
0.2 0
**
0.0 * *

**
* *

**

**
TGF-β - + - +

**

***
SB-431542 - - + + -5

**
5

A1

E2

K4

A
P1

P2

N
D

PC
lin

lin

C
yc

yc
C

C
Figure 6. TGF-β signaling modulates SOX9+ BC proliferation. (A) Direct fluorescence image of mouse lung transplanted with
1 × 106 GFP-labeled SOX9+ BCs under dissection microscope. Each lung was from mouse with indicated treatment and harvested 7
days after transplantation. The left lobes were analyzed and the GFP+ cell numbers (×106) were counted by flow cytometry analysis.
Biological replicates, n = 3. PFD, Pirfenidone. (B) SOX9+ BCs were stimulated with 10 ng/mL TGF-β for 2 h, with or without 1 mg/mL
Pirfenidone treatment overnight. Western blotting of cell lysates with anti-phosphated-Smad2/3 and anti-total Smad2/3 antibodies
was performed to examine the activation of TGF-β pathway. (C) Direct fluorescence imaging of GFP-labeled SOX9+ BCs cultured in a
6-well plate in the absence or presence of 10 ng/mL TGF-β. Scale bar, 200 μm. (D) Quantification of clonogenicity of SOX9+ BCs in
the presence of 10 ng/mL TGF-β or 10 mmol SB. SB, TGF-β type I receptor inhibitor SB-431542. Technical replicates n = 3. (E) WST
viability assay of SOX9+ BCs treated by 10 ng/mL TGF-β or 5 mmol TGF-β inhibitor SB-431542, or their combination. Technical
replicates n = 3. (F) qPCR showing cell cycle-related gene expression level of SOX9+ BCs with 10 ng/mL TGF-β treatment for
indicated h. Biological replicates, n = 3.

rebuild the respiratory tree by proliferation and differentiation. whether SOX9+ BCs are the remains of previously docu-
We and previous studies found that there are P63+ and/or mented SOX9+ progenitors in embryonic lung. In embryonic
KRT5+ cells enriched in the damaged alveolar region of lung, SOX9+ embryonic progenitors are enriched in distal
many lung disease patients (including those with COPD, bud tips of respiratory tree at canalicular stage (Perl et al.,
pulmonary fibrosis and bronchiectasis) (Chilosi et al., 2002; 2005). Interestingly, previous report demonstrated that
Asano et al., 2011; Smirnova et al., 2016), which could be intravenous transplantation of human canalicular-stage
the progeny of endogenous SOX9+ BCs in the middle of embryonic lung cell mixture into NOD-SCID mice can give
differentiation process. Interestingly there was almost no rise to chimeric lung (Rosen et al., 2015). Similarly, a more
SPC+ AEC2 generated in our xeno-transplantation model, recent report showed mouse SOX9+ progenitors in embry-
which suggested the limitation of the SOX9+ progenitor onic lung can be grown in vitro as organoid and transplanted
potency and also the possibility that the traditional concept of to generate mouse alveoli (Nichane et al., 2017). Altogether
human AEC2 as the progenitor of AEC1 may not be correct these work supported the concept that SOX9+ cells are lung
in this circumstance. progenitors in both embryonic and adult lung.
The pattern that SOX9+ BCs adopt to regenerate the One important technical advance we bring out in this work
respiratory tree resembles the natural development process is the system to selectively expand SOX9+ BCs in a feeder-
of lung in gestation, which raises an open question as free condition. Previously through bronchoscopic brushing

© The Author(s) 2018. This article is an open access publication

 RESEARCH ARTICLE Qiwang Ma et al.

A B
Patient 1 Patient 2

1.6/0.67
2.0/1.1

1.6/0.67
3.2/ -/0.67
1.6

C
Before
Protein & Cell

After

D
FEV1 (L) FVC (L)
Months post 0 1 3 12 0 1 3 12
transplantation
Patient 1 1.07 (36.6%) 0.94 1.40 1.29 2.56 (69.4%) 2.15 2.93 2.73
Patient 2 0.51 (20.8%) 0.85 0.81 0.71 0.73 (24.3%) 1.85 1.88 1.65
DLCO/VA (mmol/min/kPa/L)
Months post 0 1 3 12
transplantation
Patient 1 1.77 (128.2%) 1.88 1.66 1.60
Patient 2 0.21 (14.9%) 1.47 1.36 1.49

Figure 7. Autologous SOX9+ BC transplantation: a pilot clinical study. (A) Cultured SOX9+ BCs from two patients of
bronchiectasis. (B) Diagram showing the number of SOX9+ BCs (×107) transplanted into each lobe of Patient 1/Patient 2. Five lobes
are labeled with different colors. Note the Patient 1 undertook left lower lobectomy a decade ago. (C) Representative images of
consecutive CT scan show the regional recovery of bronchiectasis (yellow square) 1 year after autologous SOX9+ BC transplantation
in Patient 2. (D) Measurement of pulmonary function and exercise capacity in both patients: FEV1 (forced expiratory volume in 1
second), FVC (forced vital capacity), DLCO/VA (diffusing capacity of the lung for carbon monoxide adjusted by the alveoli volume)
were shown as absolute value and percentage to predicted level. For each parameter, percentage >80% was regarded as clinically
normal.

followed by routine basal cell culture, Crystal et al., obtained
P63+/KRT5+/SOX7/15/4+ basal cells but not the rare SOX9+
subpopulation (Hackett et al., 2011) . Here from as few as
one single SOX9+ BC, we can expand it to 5 × 107 purely
undifferentiated cells within 3–4 weeks. Therefore we can
acquire a homogeneous population of regenerative cells with
uniform characteristics, which is crucial for cell quality control
in further clinical application.
Most importantly, we showed that SOX9+ BCs cultured
under GMP guidelines can be applied clinically in order to
reconstitute human lung for devastating chronic lung disease
treatment. To the best of our knowledge, this is the first
successful attempt to regenerate human large inner organ
based on cell replacement strategy. As demonstrated in
hematopoietic, skin and corneal regeneration field, autolo-
gous stem/progenitor cell transplantation strategy has been
successfully applied to treat multiple devastating diseases.

© The Author(s) 2018. This article is an open access publication

Regeneration of human lung
We show here that supplement of expandable SOX9+ BCs
by autologous transplantation could repair the damaged lung
in two bronchiectasis patients in both pulmonary structure
and function. Generally the bronchiectasis patients without
medical intervention will deteriorate over time, thus the
recovery of lung function and structure after cell therapy
suggested the efficacy of this strategy, and established a
basis for future trials. As the SOX9+ BCs are derived from
patients’ own airway, it is a recapitulation and augmentation
of naturally occurring lung repair process. However after all,
our pilot clinical trial is a very preliminary exploratory study
so the safety and efficacy of the current strategy still need
additional verification in a much larger cohort. Although we
have never observed aberrant growth of SOX9+ BCs in
NOD-SCID mouse model, longer time follow-up on the
patients is still necessary to fully eliminate the tumorigeneic
possibility. We have demonstrated the differentiation poten-
tial of SOX9+ BCs in mouse model, but the exact fate of
transplanted SOX9+ BCs in human lung remains to be pro-
ven with future development of non-invasive cell tracking
techniques. Furthermore, we have demonstrated that SOX9+
BCs derived from normal or diseased people can both give
rise to multiple lineages of lung epithelial cells, but detailed
quantitative comparisons of the regenerative capacity
between normal and diseased persons would require a lar-
ger sample size investigation in future.
In conclusion, our study clearly shows the capability of
SOX9+ BCs to regenerate human lung, proves the concept
of chronic lung disease treatment by SOX9+ BC transplan-
tation and provides exciting translational opportunities in
near future.
METHODS
Human tissue collection
Patients without or with chronic lung diseases (COPD, bronchiec-
tasis and ILD) were diagnosed by ATS/ERS criteria. All individuals
went through thorough medical examination before sampling. The
bronchoscopic procedure for sampling was performed by board-
certified respiratory physicians using a flexible fiber-optic broncho-
scope (Olympus, Japan). Before the bronchoscopy, oropharyngeal
and laryngeal anesthesia was obtained by administration of 2 mL of
nebulized 4% lidocaine, followed by 1 mL of 2% topical lidocaine
sprayed into the patient’s oral and nasal cavities. After the bron-
choscope was advanced through the vocal cords, 2 mL of 2%
lidocaine solution was instilled into the trachea and both main
bronchi through the working channel of the bronchoscope. Then a
disposable 2-mm brush was advanced through the working channel
of the fiberoptic bronchoscope and used to collect airway epithelial
cells by gently gliding the brush back and forth 1 or 2 time in random
regions of trachea or 3–4 order bronchi in the right or left lobe. No
obvious differences were observed between the BC clones isolated
from 3rd vs. 4th order bronchi, or from different lung lobes. For bulk
lung sampling, normal human lung bulk samples were collected from
unaffected lung area of lung cancer patients with open chest sur-
gery. All the human tissues were obtained following clinical SOP
© The Author(s) 2018. This article is an open access publication
RESEARCH ARTICLE
under patient’s consent and approved by Southwest Hospital Ethics
Committee (Chongqing, China) and Shanghai East Hospital Ethics
Committee (Shanghai, China).
Isolation and culture of human SOX9+ BCs
To isolate the SOX9+ BCs, 2 mm brush with samples were cut with
scissors into 1 cm pieces. After removing sputum, the brush pieces
were directly digested with dissociation buffer including DMEM/F12
(Gibco, USA), 2 mg/mL protease XIV (Sigma, USA), 0.01% trypsin
(Gibco, USA) and 10 ng/mL DNase I (Sigma, USA). Specimens
were incubated at 37°C for an hour with gentle rocking. Alternatively,
human small airway were dissected from a bulk of lung tissue and
digested in the same dissociation buffer at 37°C overnight. Disso-
ciated cells were passed through 70-μm Nylon mesh (Falcon, USA)
to remove aggregates and then washed twice with cold F12 medium.
Cell viability was assessed by exclusion of trypan blue dye. Cell
pellets were collected by centrifuge of 200 ×g and plated onto mit-
Protein & Cell
omycin-inactivated 3T3 feeder cells in BC culture medium for lung
(BCM-L) including DMEM/F12 (Gibco, USA), 10% FBS (Hyclone,
Australia), antibiotics, amphotericin and growth factor cocktail as
previously described (Zuo et al., 2015). Under 7.5% CO2 culture
condition, the SOX9+ BC colonies emerged 3–5 days after plating,
and were digested by 0.25% trypsin-EDTA (Gibco, USA) for 3–5 min
for passaging. Typically, SOX9+ BCs are passaged every 5 to 7 days
and split at 1:7 ratio. To obtain single cell-derived clone, cells are
digested into single cells, loaded through 40-μm Nylon mesh and
seeded with extremely low density, then a single colony grown up
from a single cell was picked up by clone cylinder (Sigma, USA) and
high vacuum grease after its neighboring colonies were cleared by
scraper to ensure the pedigree purity. For feeder-free culture of
SOX9+ BCs, feeder cells were removed by differential trypsinization
with 0.05% trypsin (Gibco, USA) and SOX9+ BCs were plated with
high density onto dishes pre-coated with 15% cold collagen type I
(Corning, USA) and 20% Matrigel (Corning, USA) for further
expansion.
For labeling of cells by GFP, pLenti-CMV-EGFP plasmid was
transfected into 293T cells together with lentivral packaging mix (Life
Technologies, USA). Lentivirus supernatant produced by 293T was
collected, filtered and cryo-preserved before use. To infect SOX9+
BCs, 0.5 mL lentivirus containing medium was directly added to
2 mL cell culture medium with 10 μg/mL polybrene and incubated for
12 h. The overall labeling efficiency of cells is above 95%. To confirm
that GFP containing virus will not spread between cells after label-
ing, we co-cultured GFP-labeled SOX9+ BCs with mCherry-labeled
(pLenti-CMV-mCherry) SOX9+ BCs for 5 days and did not observe
any yellow color cells.
Immunofluorescence staining
For immunofluorescence staining, cells were fixed by 3.7%
formaldehyde, and then incubated with 0.3% Triton X-100 to
improving the cell permeability for 10 min. Paraffin- or cryo-embed-
ded tissues were sectioned and subjected to antigen retrieval in
citrate buffer (pH 6.0, Sigma, USA) in microwave oven for 20 min
before staining. 10% normal donkey serum (Jackson ImmunoRe-
search) was used to block the non-specific antigen. Primary anti-
bodies used in this work include BC markers:KRT5 (1:200,
 RESEARCH ARTICLE
EP1601Y, Thermo), P63 (deltaN, 1:200, 4A4, Abcam), E-cadherin
(1:200, H-108, Santa cruz), SOX9 (1:200, ERP14335-78, Abcam),
SOX9 (1:200, AF3075, R&D); AEC markers: AQP5 (1:1000,
EPR3747, Abcam), HOPX (1:500, E-1, Santa Cruz), PDPN (1:500,
FL-162, Santa Cruz), SPC (1:200, M-20, Santa Cruz), SPC (1:200,
FL-197, Santa Cruz), LAMP3 (1:200, 12632-1-AP, Proteintech);
bronchiolar cell markers: CC10 (1:200, T-18, Santa Cruz), acety-
lated-α-Tubulin (1:1000, 6-11B-1, Abcam), MUC5AC (1:500, 45M1,
Thermo), FOXJ1 (1:200, 2A5, eBioscience); vasculature markers:
CD31 (1:100, M-20, Santa Cruz), CD34 (1:1000, EP373Y, Abcam);
myofibroblast marker: α-SMA (1:500, 1A4, DAKO), Fibronectin
(1:500, F14, Abcam), others: KI67 (1:200, RM-9106, Thermo), GFP
(1:200, B-2, Santa Cruz), GFP (1:200, FL, Santa Cruz), GFP (1:200,
T-19, Santa Cruz), ITGB1 (1:500, ERP16895, Abcam), Human
specific Lamin A+C (1:200, EPR4100, Abcam). Alexa Fluor-conju-
gated Donkey 488/594/647 (1:200, Life Technologies, USA) were
used as secondary antibodies. For antibodies of low reactivity, Bio-
tin-Streptavidin signal amplification system (Life Technologies, USA)
Protein & Cell
was used. After counterstaining with DAPI (Roche, USA), samples
were treated with 0.1% Sudan Black (Sigma, USA) for 1 min to
remove autofluorescence and then mounted with VECTASHIELD®
Mounting medium (Vector labs, USA). Images were visualized under
fluorescence microscope (Nikon 80i and Eclipse Ti, Nikon, Japan) or
fluorescence stereomicroscope (MVX10, Olympus, Japan). Confo-
cal images were taken under Nikon A1R microscope (Nikon, Japan).
RNA-sequencing and bioinformatics
SOX9+ BCs isolated from two donors and their corresponding brush-
off specimens were subjected to RNA-Seq analysis. The total RNA
concentration and RIN were measured by Agilent 2100 Bioanalyzer
(Agilent). For human SOX9+ BCs, 200 ng total RNA sample was
purified, and the first-strand cDNA was synthesized using first strand
master mix and super script II (Life Technologies). Second strand
master mix (Life Technologies) was then used to synthesize the
second-strand cDNA. After cDNA purification and adapter ligation,
PCR amplification was performed to enrich the cDNA fragments. For
brush-off samples, after RNA extraction and quality control, cDNA
was prepared using the SMARTer Ultra Low RNA Kit (Clontech) for
Illumina sequencing. Low Input Library Prep Kit (Clontech) was then
used for library construction. The library quantity and quality was
verified by Agilent 2100 Bioanalyzer and real-time quantitative PCR.
Then the library is sequenced using Illumina HiSeq 4000. Clean data
were acquired from raw data (fastq format) using the NGSQC Toolkit
by removing low-quality reads. Clean RNA-seq reads were mapped
to the reference genome (Ensembl, GRCh37) using Tophat v2.0.049
using default settings.
With genome mapping result, gene expression level was calcu-
lated with RSEM software (v1.2.12). Transcript levels were quanti-
fied as fragments per kilobase of transcript per million mapped reads
(FPKM). Pearson correlation coefficient between samples was cal-
culated by R scripts (3.2.3). Heatmap was generated using R scripts.
The protein-protein interactions were retrieved from Human Protein
Reference Database (HPRD, release 9) and visualized with Cytos-
cape (v3.3.0). SNPs were called by GATK (v3.4-0).
Karyotyping
Qiwang Ma et al.
To arrest SOX9+ BCs in mitosis metaphase, cells of 75% confluence
were treated with 1 μg/mL colchicines for 7 h and digested into
single cells by 0.25% trypsin. Then the cells were incubated by 0.4%
KCl at 37°C for 40 min and fixed by 10 mL fixation solution including
methanol and glacial acetic acid (3:1) at room temperature for
30 min. Suspension with chromosomes was dropped and spread on
slides. Samples on slides were treated by 0.0005% trypsin for 5 min
and stained with 15% Giemsa (Sigma-Aldrich, USA). Banding pat-
terns on chromosome spreads were checked for more than 15
mitotic phases and all of them are normal human cells. All cells used
for clinical purpose were subjected to karyotyping in prior to
transplantation.
Quantitative reverse transcription PCR
Total RNA from tissues or cells were isolated using the RNeasy mini
kit with DNase digestion according to the manufacturer’s instructions
(Qiagen). RNA quality was determined by SimpliNano (GE Health-
care). 1 μg total RNA was reverse-transcribed into cDNA with Pri-
meScript™1st Strand cDNA synthesis Kit (TaKaRa). The real-time
PCR assays were performed on an ABI 7500 real-time PCR system
(Applied Biosystems) according to the instructions of SYBR® Premix
Ex Taq™II (Tli RNaseH Plus, Takara). qPCR reactions were set as
following: 95°C for 2 min, then 40 cycles of 95°C for 10 s, and 60°C
for 40 s. Melt curve stage was added after PCR amplification stage.
The threshold crossing value (Ct) of each transcript was normalized
to reference genes (β-Actin or GAPDH). The relative expression
level of each genes was calculated using the 2−ΔΔCt method.
Sequence of primer pairs for qPCR was listed in Supplementary
Table.
Animal tissue histology
All animal experiments were conducted according to guidelines
approved by University Association for Laboratory Animal Science.
NOD-SCID mice (female or male, 6–10 weeks, The Jackson Lab-
oratory, USA) were euthanized at proper time points and the dia-
phragm was carefully cut open without touching the lung. In situ
fixation by injecting 3.7% formaldehyde (Sigma, USA) through tra-
chea was performed using 29 G needle. Then the lung was dis-
sected and fixed in 3.7% formaldehyde at 4°C overnight. For
cryosection, the fixed lung was settled by 30% sucrose before
embedding into the Tissue-Tek O.C.T compound (Sakura, Japan),
the 5–10 μm sections were cut using a cryotome (Leica microsys-
tem, Germany). For paraffin section, the lung was dehydrated by
gradient ethanol and processed in an automatic tissue processor,
then embedded into the paraffin blocks. All the samples were sliced
into 5–7 μm thickness using microtome (Leica microsystem, Ger-
many). Haematoxylin and eosin (H&E) staining was performed fol-
lowing standard protocol. Masson trichrome staining was performed
following the manual of Trichrome Staining Kit manual.
Small animal micro-CT
Micro-CT was used to monitor mouse lung damage before trans-
plantation. Mice were anesthetized through intraperitoneal injection
© The Author(s) 2018. This article is an open access publication
Regeneration of human lung
with chloral hydrate and fixed with tap. Lung image was obtained
using a volumetric micro-CT scanner without respiratory gating
(TriumphTM, Gamma medica-ideas, Northridge, USA). Scanning
was performed at 70 kV, 350 μA. The number of projections were
512 slices and total acquire time was around 4.27 min. All data were
converted into digital imaging by TRIUMPH ‘X-O’ CT system
software.
SOX9+ BC transplantation in mouse
Adult NOD-SCID mice (female or male, 6–10 weeks, The Jackson
Laboratory, USA) maintained in SPF animal facilities were used for
xeno-transplantation experiments. Mouse lung was injured by
intratracheally instilling with 3 U/kg body weight bleomycin (Sel-
leckchem, USA) eight days prior to transplantation. Mouse lung was
monitored by small animal micro-CT before transplantation to verify
lung damage. Then mice were anesthetized by I.P injection of 3%
chloral hydrate and rested on a stand gesture. One million GFP-
labeled cells were suspended in 50 μL PBS and used for trans-
plantation of each mouse. Intratracheal aspiration was performed by
injecting the cells into trachea via mouth. Three weeks after trans-
plantation, the lung samples were collected for analysis.
Fluorescence compatible optical clearing of lung sample
Optical clearing of SOX9+ BC transplanted lung was performed
following the SeeDB protocol of Meng-Tsen Ke et al., (2013) with
minor modification. Briefly, a whole lobe of lung was fixed by 3.7%
formaldehyde, and then transferred into 20%, 40%, 60%, 80% and
100% (w/v) fructose solution containing 0.5% α-thioglycerol. In each
gradient solution lung was incubated for 12 h at RT. In the end, lung
was transferred into SeeDB solution (80.2% w/w fructose) for 48 h to
72 h. The GFP fluorescence and blood vessels were directly visu-
alized by fluorescence stereomicroscope.
Tracing intravascular transport by nanoparticles
Water soluble 5 nm gold nanoparticles (AuNPs) were synthesized as
described previously (Cheng et al., 2008) with minor modifications:
0.25 mmol tetra-n-octylammonium bromide (TOAB) and 0.6 mmol
dodecylamine (DDA) dissolving in 5 mL of toluene was mixed with
0.53 mmol HAuCl4 solution (30% in HCl solution). A 2 mmol cold
NaBH4 aqueous solution was added into the organic phase and
stirred vigorously for 2 h. The DDA-AuNPs were collected by pre-
cipitation in 40 mL of ethanol and then redispersed in 3 mL of
chloroform. Next, MeO-PEG-SH (MW = 5000) and the DDA-stabi-
lized Au nanoparticles were mixed in chloroform and stirred over-
night. The organic phase was washed twice by water and then
evaporated under vacuum. The residues were washed three times
by water and purified by centrifugation.
To trace the intravascular transport route into lung, 10 mmol/L
AuNPs were dissolved in 50 μL PBS and injected intravenously into
mouse tail. To trace the aspiration route into lung, 10 mmol/L AuNPs
were dissolved in 50 μL PBS and instilled intratracheally into mouse
lung. One hour later, the lung was collected and then briefly fixed in
3.7% formaldehyde for 30 min on ice, and then embedded into the
Tissue-Tek O.C.T compound followed by cryosections. GFP signal
on tissue slide was captured by direct immunofluorescence and then
© The Author(s) 2018. This article is an open access publication
RESEARCH ARTICLE
the same slide was stained with LI Silver Enhancement Kit following
manufacturer’s instruction (Thermo, USA). Brown color indicates the
precipitation of AuNPs after reaction with silver
Western Blotting
Cells were washed in cold PBS and harvested by plastic scraper.
Collected lung tissues were washed in cold PBS, ground and lysed
by electric tissue grinder in RIPA buffer (150 mmol/L sodium chlo-
ride, 0.5% Triton-X100, 0.5% sodium deoxycholate, 5 mmol/L EDTA,
0.1% SDS, 50 mmol/L Tris-HCl, pH 7.5) with protease inhibitors
cocktail (Roche, USA). Approximately 30 μg total protein from each
sample was loaded. Samples were separated on a 10% SDS poly-
acrylamide gel and transferred to PVDF membranes (Roche, USA)
with electrophoresis blotting transfer apparatus. The membranes
were blocked with 5% dehydrated milk for 1.5 h and then incubated
with primary antibodies overnight. The next day, the membranes
were incubated with horseradish peroxidase-conjugated secondary
Protein & Cell
antibody. The specific signals were detected by ECL plus western
blotting detection reagents and X-ray film system.
Flow cytometry analysis
For immunostaining, cells were fixed by 3.7% formaldehyde for
30 min and permealized by 0.2% Triton X-100 for 5 min. 0.5%
donkey serum was used to block the non-specific signals at room
temperature for 30 min. Then the samples were incubated
sequentially with primary antibody and FITC/APC-Cy7-conjugated
secondary antibody (1:400, Life technologies, USA) at room tem-
perature for 1–2 h. BD FACS Verse (BD, USA) equipped with
488 and 647 lasers was used to detect the fluorescence signals for
samples. Single cell suspensions went through 40 μm strainer
before test. FSC-A and SSC-A parameters were used to exclude the
debris and FSC-H, FSC-W, SSC-W parameters were used to
exclude the clusters in the cell suspension. IgG control sample was
used to set the bottom-line of the positive signals.
SOX9+ BC transplantation clinical trial
A prospective, single-center, non-randomized clinical study was
conducted to evaluate the feasibility, safety and efficacy of SOX9+
BC transplantation in patients with bronchiectasis. The trial was
approved by Southwest Hospital Ethics Committee (Chongqing,
China, 2016-Research-#19,ClinicalTrials.gov: NCT02722642), con-
ducted in compliance with Good Clinical Practice (GCP) standard
and the most recent version of the Declaration of Helsinki. Two
patients who had signed informed consent form were admitted to
hospital twice for SOX9+ BC isolation and transplantation, respec-
tively. Diagnosis was established based on ATS/ERS guidelines.
Patient medical history, vital signs, routine laboratory tests (regular
blood counts, biochemical measurements, coagulation test, liver/
renal function tests, myocardiozymogram measurements), electro-
cardiogram, arterial blood gas, pulmonary function tests and HRCT
scan were conducted based on standardized clinical SOP of hospital
1 day before and different times after SOX9+ BC transplantation.
More detailed information for the clinical trial was described in
Supplementary Materials.
 RESEARCH ARTICLE
Statistics
Block randomization was used to randomize samples/mice into
groups of similar sample size. No samples, animals or patients were
excluded from all analysis. Statistical analysis was performed by
Student’s t-tests (two-tail comparisons) or Wilcox test and significant
difference was defined as P < 0.05. Values in text were presented as
means with S.E.M. Microsoft Excel 2011 (Microsoft, USA) or R
programming was used for data management, statistical analysis
and graph generation. Statistical power analysis was used to ensure
adequate sample size for detecting significant difference between
samples. The variance is similar between groups that are being
statistically compared. All experiments (except the clinical trial) were
replicated for at least three times with consistent results in the lab-
oratory. All experimental including the clinical trial outcomes were
assessed by at least one blinded participating investigator.
ACKNOWLEDGEMENTS
Protein & Cell
This work was funded by The National Key Research and Develop-
ment Program of China (2017YFA0104600), Youth 1000 Talent Plan
of China to W. Zuo, Tongji University (Basic Scientific Research-In-
terdisciplinary Fund and 985 Grant to W. Zuo), Shanghai Pulmonary
Hospital (Annual Grant to W. Zuo), National Science Fund Committee
of China (81570091 and 81770073 to W. Zuo), Academic Leadership
Fund of Shanghai (16XD1403100 to T. Ren), Postdoc Grant of China
(2016M601651 to Y. Ma and 2016M591716 to Q. Ma) and Kiangnan
Stem Cell Institute. We thank Prof. Zhongwei Li for comment on the
manuscript, and thank Profs. Frank McKeon, Wa Xian and Nanshan
Zhong for helpful discussion of this work, and thank Profs. Xiaoqing
Zhang, Long Zhang and Huiping Li for reagents help.
COMPLIANCE WITH ETHICS GUIDELINES
Qiwang Ma, Yu Ma, Xiaotian Dai, Tao Ren, Yingjie Fu, Wenbin Liu,
Yufei Han, Yingchuan Wu, Yu Cheng, Ting Zhang and Wei Zuo
declare that they have no conflict of interest. All institutional and
national guidelines for the care and use of laboratory animals were
followed. All procedures followed were in accordance with the ethical
standards of the responsible committee on human experimentation
(institutional and national) and with the Helsinki Declaration of 1975,
as revised in 2000 (Mannino 2002). Informed consent was obtained
from all patients for being included in the study.
OPEN ACCESS
This article is distributed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to
the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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https://doi.org/10.1186/s12931-018-0837-5

REVIEW Open Access

Understanding alveolarization to induce

lung regeneration
José Alberto Rodríguez-Castillo1, David Bravo Pérez1, Aglaia Ntokou1, Werner Seeger1,2, Rory E. Morty1,2
and Katrin Ahlbrecht1,2*

Abstract
Background: Gas exchange represents the key physiological function of the lung, and is dependent upon proper
formation of the delicate alveolar structure. Malformation or destruction of the alveolar gas-exchange regions are
key histopathological hallmarks of diseases such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary
disease (COPD), and pulmonary fibrosis; all of which are characterized by perturbations to the alveolo-capillary barrier
structure. Impaired gas-exchange is the primary initial consequence of these perturbations, resulting in severe clinical
symptoms, reduced quality of life, and death. The pronounced morbidity and mortality associated with malformation
or destruction of alveoli underscores a pressing need for new therapeutic concepts. The re-induction of alveolarization
in diseased lungs is a new and exciting concept in a regenerative medicine approach to manage pulmonary diseases
that are characterized by an absence of alveoli.
Main text: Mechanisms of alveolarization first need to be understood, to identify pathways and mediators that may be
exploited to drive the induction of alveolarization in the diseased lung. With this in mind, a variety of candidate cell-types,
pathways, and molecular mediators have recently been identified. Using lineage tracing approaches and lung injury
models, new progenitor cells for epithelial and mesenchymal cell types – as well as cell lineages which are able to
acquire stem cell properties – have been discovered. However, the underlying mechanisms that orchestrate the complex
process of lung alveolar septation remain largely unknown.
Conclusion: While important progress has been made, further characterization of the contributing cell-types, the cell
type-specific molecular signatures, and the time-dependent chemical and mechanical processes in the developing, adult
and diseased lung is needed in order to implement a regenerative therapeutic approach for pulmonary diseases.
Keywords: Alveolarization, Neo-alveolarization, Regeneration

Background key molecular and cellular drivers of alveolarization and

Therapeutic options for diseases that cause perturba- neo-alveolarization would be used to induce regener-
tions to the lung structure such as bronchopulmonary ation of alveoli in the diseased lung. The aim of this
dysplasia (BPD), chronic obstructive pulmonary disease review is to provide an overview of recent developments
(COPD), and pulmonary fibrosis, are limited; and as in the underlying concepts of alveolarization and
such, new therapeutic concepts are needed [1–4]. A neo-alveolarization, and to explain how this knowledge
translational regenerative approach represents one fu- might be used to induce regeneration of alveoli. Further-
ture promising option for the development of new thera- more, techniques currently available to approach this
peutic concepts. In this approach, the identification of question are highlighted. Current knowledge of alveolar-
ization and neo-alveolarization includes consideration of
the contributing cell-types, extracellular matrix (ECM)
* Correspondence: katrin.ahlbrecht@mpi-bn.mpg.de
1
Member of the German Lung Research Center (DZL), Department of Lung
components and selected molecular mediators [5–14].
Development and Remodelling, Max Planck Institute for Heart and Lung However, studies that have assessed cell-lineage specifi-
Research, Parkstrasse 1, 61231 Bad Nauheim, Germany cation, progenitor- or stem-cell characteristics, and mo-
2
Member of the German Lung Research Center (DZL), Department of
Internal Medicine (Pulmonology), University of Giessen and Marburg Lung
lecular signatures in relation to the localization of a cell
Center (UGMLC), Klinistrasse 33, 35392 Giessen, Germany are limited. Future directions for research supporting
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Rodríguez-Castillo et al. Respiratory Research (2018) 19:148
this regenerative approach remain a crucial topic to be
discussed, and likely directions are highlighted in the last
paragraph of this review.
Main text
Alveolarization
Alveolarization represents a process during lung develop-
ment that leads to the formation and maturation of the
distal parts of the lung: the alveoli. In rats and mice, alveo-
larization takes place postnatally, whereas in humans,
alveolar development begins prior to birth [3, 15–20]. Due
to the limited availability of human tissue, most of the
studies dissecting the principles of alveolarization have
been conducted in rodents. At birth, the murine lung is in
the saccular stage, which lasts from embryonic day (E)18.5
until postnatal day (P)5 [5], and which is comparable to
the stage of lung development in which most pre-term
born human infants are undergoing at the time of prema-
ture rupture of membranes. As such, term-born mouse
and rats are often used to model the lung in pre-term
born infants, with the important caveat that these lungs
are perfectly competent for effective gas exchange at birth,
contrasting with the situation in pre-term born human
infants.
Fig. 1 Alveolar epithelial cells during alveolarization. During the saccular stage, alveolar epithelial type I cells (AECI) and alveolar epithelial type II
cells (AECII) are derived from a common bipotent progenitor cell. After differentiation single AECl can cover multiple alveoli during alveolarization
and in the adult lung
Page 2 of 11
Nature of alveolar epithelial cells during the saccular
stage and alveolarization
During the saccular stage of lung development, distal parts
of the lung contain air sacs (sacculi or saccules) which are
lined with an epithelial layer that originates from the fore-
gut endoderm, and consists of differentiated alveolar epi-
thelial type I cells (AECI) and alveolar epithelial type II
cells (AECII) [5]. During the saccular stage, AECl and
AECII are derived from a common bipotent progenitor
cell (Fig. 1) [21]. As described in a later section of this re-
view, mechanical forces and fibroblast growth factor
(FGF) 10 are amongst recently-identified regulators of this
differentiation process [22–24]. After alveolarization, and
in the adult lung, AECII acquire stem cell properties and
are capable of self-renewal to replace AECl after injury
[21, 25]. Single-cell sequencing of distal lung epithelial
cells during the saccular stage confirmed a bipotent pro-
genitor for AECl and AECll and revealed further cell-type
specific markers and subpopulations during the process of
differentiation [26]. Cuboidal AECII cells are capable of
producing surfactant proteins and lipids which decrease
the surface tension of the alveoli [5, 27]. The cell surface
of squamous AECl expands drastically during the alveolar
stage [28]. One AECl covers multiple alveoli during
Rodríguez-Castillo et al. Respiratory Research (2018) 19:148
alveolarization and in the adult lung (Fig. 1) [28–30]. Due
to the close proximity of AECI to the capillary network,
and the comparatively larger surface area when compared
to AECII, AECl represent the site of gas exchange [30].
Role of mesenchymal cells and extracellular matrix during
alveolarization
Further components of the alveolar air-sac walls (primary
septa) are endothelial cells that originate from the lung
endoderm [8, 31, 32], and a variety of interstitial cell-types
such as fibroblasts, which originate from the lung meso-
derm [8, 33–35]. In rat lung fibroblasts, an increase in ret-
inoic acid levels has been demonstrated during secondary
septation [36], and retinoic acid has been proposed to
impact elastin production [36]. These studies highlighted
a potential regenerative activity of retinoids in the
lung. Retinoic acid administration to rats has been dem-
onstrated to promote postnatal alveolarization, and to at-
tenuate elastase-induced pulmonary emphysema [37, 38].
During the saccular stage, elastin expression increases,
and elastin deposition by fibroblasts takes place [39]. In
rodents, by P4, so-called secondary septa appear in the
primary septa at sites of elastin deposition [5, 31]. At the
tip (secondary crest) of these still-immature secondary
septa, α-smooth muscle actin (αSMA)+ myofibroblasts ap-
pear, and the expression of ECM components such as
Fig. 2 Mesenchymal cell-types during alveolarization. Alveolar mesenchymal fibroblasts such as myofibroblasts and lipofibroblasts differentiate
from mesenchymal progenitor cells. Key cell lineages involved are the platelet-derived growth factor receptor (PDGFR)α lineage, the fibroblast
growth factor (FGF)10 lineage and the GLI-Kruppel family member (Gli-1) lineage. Elastin deposition by myofibroblasts takes place at the so called
“secondary crests” visualized in 2D lung sections
Page 3 of 11
elastin further increases (Fig. 2) [5, 6, 33, 39]. One recent
study has carefully dissected structural changes in the
developing alveoli, and elastin localization during alveolar-
ization, using 3D imaging techniques [14]. These analyses
pointed out that secondary crests arise as ridges into the
alveolar air-sac lumen. An organized network resembling
a “fishnet” composed of αSMA and the ECM component
elastin runs within the ridges [14]. Quite similar observa-
tions were made in a further study that analyzed the spatial
and temporal changes in elastin and laminin distribution
during alveolarization [40]. That study demonstrated that
elastin fibers formed ring-like structures which were local-
ized to the saccular openings, and later on were intercon-
nected by further elastin fibers [40]. Crosslinking of ECM
components has been demonstrated to be altered during
aberrant lung development [41]. The downstream signaling
molecule of the sonic hedgehog pathway, Gli-1, has been
demonstrated to label a cell-lineage which gives rise to sec-
ondary crest myofibroblasts (Fig. 2) [42–44]. The depos-
ition of ECM components and the presence of alveolar
myofibroblasts seems to be an attribute for secondary sept-
ation and has been demonstrated to be dependent on
platelet-derived growth factor (PDGF)-A signaling [6, 45].
The ligand PDGF-A is produced by epithelial cells and
signals via the cognate receptor PDGF receptor (PDGFR)α,
which is expressed by mesenchymal cells [6, 46, 47]. In vivo
Rodríguez-Castillo et al. Respiratory Research (2018) 19:148 Page 4 of 11

labeling and lineage-tracing studies of PDGFRα+ cells have
demonstrated that pulmonary PDGFRα+ cells serve as pro-
genitor cells for peribronchial and alveolar myofibroblasts,
as well as for a proportion of the pulmonary lipofibroblasts,
and are present in the primary and secondary septa (Fig. 2)
[6, 45, 48, 49]. Further studies have highlighted the pro-
genitor nature of PDGFRα+ cells for peribronchial smooth
muscle cells, myofibroblasts and lipofibroblasts during pre-
natal lung development and alveolarization using time-series
RNA-Seq analyses and immunophenotyping [50, 51]. An-
other recent study has described the spatiotemporal distri-
bution of PDGFRα and the two PDGF ligands PDGF-A and
PDGF-C over the course of lung development [52]. Ex-
pression of the ligands was detected in epithelial and
smooth muscle cells, whereas the expression of PDGFRα
was located to different mesenchymal cell populations
[52], which is consistent with the prevailing view that
epithelial-mesenchymal interactions are key mediators of
lung development.
Possible role for lipogenic versus myogenic fibroblast
phenotypes during alveolarization
Lipofibroblasts are also located within the primary and
secondary septa in close proximity to AECII [48, 49, 53].
Lipid droplets and the expression of adipocyte differenti-
ation related protein (ADRP, encoded by the Plin2 gene)
represent phenotypic characteristics of lipofibroblasts
[53–57]. Further molecules such as peroxisome
proliferator-activated receptor (PPAR)γ, cellular retinoic
acid binding protein (CRABP), and the transcription fac-
tor TCF21 are expressed by lung mesenchymal lipofibro-
blasts (Fig. 3) [36, 58–61]. Lineage-tracing of FGF10+ cells
revealed labeling of a subset of the lipofibroblast population
[62]. Furthermore, lipofibroblasts have been demonstrated
to support the synthesis of surfactant in AECII by providing
triacylglycerols to AECII in a leptin- and stretch-dependent
manner (Fig. 3) [53, 63]. During the period of secondary
septation, the number of lipofibroblasts has been demon-
strated to increase and peak at the same time as the peak of
secondary septation, at P7 [64]. Activation of PPARγ using
rosiglitazone (which promotes the lipofibroblast phenotype)
in rat pre- and post-natally has been demonstrated to be
protective against the structural changes that occur during
the development of hyperoxia-induced lung injury [65, 66].
However, the presence of lipofibroblasts in the human lung
remains controversial [54, 57, 67]. In contrast to rosiglita-
zone, which induces a lipogenic phenotype; Other reagents,
stimuli and factors such as nicotine, mechanical forces,
PDGFRα and transforming growth factor (TGF)-β have
been demonstrated to induce a myogenic phenotype: nico-
tine treatment of isolated fibroblasts in vitro led to a myo-
genic phenotype (differentiation from lipofibroblasts to
myofibroblasts) and could be reversed by rosiglitazone
treatment [68]. Furthermore it has been demonstrated that

Fig. 3 Lipogenic versus myogenic fibroblast phenotype. Lipogenic (lipofibroblast) and myogenic (myofibroblast) fibroblasts differentiate during
early lung development and the saccular stage. Lipofibrobasts support alveolar epithelial type II cells (AECII) cell function via an intercellular
crosstalk mediated by stretch, parathyroid hormone-related peptide (PTHRP), prostaglandin E2 (PGE2) and leptin while myofibroblasts produce
extracellular matrix molecules such as elastin. Activation of peroxisome proliferator activated receptor (PPAR)γ by Rosiglitazone promotes the
lipogenic phenotype. Stretch and transforming growth factor (TGF)-β induce the myogenic phenotype
Rodríguez-Castillo et al. Respiratory Research (2018) 19:148
mechanical forces could stimulate the differentiation of fi-
broblasts towards myofibroblasts (Fig. 3) [69]. Expression
and activation of PDGFRα has also been demonstrated be
involved in driving fibroblast differentiation towards a myo-
genic phenotype [6, 49, 70]. The expression of the PDGFRα
chain has been demonstrated to be regulated by TGF-β
(Fig. 3) [71]. A very recent study has also carefully dissected
the role of the ligand PDGF-A using mice carrying a floxed
Pdgfrα allele in combination with a Sftpc-Cre mouse strain
[72]. The authors demonstrated that PDGF-A was needed
for myofibroblast formation and proliferation as well as for
the regulation of AECII proliferation [72]. Taken together,
tightly regulated differentiation of alveolar fibroblasts to-
wards the myogenic or the lipogenic phenotype seems to
be relevant for secondary septation. Furthermore, neuropil-
lin 1 has been demonstrated to impact the PDGF-A axis
via activation of Src kinases and to be required for alveolar
mesenchymal cell migration [73]. Following this line, a re-
duced expression of PDGFRα and PDGFRβ has been dem-
onstrated in mesenchymal cells of infants who develop
BPD [74]. Furthermore, the signaling via FGF members has
been demonstrated to impact the formation of myofibro-
blasts from PDGFRα+ cells, as well as alveolar regeneration
per se [75]. Deficiency of FGF10 has been demonstrated to
be causative for the lethality in a mouse model of BPD [76].
Another mediator, Thy-1, which is expressed on lympho-
cytes and fibroblasts, has been demonstrated to severely
impact alveolarization: an arrest of alveolarization itself has
been demonstrated upon global loss of Thy-1 (CD90) [7].
The glycoprotein Thy-1 inhibits TGF-β activation, which
leads to a reduced myogenic phenotype [7]. Since Thy-1
also is expressed on lipofibroblasts it might represent a
molecule impacting on the balanced appearance of lipo-
genic and myogenic fibroblasts during secondary septation
(Fig. 3) [7, 48]. However, the role of leucocytes might be of
relevance since inflammatory cells have been proposed to
play a role – and to be present – during lung development
[77], and resident alveolar macrophages have recently been
implicated as master regulators of arrested lung devel-
opment [78]. Apart from the role of lipogenic and myo-
genic phenotypes of fibroblasts in lung development,
lipogenic and myogenic fibroblast phenotypes are also
involved in the progression and resolution of pulmon-
ary fibrosis, which has been demonstrated in a murine
model of bleomycin-induced lung fibrosis [79]. This
mechanism supports the hypothesis that understanding
the nature and development of lipogenic versus myogenic
fibroblast phenotypic transformation might help to de-
velop new therapeutic strategies for pulmonary diseases.
Epithelial-mesenchymal interactions during
alveolarization
The Interaction between mesenchymal and epithelial
cells has been demonstrated to be essential for cellular
Page 5 of 11
differentiation and function during alveolarization. The
formation of alveolospheres by alveolar epithelial cells
(AEC) has been demonstrated to be supported by
PDGFRα+ cells in vitro [25]. In line with these findings, a
mesenchymal cell population from the Axin2 cell-lineage
which expressed PDGFRα has been demonstrated to sup-
port AECI and AECII differentiation and function [12]. In
contrast, a rare population of AECII which express
Axin2 has been demonstrated to have alveolar stem cell
activity in the adult lung and to be supported by juxta-
crine Wnt signals from neighboring fibroblasts during
homeostasis, and autocrine Wnt signals upon severe in-
jury [80]. Further evidence for mesenchymal-epithelial
interactions being key drivers of cell differentiation during
alveolar development has emerged from analyses compar-
ing two distinct mesenchymal cell populations of the
leucine-rich repeat-containing G protein-coupled receptor
(LGR)5 and LGR6 cell lineage: the LGR5 and LGR6 lin-
eages have been demonstrated to support cell function
and differentiation of either alveolar or bronchiolar epi-
thelial cells [13]. Furthermore human alveolar fibroblasts
have been demonstrated to exhibit direct intercellular
contact with AECl, AECll, capillaries and pericytes [81].
This strategic localization might position fibroblasts to
mediate crosstalk between the epithelial and the capillary
endothelial cells, as well as epithelial-mesenchymal inter-
actions which are key drivers of cellular differentiation
and function during alveolarization.
Significance of mechanical forces in alveolarization
Further key drivers of cellular processes that promote
alveolarization are mechanical forces. An understanding
of lung alveolarization is based primarily on understand-
ing processes such as cellular differentiation, localization,
production of ECM molecules and underlying signaling
cascades. However, the impact of mechanical forces on
these processes has been analyzed in the context of
intercellular crosstalk and the differentiation of alveolar epi-
thelial cells [22–24, 63, 82]. Surfactant production of AECII
has been demonstrated to be stretch-dependent via a
stretch-induced de novo synthesis of phosphatidyl choline
by AECII [63]. Furthermore, stretch-dependent interactions
with fibroblasts via parathyroid hormone-related peptide
(PTHRP), prostaglandin E2 (PGE2) and fibroblast-derived
leptin increased surfactant synthesis (Fig. 3) [63, 82]. A very
recent study carefully dissected the role of mechanical
forces on alveolar epithelial cell differentiation using live
imaging techniques [23]. Mechanical forces generated
by inhalation of amniotic fluid by prenatal breathing
movements were essential for the differentiation of AECI
[23]. Furthermore FGF10/FGFR2 signaling has been dem-
onstrated to prevent flattening of alveolar progenitor cells,
protecting AECII differentiation [23]. Mechanical forces
have also been demonstrated to impact airway tube
Rodríguez-Castillo et al. Respiratory Research (2018) 19:148
morphogenesis by controlling cell shape and orientation
of cell division of the airway epithelium [22]. Taken to-
gether, mechanical forces induced by pre- and post-natal
breathing movements are essential for cellular differenti-
ation processes of epithelial and mesenchymal cells during
alveolarization.
Different types of alveolarization and maturation of the
secondary septum
Analyses of the temporal dynamics of the lung transcrip-
tome during lung development revealed that changes in
the transcriptome profiles confirmed previously defined
stages of lung development [83]. Additionally, four stages
of postnatal alveolar development were suggested based
on the temporal changes in the lung transcriptome [83].
Similarities and contrasts of developmental processes and
processes regulating disease and tissue homeostasis in the
adult lung await to be dissected. For example, in contrast
to the “classical or bulk alveolarization” which is initiated
from an immature primary septum, “continued alveolari-
zation” has been demonstrated starting from a more ma-
ture septum from P14 [31]. Maturation of the immature
secondary septa by thinning of the septa and remodeling
of double capillary networks to a single capillary network
occurs during the stage of microvascular maturation start-
ing at P12 [31]. During the stage of microvascular matur-
ation, the laminin network has been demonstrated to be
simplified, to ensure septal thinning [40]. During the last
period of septal maturation by P21 mature septa appear
next to continued alveolarization until young adulthood
[31]. Taken together, understanding processes driving dif-
ferent types of alveolarization and the maturation of the
alveolar septum might help to identify cellular and
molecular drivers for each period, which might be used to
induce regeneration of the alveolus in the diseased lung in
which either regrowth or thinning of the alveolar wall is
required.
Conclusion alveolarization
Different cell-types contributing to secondary septation
have been identified, but distinct cell-type specific func-
tions against the background of chemical and mechan-
ical conditions during lung development still need to be
understood. Identification of progenitor cells and a de-
tailed characterization of the differentiation and function
of mesenchymal lineages are needed to better under-
stand secondary septation. Furthermore, understanding
the function of AECll and AECl during secondary sept-
ation, and mapping the molecular signatures of the
interactions between alveolar mesenchymal and epithe-
lial cells might provide further insights into the nature
of alveolar septation.
Page 6 of 11
Neo alveolarization
The formation of new alveoli in the adult lung has been
demonstrated in a variety of species including humans,
after removal of a part of the lung (by pneumonectomy,
PNX) [84–87]. In mice, left-sided PNX leads to a complete
restoration of the mass-specific lung volume and total
alveolar surface area within 21 days after the operation
[86]. In humans, there is evidence for compensatory lung
growth after PNX, but the time-course is months to years
and a complete restoration of the lung capacity has not
been demonstrated [84]. Some underlying mechanisms
contributing to compensatory lung growth have been
demonstrated in rodents, such as mediators of the alveolar
stem cell niche and of vascular- epithelial interactions and
will be discussed in the following paragraphs.
Nature of alveolar epithelial cells during neo-alveolarization
In adult mice it has been demonstrated that HOPX1
lineage-labeled AECl expressed surfactant protein C
(SPC) 21 days after PNX suggesting the generation of
AECll from AECl during neo-alveolarization after PNX
[10]. Furthermore a SPC−AEC progenitor cell pool has
been identified in an in vivo embryonic lung organoid
assay in mice suggesting a further progenitor cell-lineage
for AECII [88]. However there is strong evidence based
on lineage tracing approaches that AECll hold stem cell
features in the adult and postnatal lung [21, 25]. A further
very recent study carefully dissected the role of bone mor-
phogenic protein (BMP) signaling during alveolar regener-
ation in organoid culture and in vivo during the PNX
model [89]. In the alveolar stem cell niche which, consists
of AECII and PDGFRα-expressing fibroblasts [90],
BMP signaling was demonstrated to regulate AECII sup-
port function of PDGFRα+ fibroblasts and differentiation
of AECI and AECII, as well as AECII proliferation and
self-renewal [89].
Role of the vascular system and vascular mediators
during neo-alveolarization
Similar to the bulk alveolarization that occurs during post-
natal lung development, crest formation arising from
preexisting septa involving capillaries and mesenchymal
cells has been demonstrated using scanning electron mi-
croscopy of vascular casts after PNX [91]. Mechanisms
driving neo-vascularization such as sprouting and intus-
susceptive angiogenesis resemble neo-vascularization dur-
ing postnatal bulk alveolarization [91]. Platelet-derived
stromal-cell-derived factor (SDF) has been demonstrated
to impact AEC expansion and neo-alveolarization after
PNX [9]. Likewise, the crosstalk between pulmonary capil-
lary endothelial cells and AEC during neo-alveolarization
after PNX has been demonstrated to involve vascular
endothelial growth factor receptor (VEGFR)2, matrix me-
talloproteinase (MMP)-14 and epidermal growth factor
Rodríguez-Castillo et al. Respiratory Research (2018) 19:148
receptor (EGFR) resulting in expansion of epithelial pro-
genitor cells [92]. Taken together, growth of the vascular
system, and vascular mediators and growth factors such as
SDF-1 and VEGF-A, are further drivers of epithelial cell
function during lung regrowth after PNX.
Impact of mesenchymal cells, mechanical forces and
innate lymphoid cells on neo-alveolarization
Further key drivers of lung growth after PNX have been
identified in mesenchymal lineages. There is evidence
for the participation of PDGFRα+ cells in compensatory
lung regrowth [93, 94]. Gene expression profiling of
postnatal and adult mouse lungs undergoing PNX re-
vealed concordantly as well as variably regulated genes
[95]. There is a growing body of evidence that features
of bulk alveolarization occur during compensatory lung
growth of the adult lung, but alternative mechanisms
need to be considered in addition. Cell proliferation,
change in mechanical forces, ECM remodeling and the
activation of different signaling pathways have been
demonstrated in response to PNX [86, 92, 95–97]. Fi-
nally, myeloid cells and type 2 innate lymphoid cells
(ICL2) have been demonstrated to hold regenerative
capacity during compensatory lung growth after PNX
[11]. A variety of cellular and molecular factors impact-
ing on neo-alveolarization after PNX has been identi-
fied. However, target molecules capable to initiate neo-
alveolarization have not been recognized. Further
combined lineage tracing, lineage ablation and cell
type specific loss or gain of function studies are needed to
identify hierarchies and functions of cell types and
signaling cascades driving neo-alveolarization in the
adult lung.
Models to approach lung regeneration
The induction of neo-alveolarization in the diseased lung
requires a detailed knowledge of the conditions which
are necessary for alveolar septum formation. Therefore,
the identification of suitable models to study alveolar
septum formation is essential. Transgenic tools [98–100]
and genome-wide screening approaches [83] have re-
vealed a variety of contributing cellular and molecular
candidates and thus represent suitable tools to elucidate
relevant candidate drivers for alveolarization. Therefore,
alveolariaztion and neo-alveolarization after PNX are
currently analyzed primarily in mice in vivo. However,
some disadvantages of both models remain, and these
are discussed in the following sections.
Analyzing alveolarization
Mice represent a valuable tool to analyze the process of
alveolarization since alveolarization largely takes place
postnatally in mice. Advantages of analyzing alveolariza-
tion in vivo in mice are the possibility to perform
Page 7 of 11
cell-type specific and inducible lineage tracing, cell and
gene modulation, as well as cell ablation based on the
CreERT2/loxP system [101]. Differentiation processes,
stem cell features, and morphological appearance can be
analyzed in combination with high quality imaging ap-
proaches. Even cell-lineage and single-cell sequencing
analyses can be performed at different time-points of
alveolarization. However, a key limitation of studying
alveolarization in mice is the need to validate identified
candidate cells and genes in human tissue, which has to
be performed to ensure the ranslational value of the
pathway identified. Furthermore, it remains to be discov-
ered which candidates relevant for alveolarization in
postnatal mice might also be relevant for lung regener-
ation. Taken together, analyzing alveolarization in mice
in vivo represents a suitable model, but validation of
candidate factors in human lung tissue and adult mouse
lung tissue has to be considered.
Elucidating neo-alveolarization using the PNX model
Exploitation of the model of compensatory lung
growth after PNX will provide a better understanding
of the cellular and molecular mechanism driving neo-
alveolarization. Beneficial aspects of this model are
represented by the possibility that the advantages of
transgenic mice can be used as well. Furthermore, alveolar
regrowth can be studied in the adult lung, which might be
more directly relevant to lung regeneration of the adult
lung. Like studying alveolarization in mice, a disadvantage
of studying regrowth after PNX in mice also remains, that
driving factors might be different in mice and humans.
Furthermore, it has been demonstrated that strongest
regrowth takes place in the cardiac lobe [86]. Therefore,
it might be necessary to restrict analyses to the
cardiac lobe.
Perspectives
A possible way to approach lung regeneration is to
better understand the composition of contributing cell
types, the molecular signature and plasticity of cells, and
the ECM composition, which together drive alveolariza-
tion and neo-alveolarization in order to identify possible
candidates for the induction of lung regeneration. Pro-
genitor cells need to be identified. Single-cell genome-wide
screening approaches lead to a broader and more complex
understanding of cell lineages of different tissue compart-
ments [26, 102]. Better characterization of endodermal and
mesenchymal cell lineages including molecular signatures
might provide new insights into the cellular hierarchy of
alveolarization, and might reveal progenitor cell candidates.
Detailed time-dependent lineage tracing and cell depletion
approaches covering prenatal and postnatal periods is
needed to discover cell type-specific commitment and func-
tion during alveolarization. Furthermore, there is need to
Rodríguez-Castillo et al. Respiratory Research (2018) 19:148 Page 8 of 11

understand if factors driving bulk alveolarization in
the developing lung might also be capable of inducing
neo-alveolarization in the adult lung. Cellular and
molecular candidates ensuring the homeostasis of the
adult lung tissue additionally might be relevant for
neo-alveolarization. A further aspect to approach al-
veolar regeneration might include the impact of aging
and senescence on lung homeostasis and repair. As
an example, telomerase activity, which impacts on
cellular senescence, has been demonstrated to be tis-
sue specifically regulated in mice during development
and aging [103]. This is relevant because lung-specific
modulation of telomerase activity during lung devel-
opment might reveal possible target candidates that
impact lung regeneration. Furthermore, the identifica-
tion of possible targets to lung-specifically modulate
the process of aging might reveal targets for future
therapeutic concepts. Regulation of the cell cycle rep-
resents a key mechanism for tissue homeostasis and
development. Signal transduction programs of cellular
senescence cause an irreversible cell cycle arrest and
thus might crucially impact lung regeneration [104].
Apart from processes which drive alveolarization and
neo-alveolarization, processes which drive pulmonary

Fig. 4 Approaches for lung regeneration. Identification of progenitors or stem cells and mediators driving cellular differentiation during
alveolarization and neo-alveolarization might be used to identify possible target candidates to induce regeneration during pulmonary diseases
such as emphysema/chronic obstructive lung dsease and fibrosis. Promising target candidates are represented by platelet-derived growth factor
receptor (PDGFR)α+ cells and fibroblast growth factor (FGF)10 during alveolarization and bone morphogenic protein (BMP))/SMAD family member
(SMAD) signaling and stromal cell derived factor (SDF)-1 during neo-alveolarization
Rodríguez-Castillo et al. Respiratory Research (2018) 19:148
diseases have to be considered. Finally, target candidates
identified in mice, need to be validated in human tissue.
Conclusion
Knowledge about alveolarisation and neo-alveolarization
will reveal cellular and molecular target candidates that
might be exploited for the development of new thera-
peutic concepts for pulmonary structural diseases. To ex-
plore cellular and molecular mediators of alveolarization
and neo-alveolarization in vivo, suitable tools such as
lineage tracing, cell-type specific cell-ablation techniques
and cell-type specific molecular modulation, single cell se-
quencing and high quality 3D imaging approaches are
currently available in mice. So far, concerning alveolariza-
tion, modulating the PDGFRα+ cell-lineage which repre-
sents a member of the alveolar stem cell niche and
modulating FGF10 which is involved in transducing
mechanical forces seem to be promising approaches to
modulate alveolarization (Fig. 4). The related candidate
of the alveolar niche is represented by AECII. The stem
cell function of AECII has been demonstrated to be es-
sential for alveolar regrowth after PNX and has also
been highlighted in this review. Identification of factors
modulating stem cell functions of AECII and epithelial
homeostasis such as BMP/SMAD signaling and SDF-1
represent the most promising approach to understand
lung regrowth after PNX and further to develop strat-
egies to induce lung regeneration during pulmonary
diseases such as emphysema/COPD and fibrosis (Fig. 4).
However, validation of identified candidates and pathways
in human material always has to be considered, to ensure
the chance of developing new therapeutic concepts for
pulmonary diseases in human.
Abbreviations
3D: Three dimensional; AECI: Alveolar epithelial type I cell; AECII: Alveolar
epithelial type II cell; BPD: Bronchopulmonary dysplasia; CD90: Cluster of
differentiation 90; COPD: Chronic obstructive lung disease; CRABP: Cellular
retinoic acid binding protein; E: Embryonic day; ECM: Extracellular matrix;
FGF: Fibroblast growth factor; Gli-1: GLI-Kruppel family member;
LGR: Leucine-rich repeat- containing G protein-coupled receptor; P: Postnatal
day; PC: Phosphatidyl cholin; PDGF-A: Platelet-derived growth factor-A;
PDGF-C: Platelet-derived growth factor-C; PDGFRα: Platelet-derived growth
factor receptor-α; PGE2: Prostaglandine E2; PNX: Pneumonectomy;
PPARγ: Peroxisome proliferator activated receptor gamma;
PTHRP: Parathyriod hormone related peptide; PTHRP: Parathyroid hormone-
related peptide; SDF-1: Stromal cell derived factor 1; SMAD: SMAD family
member; SPC: Surfactant protein C; TCF21: Transcription factor 21;
TGF: Transforming growth factor; Thy-1: Thymus cell antigen 1;
αSMA: α−Smooth muscle actin
Funding
This study was supported by the Max Planck Society (all authors, DB, LN,
REM, KA); the German Center for Lung Research (Deutsches Zentrum für
Lungenforschung; DZL; all authors); the Federal Ministry of Higher Education,
Research and the Arts of the State of Hessen LOEWE Programme through
grant UGMLC, (all authors); Rhön Klinikum AG, through grants FI_66 (to
REM) and FI_71 (to K.A.); and the German Research Foundation
(Deutsche Forschungsgemeinschaft, DFG) through: Excellence Cluster
EXC147 “Cardio-Pulmonary System” (to REM), Collaborative Research
Page 9 of 11
Center SFB1213 (to REM), Clinical Research Unit KFL309 (to REM) and
individual research grant Mo 1879/1 (to REM).
Availability of data and materials
Not applicable.
Authors’ contributions
JARC, DPB, LN, REM, KA drafted manuscript; JARC, DPB, LN, KA prepared
figure; JARC, DPB, LN, WS, REM, KA edited and revised manuscript; JARC,
DPB, LN, WS, REM, KA approved final version of the manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Received: 14 February 2018 Accepted: 2 July 2018
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