ARTICLESPUBLISHED ONLINE: 4 AUGUST 2013 | DOI: 10.1038/NNANO.2013.147

Tracking the engraftment and regenerative
capabilities of transplanted lung stem cells
using ﬂuorescent nanodiamonds
Tsai-Jung Wu1,2,3†, Yan-Kai Tzeng4,5†, Wei-Wei Chang2,3, Chi-An Cheng4, Yung Kuo4,5,Chin-Hsiang Chien1, Huan-Cheng Chang2,4,5 * and John Yu1,2,3,6*
Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost
lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate
and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity andinterference from background tissue autoﬂuorescence. Here we show that ﬂuorescent nanodiamonds, in combination withﬂuorescence-activated cell sorting, ﬂuorescence lifetime imaging microscopy and immunostaining, can identify
transplanted CD45–CD541CD1571 lung stem/progenitor cells in vivo, and track their engraftment and regenerative
capabilities with single-cell resolution. Fluorescent nanodiamond labelling did not eliminate the cells’ properties of selfrenewal and differentiation into type I and type II pneumocytes. Time-gated ﬂuorescence imaging of tissue sections of
naphthalene-injured mice indicates that the ﬂuorescent nanodiamond-labelled lung stem/progenitor cells preferentiallyreside at terminal bronchioles of the lungs for 7 days after intravenous transplantation.

The lung is a complex organ consisting of at least 40 different
cell lineages1. Following postnatal growth, the lung epithelium
slowly turns over under normal conditions, but its regenerative capacity can be activated substantially after injury2. Resident
lung stem/progenitor cells (referred to collectively as LSCs throughout this Article) are responsible for this epithelial repair and tissue
homeostasis. Several stem cell niches have been identiﬁed as being
located in terminal bronchioles or bronchoalveolar junctions3–6.
These putative LSCs are susceptible to infection by severe acuterespiratory syndrome (SARS) associated coronavirus6,7, leading todeterioration of lung tissue and loss of lung repair capacity. LSCs
have also been found to be able to renew and proliferate to form
epithelial cells in vivo4. They are potentially useful in regenerative
therapy, for example, in repairing damaged lung tissue in patients.
The advances and obstacles of using various strategies and assays
to establish the regenerative potential of endogenous LSCs havebeen reviewed recently8,9.
To enable the aforementioned therapeutic applications of LSCs,
it is crucial to determine their tissue-speciﬁc engraftment and regenerative capacity in animals. Achieving this goal requires the use of ahighly sensitive imaging modality, together with a highly stable(both biologically and photophysically) imaging probe, to allow
tracking of the transplanted cells in vivo over an extended periodof time. Compared with magnetic resonance imaging (MRI),
optical imaging offers the advantages of higher sensitivity, larger
in vitro and in vivo throughputs, and greater potential for clinicaltranslation10. However, existing ﬂuorescence-based technologies
such as dye labelling11 and in vitro transfection of cells with plasmids to express ﬂuorescent proteins (for example, GFP)12,13 or
non-ﬂuorescent compounds (for example, luciferase)14 often

suffer from the problems of photobleaching, interference frombackground tissue autoﬂuorescence and/or low transfection efﬁciency for primary cells. Although quantum dots have been
adopted and highly regarded as alternatives15,16, their applicationis hampered by their potential toxicity and facile degradationin vitro17 as well as in vivo18.
Fluorescent nanodiamonds (FNDs)19, a new member of thenanocarbon family20, have emerged recently as a novel nanotechnology imaging probe for biological applications21,22. The nanoscalediamond material is chemically robust, biologically inert, andnon-toxic at both cellular23,24 and whole-organism levels25,26. A
number of experiments have demonstrated that bare FND particles
can be readily taken up by cells through clathrin-mediated endocytosis27, but with an exceptionally low exocytosis activity28,29. When
excited by green-yellow light, the ﬂuorophores (for example, negatively charged nitrogen–vacancy centres, NV2) built into the FND
emit perfectly stable far-red emission (600–800 nm) with a ﬂuorescence lifetime (t) greater than 15 ns, even in cells30,31. This lifetime
is substantially longer than that (t ≈ 1–4 ns) of the endogenous andexogenous ﬂuorophores commonly used in cell biology32, making it
possible to separate FND emission from the strong autoﬂuorescencebackground of host tissue using ﬂuorescence lifetime imaging
microscopy (FLIM) and various nanosecond time-gating techniques31,33. In this work, we show that primary cells such as LSCs
can be spontaneously labelled with FNDs by endocytosis, and thatthe labelling does not eliminate cellular functions such as division,
proliferation and differentiation. By combining FND–FLIM withﬂuorescence-activated cell sorting (FACS) and immunostaining
analysis, we have been able to identify, unequivocally, transplanted
cells in histological lung sections after intravenous (i.v.) injection of1

Institute of Biochemistry and Molecular Biology, Program in Molecular Medicine, School of Life Sciences, National Yang-Ming University, Taipei 112, Taiwan,
Genomics Research Center, Academia Sinica, Taipei 115, Taiwan, 3 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, 4 Instituteof Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan, 5 Department of Chemistry, National Taiwan University, Taipei 106, Taiwan, 6 Centerof Stem Cells and Translational Cancer Research, Chang Gung Memorial Hospital, Linkou, Taoyuan County 333, Taiwan; † These authors contributed equallyto this work. * e-mail: johnyu@gate.sinica.edu.tw; hchang@gate.sinica.edu.tw
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NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2013.147
a

b

Oct-4

Cytokeratin-7

SP-C

SP-C

Nanog

CCSP

Aqp-5

Aqp-5

Figure 1 | Phenotypes of LSCs before and after induction for differentiation. a, Immunoﬂuorescence staining of the sorted CD452CD54þCD157þ cells at
the time of isolation against various types of undifferentiated and differentiated markers, including Oct-4 and Nanog (red), and cytokeratin-7, CCSP, SP-C andAqp-5 (green). The cells expressed neither type II nor type I pneumocyte markers (SP-C and Aqp-5, respectively). Cell nuclei were stained in blue by DAPI.
Scale bar, 10 mm. b, Examination for the differentiation of cultured CD452CD54þCD157þ cells in vitro. The cells were induced to undergo differentiation by
incubation in MCDB201 for 7 days, before being stained with antibodies against the type II pneumocyte marker, SP-C (green). Cells were then stained with
antibodies against the type I pneumocyte marker, Aqp-5 (green), after incubation in MCDB201 for 14 days. Scale bar, 50 mm. Data are representative images
from three independent experiments.

the FND-labelled LSCs into mice for more than a week, with singlecell resolution. The tissue-speciﬁc engraftment capacity of thesecells is further elucidated with lung injury models.

Isolation and characterization of LSCs
Single-cell suspensions of pulmonary cells were prepared from theproteolytic digestion of neonatal mouse lung tissues, and removalof red blood cells by lysis6. Viable LSC candidates were isolated by
FACS through staining with two speciﬁc cell surface markers(CD54 and CD157), propidium iodide and the CD45 antibody to
exclude haematopoietic lineage cells. Flow cytometric analysis
with CD31 and immunostaining with von Willebrand factor andisolectin showed that the sorted CD452CD54þCD157þ cells did
not express any of these endothelial markers, indicating that they
were not contaminated with endothelial cells (Supplementary
Fig. S1). Only a few cells of the subpopulation expressed a6-integrin, CD24 and EpCAM (Supplementary Fig. S2). The expression
pattern of these epithelial markers suggests that the presently isolated CD452CD54þCD157þ cells markedly differ from the
LSC cohorts reported in the literature34,35 (see Supplementary
section, ‘Results and Discussion’ for details).
To fully characterize the sorted CD452CD54þCD157þ cells,
immunoﬂuorescence analysis was ﬁrst performed at the time of isolation. Through a careful assessment for the speciﬁcity of the detection using isotype control antibodies (Supplementary Figs S3, S4),
we found that these cells expressed the undifferentiated embryonicstem cell markers (Oct-4 and Nanog), the epithelial cell marker(cytokeratin-7), and the club cell marker (CCSP), but not the differentiated type II and type I pneumocyte markers (SP-C and Aqp-5,
respectively) (Fig. 1a). There were no changes in the expression ofthese markers after cell culture on collagen I-coated plates using
DMEM medium supplemented with 10% fetal bovine serum(FBS), 1% insulin–transferrin–selenium (ITS) and 10 ng ml21 epithelial growth factor (EGF) for 10 days (Supplementary Fig. S5).
We next examined whether the cultured CD452CD54þCD157þ
cells had the capacity for cell differentiation. Cells were incubated in
MCDB201 medium supplemented with 1% FBS, 1% ITS and
10 ng ml21 EGF for 1–2 weeks. After 7 days of incubation, thecells became ﬂattened and larger, and SP-C expression was detectedin the cytoplasm close to the perinuclear region, which is indicativeof cell differentiation into type II pneumocytes (Fig. 1b). Extensionof the incubation to 14 days led to further ﬂattening and enlargement of the cells (approximately ﬁvefold greater than that of theoriginal undifferentiated cells). These morphological changes wereaccompanied with a decrease in SP-C expression and an increasein Aqp-5 expression, an indication that the prospectively isolated
CD452CD54þCD157þ cells have the potential to differentiate
into type II and then type I pneumocytes in a sequential manner.

Labelling of LSCs with FNDs

FNDs with dimensions of 100 nm were produced by ion
irradiation and thermal annealing of type Ib diamondpowders21,30. To carry out FND labelling, the cultured
CD452CD54þCD157þ cells (identiﬁed hereafter as LSCs) wereincubated with 100 nm FNDs in DMEM at 37 8C with 5% CO2
for 4 h, after which the cells were washed thoroughly with phosphate-buffered saline (PBS) to remove free FNDs, and then collectedby trypsin treatment. Flow cytometric analysis showed that theamount of 100 nm FNDs taken up by the LSCs reached a plateau
at a concentration of 100 mg ml21 (Supplementary Fig. S6). Thisbehaviour closely resembles that of many other cell types including
HeLa cells, 3T3-L1 pre-adipocytes and 489-2.1 multipotent
stromal cells27,29. An extraordinary feature of the FND labelling isthat in addition to far-red ﬂuorescence, data collected in the side
scatter (SSC) channel also provide useful information on FND
uptake (Fig. 2a)29. This is attributable to the fact that diamondhas the highest refractive index of all transparent minerals36, anddiamond nanocrystallites scatter light strongly in the visible
region and thus produce strong SSC signals. By properly gating
the bivariate plot (SSC versus Far-Red) in quadrants, two distinct
separate populations of FND-labelled and unlabelled cells could
be detected in these two channels (Fig. 2a). The double-positive
cell population, denoted SSCþFar-Redþ, was subsequently identiﬁed as FND-labelled cells without additional staining.
Primary cells like LSCs divide slowly (2 days for one division).
Moreover, they are sensitive and fragile, and properties such as
growth rate and differentiation capacity are likely to be affected by
the nanoparticle-based labelling. To address this issue, we tracked

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NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2013.147

a 104

b 150
120

Counts103

SSC

Control
D0
D2
D6
D15

102

101

Control
FND-LSC

90
60
30

100

0
100

102

101

103

104

101

102

103

104

Fluorescence intensity

Far-Redc100

d

DAPI/SP-CDAPI/SP-C

FND

Figure 2 | FND-labelled LSCs in vitro. a,b, Flow cytometric analysis of FND-labelled LSCs (red) and control LSCs without FND labelling (grey), showing two
distinct populations in the side scatter (SSC) and far-red ﬂuorescence channels (a), and that the FND-labelled LSCs cultured in DMEM could be continuously
monitored for 15 days post-labelling (b). D0, D2, D6 and D15 in b denote days 0, 2, 6 and 15 after FND labelling, respectively. c,d, Representative wide-ﬁeld
ﬂuorescence images of differentiated type II pneumocytes derived from the control LSCs (c) and FND-labelled LSCs (d) after induction in MCDB201 medium
for 7 days, followed by immunostaining with SP-C (DAPI in blue, SP-C in green and FND in red). The FND image was acquired by excitation at 515–560 nm
and collection of emission at .590 nm. No FNDs were found in cell nuclei. Scale bars, 10 mm. Experiments were repeated in triplicate.

the proliferation of the FND-labelled LSCs continuously over two
weeks by ﬂow cytometry (Fig. 2b). Immediately after FND labelling,
the mean ﬂuorescence intensity of the LSCs was 45-fold greaterthan that of the unlabelled control cells. A successive decrease inﬂuorescence intensity by half occurred approximately every 48 h
due to cell division. LSCs labelled with FNDs at 100 mg ml21 could
be identiﬁed for more than a week (approximately four divisions),
after which the cells became conﬂuent and stopped proliferating.
The identiﬁcation of LSCs in the cell culture up to day 15 revealed
that the extent of FND exocytosis was low29. Further analysis by ﬂuorescence microscopy showed that both untreated and FND-labelled
LSCs were able to undergo morphological alterations and express
SP-C on day 7 after induction (Fig. 2c,d). The expression of SP-C
in the cytoplasm of the FND-labelled cells implies that the endocytic
FNDs do not eliminate the apparent biological functions of the LSCs.

Tracking of FND-labelled LSCs in vivo
The performance of this nanodiamond-based optical imaging platform for in vivo stem cell tracking was ﬁrst tested with healthy
normal mice. Approximately 5 × 105 FND-labelled LSCs were
injected into the tail veins of adult mice (four weeks old). Mice
injected with saline served as controls. Organs and tissues including
lungs, kidneys, liver and spleen were collected for examination ondays 1, 4 and 7 after injection. Flow cytometric analysis conﬁrmedthat the injected LSCs preferentially resided in the lungs, and notin other organs (Supplementary Fig. S7). On day 1, 1.64% of thetotal population of viable pulmonary cells appeared as FND-labelled
LSCs (Fig. 3a). This fraction, however, markedly decreased to 0.22%684

and 0.12% on days 4 and 7, respectively. In this analysis, the gating
thresholds in the bivariate plots were carefully chosen by referringto the in vitro result (Fig. 2a) as well as the proﬁles of the saline controls (Supplementary Fig. S8) to ensure good reliability. With a false
positive rate of less than 0.05%, as determined from the controls, theobserved approximately tenfold decline in the SSCþFar-Redþ subpopulation was a reﬂection of the fact that most of the transplanted
cells were not functionally engrafted. It is most likely that they were
only initially trapped in the lung microvasculature and were eventually lost during the ﬁrst week following transplantation.
To exclude the possibility that the sorted SSCþFar-Redþ cells
were false positives, lung tissue sections were stained with haematoxylin and eosin (H&E) for morphological analysis, and then ﬂuorescently imaged. However, when searching for FND-labelled LSCs
in the tissue sections using confocal ﬂuorescence microscopy, theimages acquired using a continuous-wave 561 nm laser as thelight source were overwhelmed by tissue autoﬂuorescence and by
the ﬂuorescence derived from photoexcitation of the stain(Fig. 3b,c). With the aid of FLIM, the background noises and
FND ﬂuorescence signals could be readily distinguished. Gating
the ﬂuorescence signal at t ¼ 9–18 ns clearly revealed the locationof FND-labelled LSCs with an enhancement in the signal-to-noise
ratio of more than an order of magnitude (Fig. 3b). The identityof the FNDs was also conﬁrmed by prolonged excitation, which
did not result in any signiﬁcant decrease in ﬂuorescence intensity,
consistent with the unique characteristic of the NV2 ﬂuorophores.
We further examined whether our observation was a consequence of FND engulfment by resident macrophages. To address

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NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2013.147
Day 4

Day 1
0.52

104

1.64

103

102

103

102
101

101
97.23

100

100

0.61
102
103
Far-Red
Day 1

104

100

102
101

98.67
100

101

0.39
102
103
Far-Red

104

Day 4

98.92
100
100
101

0.29
102
103
Far-Red

104

Day 7

255
Intensity

FLIM

b

101

0.12

0.67

Confocal fluorescence

SSC

SSC

103

c

Day 7
104

0.22

0.73

Anti-F4/80

104

SSC

a

0

FLIM

3

Lifetime (ns)

TGF

18

Anti-F4/80/TGF

H&E/TGF

0

Figure 3 | FND-labelled LSCs in uninjured mice. a, Flow cytometric analysis of total lung cells collected from uninjured mice receiving an i.v. injection of
FND-labelled LSCs for 1, 4 and 7 days (n ¼ 3 per group). b, Representative FLIM, TGF and bright-ﬁeld H&E staining images of the same lung tissue sections
from mice in a. The merged H&E and TGF images show that the FND-labelled cells (denoted by black arrows) are primarily located in the subepithelium of
bronchiolar airways. Scale bar, 50 mm. c, Co-localization examination of FND-labelled LSCs and macrophages in a typical lung tissue section immunostainedwith macrophage-speciﬁc antigen F4/80 and haematoxylin counterstain. The enlarged view in the last panel shows that the transplanted FND-labelled cells(red, denoted by red arrows) and alveolar macrophages (brown, denoted by black arrows) are located at different positions. Scale bar, 10 mm. Experiments
were repeated in triplicate.

this issue, lung tissue sections were stained with the macrophagespeciﬁc antibody, F4/80, followed by haematoxylin counterstaining
and ﬂuorescence imaging. Overlapping of the bright-ﬁeld and timegated ﬂuorescence images (Fig. 3c) showed no sign of FND co-localization with the F4/80-stained macrophages, suggesting that theobserved FND-labelled LSCs were not phagocytosed after i.v. injection. Such identiﬁcation could not have been made using organicdyes such as carboxyﬂuorescein succinimidyl ester (CFSE)37,
because of the similarity in lifetime between CFSE and the background ﬂuorescence (Supplementary Fig. S9).

Engraftment of FND-labelled LSCs in lung injury models
It is known that the regenerative capacity of LSCs is determined not
only by their intrinsic developmental potential, but also by theirinteraction with other cell elements in their niches38. This capacity
could be substantially activated after tissue injury2. To illustrate
this effect, we tracked LSCs in vivo using mice pretreated withnaphthalene, which selectively ablated club cells in the epitheliumof terminal and respiratory bronchioles39. Club cells (or Claracells) are secretory cells that play a protective role in the bronchial
tissue against damage. In this experiment, 5 × 105 FND-labelled
LSCs were injected into the mice after lung injury for 2 days.
Because LSCs express CCSP (Fig. 1a), the extent of the injury andthe repair of the bronchiolar epithelium could be examined byimmunostaining against CCSP (club cell secretory protein). On
day 1, the bronchiolar epithelium in the lung-injured mice was sparsely surrounded by CCSPþ cells in both the control and treatmentgroups (Fig. 4a), showing low degrees of lung repair. Although

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Injured: Day 7b

Injured: Day 1
104

Injured: Day 7

SSC

102
101
98.76

FND-LSC

100
104

103

102
Far-Red

103

100

104
104

Injured: Day 7
101

FLIM

103

104

0.80

5.11

102
101

93.25
100

2.50

101

102
Far-Red

FLIM

103

104

100

93.21
100

0.88
101

102
Far-Red103

104

H&E /TGF

H&E/TGF

d

SSC

102

100

0.29
102
Far-RedFND-LSC

Injured: Day 1

101

103
FND-LSC

SSC

103

c

97.88
100

0.91

3.34

102
101

0.40

101

0.03

1.80

Control (saline)

Control (saline)

103

100

104

0.00

0.84

SSC

Injured: Day 1

Control (saline)a

NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2013.147

Figure 4 | FND-labelled LSCs in lung-injured mice. a,b, Immunohistochemical analysis of lung tissue sections (a) and ﬂow cytometric analysis of total lungcells (b) collected from naphthalene-injured mice receiving an i.v. injection of saline (control) or FND-labelled LSCs for 1 and 7 days (n ¼ 3 per group). The
tissue sections in a were stained with CCSP for club cells (brown). c, Representative FLIM, TGF and bright-ﬁeld H&E staining images of the same lung tissue
sections, showing the location of FND-labelled LSCs (denoted by white and black arrows) in terminal bronchioles of the lungs. d, FLIM and H&E/TGF imagesof the lung tissue section of a naphthalene-injured mouse on day 7, showing engraftment of the transplanted FND-labelled LSCs (denoted by white and blackarrows) to terminal bronchioles in cluster form. Scale bars, 50 mm. Experiments were repeated in triplicate.

some progress in club cell regeneration occurred in the control on
day 7, the bronchiolar epithelium of the mice injected with FND–
LSCs displayed a signiﬁcantly greater repopulation of CCSPþ
cells, that is, a greater regenerative capacity or a more rapid
restoration of the lung epithelium (Fig. 4a).
The almost complete restoration of the CCSPþ cells strongly
suggests that both endogenous LSCs and the transplanted FNDlabelled LSCs were involved in lung repair and homeostasis. The
suggestion was corroborated by ﬂow cytometric analysis, where
0.91% and 0.80% of the viable pulmonary cells were identiﬁed
as SSCþFar-Redþ on days 1 and 7, respectively (Fig. 4b). In comparison, the percentages of transplanted non-LSCs (such as
mouse embryonic ﬁbroblasts, MEFs) in the recipient lungs of the
injured mice were signiﬁcantly lower (0.33% and 0.17% on days
1 and 7, respectively) and should be attributed to non-speciﬁc
entrapment (Supplementary Fig. S10). Further analysis by compound imaging with H&E staining and time-gated ﬂuorescence(t ¼ 9–18 ns) revealed that the FND-labelled LSCs were preferentially localized in the terminal bronchioles of the lung-injured
mice (Fig. 4c), in contrast to the localization of the cells in the
subepithelium of bronchiolar airways in uninjured mice (Fig. 3b).
In the lung-injured mice, although most of the transplanted
FND-labelled LSCs were monodispersed or in singlet form
(Fig. 4c), they also exhibited different degrees of clustering in the686

terminal bronchioles (Fig. 4d). It is therefore speculated that thepattern of the distribution of these FND-labelled cells may reﬂect
different degrees of injury in the lung tissues. For instance, the clustered cells may represent regions that have sustained more injury
from the naphthalene. A close examination for the co-localizationof the FND-labelled cells with the CCSP (Fig. 5a) and pan-cytokeratin (Fig. 5b) epithelial markers by high-power ﬂuorescence imaging
with single-cell resolution conﬁrmed that the transplanted LSCs
were not entrapped in the microvascular network of the lung, butrather had been functionally engrafted and integrated in the
bronchiolar epithelium.
Our results have shown that the lung epithelia of the injured mice
were restored more rapidly after transplantation of the FND-labelled
LSCs than with saline control (Fig. 4a). To provide a semi-quantitative estimate40, we calculated the percentage (Ph) of the transplanted
LSCs engrafted to the lung based on the total number (Nv) of viable(PI2) cells isolated from the mouse lung tissue, the percentage (Ps)of the FND-labelled cells identiﬁed in FACS analysis, and thenumber (Nt) of the cells actually transplanted, as Ph ¼ NvPs/Nt.
With Nv ≈ 7 × 106, Ps ¼ 1.64% and Nt ≈ 5 × 105, the Ph fornormal mice was estimated to be 23% on day 1. This percentage,
however, markedly declined to 1.7% on day 7 (Fig. 3a). In contrast,
the Ph for the lung injury model exhibited a much smaller decrease,with Ph ≈ 13% on day 1 and Ph ≈ 11% on day 7 (Fig. 4b). The

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NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2013.147
aAnti-CCSP

FLIM

Anti-CCSP/TGF

c 100
Bronchioles
Alveolib

Anti-pan-cytokeratin

FLIM

Anti-pan-cytokeratin/TGF

Fraction (%)

80

60

40

20

0
Uninjured: D7

Injured: D7

Figure 5 | Identiﬁcation of transplanted FND-labelled LSCs in bronchiolar epithelia and their histomorphometric distribution. a,b, Club cell markers (CCSP, a)and epithelial markers (pan-cytokeratin, b) examined by FLIM and immunostaining of lung tissue sections prepared from naphthalene-injured mice on day 7
after i.v. injection of FND-labelled LSCs. The enlarged, merged immunostaining and TGF images in the red dashed boxes show that the FND-labelled LSCs
(red) co-localize with CCSPþ (brown) and pan-cytokeratinþ (brown) cells and reside at the bronchiolar epithelial region of the lung. Scale bars, 10 mm.
c, Histomorphometric distribution measured by visually counting the numbers of FND-labelled LSCs nested in terminal bronchioles (black) or alveoli (white)of the lungs of uninjured and injured mice on day 7 after i.v. injection. Results are averages of more than 30 visual ﬁelds per section, with error bars
representing standard deviations of the measurements (n ¼ 6 per group).

distinct contrast between these two results provides compelling evidence that the lodgement of the transplanted FND-labelled cells in
the injury models represents a ‘pro-active’ tissue-speciﬁc engraftment, instead of non-speciﬁc (or passive) entrapment.
A merit of the FND labelling technique is that it allows quantitative assessment of the distribution of transplanted LSCs in tissue
due to the excellent chemical stability and photostability of thenanomaterial. We conducted this analysis by acquiring images of
H&E staining using a bright-ﬁeld section scanner. From theseimages, the cells showing FND labelling were counted visually inrandomly selected regions. To reduce potential biases in the quantitative assessment, measurements were performed for more than
30 visual ﬁelds per section for six randomly selected sections ofeach sample, by independent workers. The results show that the percentages of FND-labelled LSCs nested in the terminal bronchioles
and alveoli of the lung-injured mice are 59+5% and 41+5%,
respectively, compared with 24+3% and 76+3% for the uninjured
mice (Fig. 5c). The marked difference in the histomorphometric distributions of the FND-labelled LSC population between these two
animal models serves as additional evidence that the transplanted
LSCs preferentially engraft to the terminal bronchioles of the mice
after lung injury. Although the mechanisms by which the injected
LSCs reach lung tissues and further engraft to the terminal bronchioles are still not yet fully understood38, our ﬁndings support thecontention that speciﬁc microenvironments (or niches) in lungs
play important roles in the regulation of tissue-speciﬁc engraftment,
structural support and the signals for self-renewal and differentiation of the stem cells41,42.

Conclusions
We have developed a novel nanodiamond-based platform thatenables long-term imaging and tracking of transplanted stem/progenitor cells in vivo. The platform consists of FND labelling,

FLIM and FACS technologies, with many unique features including
simple labelling, high throughput and unequivocal detection of ﬂuorescence signals that are not confounded by tissue autoﬂuorescence
and/or ﬂuorescence derived from exogenous stains. Moreover, theFND labelling does not eliminate the cells’ in vitro differentiationand in vivo migration. Using the combined techniques, we havebeen able to follow the fate of FND-labelled LSCs in vivo after transplantation, precisely locate their position in tissue with single-cellresolution, and quantify their engraftment capacity over a week
for the ﬁrst time. The platform is particularly suitable for the
study of rare stem cells that are difﬁcult to transfect to express ﬂuorescent proteins or non-ﬂuorescent compounds. The method is
general and also has broad applicability to other stem cell systems(such as haematopoietic stem/progenitor cells43,44).

Methods
Cell isolation and immunoﬂuorescence staining. Viable LSCs were isolated
from neonatal CD-1 (ICR) mice by proteolytic digestion of lung tissues and lysis of
red blood cells, followed by sorting with FACS after staining with propidium
iodide, CD54, CD157 and CD45 antibodies. Cells were then cultured throughseveral passages and seeded at a density of 2 × 105 cells ml21 in a collagen I-coatedplate using DMEM supplemented with 10% FBS, 1% ITS and 10 ng ml21 EGF.
To perform immunoﬂuorescence staining, cells in primary culture were ﬁrst ﬁxed
in 4% paraformaldehyde/PBS at room temperature, permeabilized, and blockedwith 0.5% Triton X-100 in 3% bovine serum albumin (BSA)/PBS. Cells werethen incubated at 4 8C with the primary antibodies against antigens including SP-C,
Aqp-5, cytokeratin-7 (all from Millipore), CCSP and Oct-4 (both from Santa Cruz).
After overnight incubation, the cells were washed and incubated withrespective FITC- or Cy3-labelled secondary antibodies (Jackson ImmunoResearch),and then counterstained with 4′ ,6-diamidino-2-phenylindole (DAPI).
Photomicrographs of the cells were immediately taken after staining with a Zeiss
ﬂuorescence microscope.
FND production and labelling. FNDs were produced by radiation damage oftype Ib diamond powders (Micronþ MDA M0.10, Element Six) using a 40 keV Heþ
ion beam, followed by thermal annealing at 800 8C, air oxidation at 450 8C
and puriﬁcation in concentrated H2SO4–HNO3 (3:1, vol/vol) solution at 100 8C

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ARTICLES

NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2013.147(ref. 30). LSCs after cell attachment were labelled by FNDs through endocytosis
at 37 8C with 5% CO2 for 4 h (ref. 29). The FND-labelled LSCs were then thoroughly
washed by PBS to remove free FNDs and then analysed by using a FACSAria
ﬂow cytometer (BD Bioscience) equipped with a 594 nm laser and a 670LP emission
ﬁlter in the far-red channel. To conduct differentiation studies, attached FNDlabelled LSCs were incubated in MCDB201 medium supplemented with 1% FBS,
1% ITS and 10 ng ml21 EGF. Cells were then ﬁxed, permeabilized, and stained withprimary antibodies against SP-C.
Confocal ﬂuorescence microscopy and FLIM. Confocal imaging was carried out
using a SP5 inverted microscope (Leica) equipped with three solid-state lasers
operating separately at 561, 488 and 405 nm for the excitation of FND, SP-C and
DAPI, respectively. Fluorescence was collected through an oil-immersion objective(×100, NA 1.4) and detected with either a photomultiplier tube (PMT) for SP-C
and DAPI or an avalanche photodiode (APD) for FND. To conduct FLIM, anexternal frequency-doubled picosecond Nd:YAG laser (IC-532-30, High Q Laser),operating at 532 nm with a pulse width of 7 ps and a repetition rate of 50 MHz,was coupled through a single-mode optical ﬁbre into the Leica microscope featuring
a FLIM system (Supplementary Fig. S11). FND ﬂuorescence was collected usinga ×63 oil-immersion objective and detected with an APD at a wavelength of
650–850 nm through a bandpass ﬁlter (OS019606-HQ750/210m, Chroma). A
time-correlated single-photon counting system (Pico-Harp 300, Pico Quant) wasused to measure the ﬂuorescence lifetimes, from which time-gated images wereobtained with the SymPhoTime software.
In vivo tracking. Mouse LSCs were labelled with bare FNDs for 4 h at 100 mg ml21.
After washing away excess FNDs, 5 × 105 cells in 100 ml PBS were i.v. injected into
adult mice (four weeks old) through tail veins. Mice were maintained until being
killed on days 1, 4 and 7 (n ¼ 3 per group), after which organs were collected
for analysis with FACS and FLIM. All assays were repeated for at least three
independent experiments. For ﬂuorescence imaging, tissues were ﬁrst ﬁxed in 10%
formalin overnight and then embedded in parafﬁn and sectioned. Sections(5 mm) were deparafﬁnized in xylene, rehydrated in a graded ethanol series, andincubated in blocking buffer. Sections were incubated overnight with antibodies
against antigens including F4/80 (eBioscience), CCSP and pan-cytokeratin(both from Santa Cruz) and then analysed immunohistochemically with aperoxidase detection kit (Vector Laboratories) using diaminobenzene as thesubstrate according to the manufacturer’s instructions. All sections werecounterstained with Mayer’s haematoxylin. Images of lung tissues after H&E
staining or immunostaining were acquired using a bright-ﬁeld scanner (Aperio
Digital Pathology System) equipped with a ×40 objective. In separate experimentswith lung injury models (n ¼ 3 per group), naphthalene dissolved in Mazolacorn oil was injected intraperitoneally at 275 mg kg21 into sex- and backgroundmatched adult (four weeks old) CD-1 (ICR) mice. FND-labelled LSCs were thenadministered 52 h after the naphthalene treatment and subsequently analysedfollowing the procedures described above. All assays were repeated in triplicate.

Received 4 October 2012; accepted 26 June 2013;
published online 4 August 2013

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Acknowledgements
This work was supported by Academia Sinica and the National Science Council, Taiwan(grants nos. 100-2119M-001-028 and 100-2321-B-001-036). The authors thank theDivision of Medical Biology, Genomics Research Center, for providing facilities andsupport for cell imaging and ﬂow cytometric analysis.

ARTICLES

Author contributions
T.J.W. and Y.K.T. conceived the study, conducted the experiments, interpreted the results,
and wrote the manuscript. T.J.W. performed the tissue-culture work, cell sorting andtracking experiments, and data analysis. Y.K.T. and Y.K. assembled the FLIM systems andproduced the image acquisition scripts. W.W.C. helped with the isolation of LSCs andcarried out data analysis. C.A.C. prepared FNDs and performed the cell tracking
experiments. C.H.C., H.C.C. and J.Y. supervised and planned the project, analysed the data,
and wrote the manuscript. All authors designed the experiments and edited
the manuscript.

Additional information
Supplementary information is available in the online version of the paper. Reprints andpermissions information is available online at www.nature.com/reprints. Correspondence andrequests for materials should be addressed to H.C.C. and J.Y.

Competing ﬁnancial interests
The authors declare no competing ﬁnancial interests.

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