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OPEN

received: 08 February 2017
accepted: 28 February 2017
Published: 30 March 2017

Fluorescent nanodiamonds
enable quantitative tracking of
human mesenchymal stem cells inminiature pigs
Long-Jyun Su1,2,*, Meng-Shiue Wu3,*, Yuen Yung Hui1,*, Be-Ming Chang1, Lei Pan1,
Pei-Chen Hsu4, Yit-Tsong Chen2, Hong-Nerng Ho5, Yen-Hua Huang6, Thai-Yen Ling3,
Hsao-Hsun Hsu4 & Huan-Cheng Chang1,7
Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells fortherapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable
methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various
animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugatedfluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of
human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs
by magnetic modulation. With this background-free detection technique and time-gated fluorescenceimaging, we have been able to precisely determine the numbers as well as positions of the transplanted
FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The
method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells inrodents and other animal models as well.
Mesenchymal stem cells (MSCs) are defined as self-renewing, multipotent progenitor cells with the capacity
to differentiate into distinct mesenchymal lineages such as osteocytes, chondrocytes, and adipocytes1. Human
MSCs are mainly found in bone marrow, adipose, and placenta tissues. These cells are one of the most promising sources of cell therapy and regenerative medicine for their multilineage differentiation potential and unique
immunomodulatory properties2. They have been applied to treat various human diseases including cardiovascular disorder, lung fibrosis, liver diseases, and graft versus host diseases following bone marrow transplantation3,4.
In light of the tremendous potential of this therapeutic approach, there is an imperative need to develop generaland reliable methods to measure the biodistribution and pharmacokinetics of these cells in vivo for preclinicalevaluation5. Such information is essential in clinical trials because it is vitally important to know whether thetransplanted MSCs completely home to the target organs or they have unwanted homing that will induce inappropriate differentiation leading to cancer development6.
A number of attempts have previously been made to track human MSCs in murine xenogeneic models by using either polymerase chain reaction (PCR) to detect human DNA or immunostaining to identify
human-specific nuclear proteins7,8. However, the data produced by these two methods provide little biodistribution information and are not quantitative enough to assess the safety and efficacy of these cells in vivo. In
1

Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan. 2Department of Chemistry,
National Taiwan University, Taipei 106, Taiwan. 3Department of Pharmacology, College of Medicine, National Taiwan
University, Taipei 100, Taiwan. 4Department of Surgery, College of Medicine and the Hospital, National Taiwan
University, Taipei 100, Taiwan. 5Department of Obstetrics and Gynecology, College of Medicine and the Hospital,
National Taiwan University, Taipei 100, Taiwan. 6Department of Biochemistry and Molecular Cell Biology, Schoolof Medicine, Graduate Institute of Medical Sciences, College of Medicine, Centre for Cell Therapy and Regeneration
Medicine, and International PhD Program for Cell Therapy and Regeneration Medicine, Taipei Medical University,
Taipei 110, Taiwan. 7Department of Chemical Engineering, National Taiwan University of Science and Technology,
Taipei 106, Taiwan. *These authors contributed equally to this work. Correspondence and requests for materials
should be addressed to T.-Y.L. (email: tyling@ntu.edu.tw) or H.-H.H. (email: ntuhsu@gmail.com) or H.-C.C.
(email: hchang@gate.sinica.edu.tw)
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Figure 1. Schematic of the experimental workflow. Major procedures include pcMSC isolation, FND labeling,in vitro assays, intravenous injection of FND-labeled pcMSCs into miniature pigs, and quantification of FNDs
extracted from organs of the xenotransplanted pigs.

particular, when applied to large animals such as pigs, which share many physiological similarities with humans9,
the PCR methods often yield unreliable results (Supplementary Table S1). More advanced methodologies (suchas nanotechnologies10) are in demand.
An ideal cell tracking method should be biocompatible and nontoxic, require no genetic modification, havesingle-cell detection sensitivity, and permit quantification of cell numbers at any anatomic location11. Although
X-ray, magnetic resonance, positron emission, gamma emission, and ultrasound-based modalities have enabled
non-invasive imaging of transplanted stem cells in vivo12, quantifying cell numbers with these methods is difficult.
A notable example is that of Templin et al.13 who used single photon emission computed tomography (SPECT)
to track human-induced pluripotent stem cells after 123I or 99mTc administration in a pig model. The method
relied on radioisotope labeling and also the expression of transgenic sodium iodide symporters. It is technicallydemanding and only semi-quantitative information on the biodistribution of the transplanted cells could beobtained.
Optical imaging utilizing organic dyes, fluorescent proteins, or nanoparticles as exogenous contrast agentsis more suitable for this purpose, although the technique is mainly used for small animal models in preclinicalexperimentation due to limited penetration depth of visible photons into tissue14,15. Among various exogenous
contrast agents, fluorescent nanodiamond (FND) has recently emerged as an attractive option because the nanomaterial is chemically inert and inherently biocompatible16. Additionally, the particle contains a high-density
ensemble of negatively charged nitrogen-vacancy (NV−) centers as built-in fluorophores, which are red fluorescent17, perfectly photostable18, and can be detected with high sensitivity in cells and tissues by fluorescence time
gating19, microwave modulation20,21, and magnetic modulation22. These distinctive characteristics together makeit possible to follow the biodistribution of human MSCs after FND labeling and subsequent transplantation in
animals such as miniature pigs.
Figure 1 exhibits a schematic diagram of the experimental workflow. We used human MSCs isolated fromplacenta choriodecidual membrane in this study for their high availability and low ethical issues23,24. The cells,
denoted as pcMSCs, were first labeled with albumin-conjugated FNDs and then transplanted into miniature pigs
by intravenous administration. After sacrificing the animals at specific time points, we extracted and quantified
the FNDs in five major organs by acid digestion and fluorescence intensity measurement. To minimize the sample
loss, we applied a magnetic modulation technique to achieve background-free detection of FNDs directly in thedigests without purification. Engraftment of the stem cells in tissues was finally identified by time-gated fluorescence imaging with single-cell resolution19.

Results

Quantification of FNDs.

Taking advantage of the unique magneto-optical property of NV− centers25, we
first developed magnetically modulated fluorescence (MMF) into a background-free detection method to quantify FNDs in aqueous solution. The development is important because it allows direct quantification of FNDs incells and tissue digests without pre-separation to avoid sample loss. The key instrument used in this quantification
is a home-built MMF spectrometer (Supplementary Fig. S1). Figure 2a displays a typical fluorescence spectrumof 100-nm FNDs suspended in water (1 mg/mL) and excited by a 532 nm laser equipped in this spectrometer. The

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Figure 2. Detection of FNDs in aqueous solution by MMF. (a) Normalized fluorescence spectrum of FNDs inwater (1 mg/mL), excited with a 532 nm laser. (b) Time trace of the peak fluorescence intensity at 687 nm undermagnetic modulation of f =​ 2 Hz. Inset shows the corresponding frequency spectrum after FFT. (c) Normalized
fluorescence spectrum of FNDs (5 μ​g/mL) in an acid digest of pig liver tissue (black) and its FFT spectrum (red)after demodulation at f =​ 2 Hz. The asterisk denotes the Raman peaks of water.

Figure 3. Quantification of FNDs in cells. (a) Dose-dependent uptakes of HSA-coated FNDs by A549 cells
and HeLa cells in culture, analyzed by MMF. (b) Comparison of the uptakes of HSA-coated FNDs by A549 cells,
analyzed by MMF and flow cytometry. Experiments were repeated in triplicate and error bars represent one
standard deviation of uncertainty.
fluorescence intensity maximizes at 687 nm, corresponding to the phonon sidebands of an electronic transitionof NV− centers. When exposed to a time-varying magnetic field with a strength of B =​ 20 mT and switching on
and off at a frequency of f =​ 2 Hz, the particles showed a modulation depth of ~5% in the fluorescence intensity(Fig. 2b). Fast Fourier transform (FFT) of the time trace yielded a distinct peak centered at 2 Hz (Fig. 2b). The
peak height corresponds to the demodulated fluorescence intensity.
To prove the utility of the MMF method, we spiked FNDs (25 μ​g) in a sample solution (5 mL) prepared bydigestion of pig liver tissue (1 g) in a concentrated mixture of HNO3 and H2O2 at 140 °C for several hours. As
shown in Fig. 2c, the fluorescence spectrum of the solution was overwhelmed by the fluorescence and Raman
signals of the digests. However, these signals exhibited no modulation when the sample solution was exposedto the time-varying magnetic field of B =​ 20 mT and f =​ 2 Hz. This characteristic allowed us to eliminate thesebackground signals by FFT of the time traces of all wavelengths using a MATLAB program developed in-housefor demodulation. The reduction of the background level was ~2 orders of magnitude, which enabled the recoveryof the entire fluorescence spectrum of FNDs (5 μ​g/mL) in such a noisy environment with good signal-to-noise
ratios (Fig. 2c).
We next applied the background-free detection method to quantify the amount of FNDs taken up by cells.
In this experiment, we first coated 100-nm FNDs with human serum albumin (HSA) by physical adsorption26
and then fed them to human lung adenocarcinoma epithelial cells (A549) or cervical cancer cells (HeLa) at theparticle concentrations of 10–200 μ​g/mL. The cellular uptake was subsequently analyzed by MMF after removalof untaken HSA-FNDs in the medium. To conduct the analysis, a standard calibration curve was prepared byplotting the measured fluorescence intensity against the FND concentration gradient. The amounts of internalized FNDs were then determined from the fluorescence intensities of the HSA-FND-labeled cells (1 ×​  106 cells/mL) in a cuvette (Supplementary Fig. S1) after sonication of these cells in water for 1 h to break up their plasma
membrane in a glass test tube. For A549 cells incubated with 100 μ​g/mL HSA-FND at 37 °C for 4 h, we determinedan average weight of 6.0 pg (or ~3.2 ×​  103 particles) per cell for the internalized FNDs. The weight decreased

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Figure 4. Characterization of pcMSCs. (a) pcMSCs in serum-free culture, displaying spindle-shaped
morphology. Scale bar: 100 μ​m. (b) FISH analysis of stem cells isolated from the placentas of male newborns. X
chromosomes are in red and cell nuclei in blue. The enlarged view shows two X chromosomes in the nucleus ofeach cell. Scale bar: 50 μ​m. (c) Karyotypical chromosome analysis of pcMSCs (n =​ 3). Only X chromosomes aredetected in all three pcMSC samples.
monotonically with the decreasing HSA-FND concentration to zero (Fig. 3a). The result is in good correlation
with flow cytometric analysis of the same cells incubated under the same conditions and detected in the far-red
channel (Fig. 3b)19. It is known that flow cytometry provides only a relative measure for the degree of cell labeling.
In contrast, MMF can reveal the absolute amounts of the labeling agents in the cells. Compared with A549, HeLa
cells can take up twice more particles (Fig. 3a), reflecting a distinctly different endocytic behavior between these
two types of human cell lines.

Characterization of pcMSCs and FND-labeled pcMSCs. The pcMSCs used in this study were isolated from the choriodecidual membrane of human placentas donated by women who had undergone cesarean
sections. Uniquely, the cells could be cultured in serum-free defined medium and they displayed fibroblast-likemorphology after attachment (Fig. 4a). More than 95% of the cell population expressed CD29, CD44, CD73, andCD90, and less than 2% of the population expressed CD14, CD34, CD45, and HLA-DR (Supplementary Fig. S2).
In vitro assays for osteogenic, chondrogenic, and adipogenic differentiation of the cells all showed positive signals
when stained with Alizarin Red S, Alcian Blue, and Oil Red O, respectively (Supplementary Fig. S3)27,28. Only XX
chromosomes were detected by fluorescence in situ hybridization (FISH) (Fig. 4b and Supplementary Fig. S4).
Further examination of the cells by karyotyping analysis found no evidence of Y chromosomes (Fig. 4c), confirming that the pcMSCs were derived from the maternal part (i.e. decidua basalis) of the placenta, irrespective of
the gender of the newborns. No abnormal chromosomes were observed over 20 serial passages, proving the highstability of the cells under serum-free culture conditions.
In our first experiments with pcMSCs, the cells were grown and labeled by 100-nm FNDs coated with HSA.
The purpose of the HSA coating is twofold: (1) to facilitate the dispersion of FNDs in cell medium during labelingand (2) to allow easy removal of free FNDs by phosphate-buffered saline washes after labeling. Similar to A549
and HeLa cells, the pcMSCs avidly took up the HSA-coated FND particles. For cells labeled at 100 μ​g/mL for 4 h,flow cytometric analysis indicated more than 98% of the pcMSCs containing HSA-FNDs (Fig. 5a). Colocalization
studies showed that the internalized HSA-FNDs were predominantly trapped in lysosomes and did not enter cell
nuclei (Fig. 5b). At 100 μ​g/mL, we determined an average weight of ~100 pg for the FNDs in one cell or an average
number of ~5.5 ×​  104 particles/cell by MMF (Supplementary Fig. S5). This amount is about 10 times as high asthat taken up by A549 and HeLa cells (Fig. 3a). Remarkably, even with this large quantity of particles accumulated
in the cytoplasm, the internalized HSA-FNDs do not seem to alter the cell viability (Fig. 5c) and proliferation(Fig. 5d) properties of the stem cells. No apparent exocytosis of FNDs was observed after 10 days of incubation incell medium (Supplementary Fig. S6)19,29. Additionally, no significant difference between FND-labeled pcMSCs
and untreated pcMSCs in the ability of suppressing the proliferation of mouse splenocytes was found by immunomodulation assays (Fig. 5e)30. All the results are ascribable to the exceptional inertness of the nanomaterial.
Quantitative tracking of FND-labeled pcMSCs in vivo. To conduct in vivo tracking, we
injected HSA-FND-labeled pcMSCs into miniature pigs via their left internal jugular veins (Fig. 6a andSupplementary Fig. S7). A total of 12 miniature pigs were used and they were randomized into 4 groups. The pigs
in each group received an injection of either HSA-FND-labeled pcMSCs (1 ×​  106 cells/kg BW) or HSA-FNDs(0.1 mg/kg BW), which served as the control. After injection for 24 h or 48 h, the pigs were sacrificed and five
major organs (including bilateral lungs, spleen, bilateral kidneys, heart, and liver) were collected for biodistribution measurement and fluorescence imaging. To enable FND quantification, we digested the organs in aqua regia/
H2O2 mixtures to release the nanoparticles into the solution. Fluorescence intensities were then measured directlyfor FNDs in the tissue digests without extraction or other separation procedures to avoid loss of the particles
during centrifugation or filtration treatment. Thanks to the chemical robustness of the nanomaterial, the uniquemagneto-optical properties of FNDs were still preserved and their fluorescence signals could be readily recoveredfrom the acid digests by magnetic modulation at the concentration as low as 1 μ​g/mL. The recovery rate, determined by spiking a known amount of FNDs into the pig tissue followed by digestion through the same process,was more than 90%. Such quantitative analysis is inaccessible with molecular fluorophores such as organic dyes
and fluorescent proteins due to the lack of chemical stabilities in strong acids.
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Figure 5. Characterization of HSA-FND-labeled pcMSCs. (a) Flow cytometric analysis of pcMSCs labeledwith HSA-FNDs at the particle concentration of 0–100 μ​g/mL. (b) Confocal fluorescence images of pcMSCs
labeled with Lysotracker and HSA-FND, and their merged image with the cell nucleus additionally stained inblue with Hoechst 33342. Scale bar: 20 μ​m. (c) Cell viability of pcMSCs labeled with HSA-FNDs at the particle
concentration of 0–200 μ​g/mL. (d) Cell proliferation and (e) immunomodulation assays of pcMSCs labeled with
HSA-FNDs at the particle concentration of 100 μ​g/mL. In the immunomodulation assays, unlabeled pcMSCs
or FND-labeled pcMSCs were co-cultured with mouse splenocytes at the cell number ratios of 1:80 to 1:5. Ctl
represents mouse splenocytes only. Experiments in (c–e) were repeated in triplicate and error bars represent one
standard deviation of uncertainty.

A challenge in this experiment is that it is difficult to completely digest the whole organs collected from thesacrificed animals. Given a total weight of 20 kg for a miniature pig (Supplementary Table S2), organs such as
the lungs have a typical wet weight of 200 g, which is about 1000 times greater than that of a mouse31,32. To overcome the challenge, in addition to using aqua regia/H2O2 mixtures, we employed acetone to dissolve lipids anda high-pressure autoclave reactor to facilitate the acid digestion of the whole organs. The combined approachhas enabled us to determine the absolute amounts of FNDs (and thus the labeled cells) in the individual organs
with both high sensitivity and accuracy. Figure 6b displays results of the experiment, where up to 70% of the
HSA-FND-labeled pcMSCs were found to reside in the lungs of the miniature pigs after intravenous injection for
24–48 h. In contrast, the percentages of the HSA-coated FNDs in the same organs were only ~25% (Fig. 6c). Next
to the lungs, the livers contained the highest amount (~3%) of FND-pcMSCs and also free HSA-coated FNDs.
The contents of FNDs in the other three organs (spleen, kidneys, and heart) are all less than 1%. To the best of
our knowledge, this is the first time that such quantitative information was obtained for stem cell tracking in a
pig model.
The high sensitivity of this optical detection method, e.g. 1 μ​g FNDs out of 100 g organs or tissues, allows
the determination of not only the biodistribution of pcMSCs in the animals but also their partition in different
sections of organs or tissues. For the lungs, we found more HSA-FND-labeled pcMSCs residing in the rightlobes than in the left lobes (Fig. 6d). Further analysis for the partition of the cells in six different lobe sections
revealed that there were more pcMSCs retained in the upper and middle lobes than in the lower lobes of thelungs (Supplementary Fig. S8). It should be stressed that the presently observed biodistribution is independent of the amounts of FNDs taken up by the pcMSCs. Even with the amounts of HSA-FNDs in the cells varied by a factor of 2, the percentages of the pcMSCs detected in the lungs stayed nearly the same (Fig. 6e andSupplementary Fig. S9), which indicates the high reliability of this method.
The large size of the pig organs does not prevent us from identifying the transplanted HSA-FND-labeled pcMSCs in their tissues. To accomplish this, we took advantage of another outstanding property of FND, that is, theNV− centers in nanoscale diamonds have a fluorescence lifetime of ~20 ns, which is substantially longer than that(~3 ns) of cell autofluorescence33. We first performed fast screening of the tissue sections before deparaffinizationby using fluorescence lifetime imaging microscopy (FLIM) or time-gated confocal fluorescence microscopy toachieve background-free detection (Fig. 7a and Supplementary Fig. S10). Subsequent imaging of FNDs in thedeparaffinized samples with hematoxylin and eosin (H&E) staining allowed us to clearly identify the cells at
the terminal bronchioles of the lungs (Fig. 7b). Further studies by immunostaining with FITC-tagged antibodies for human-specific nuclei confirmed that the two fluorescent labels (FITC and FND) are colocalized within
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Figure 6. Quantitative tracking of HSA-FND-labeled pcMSCs in miniature pigs. (a) Central venous catheter
insertion (top) and intravenous injection (bottom) of HSA-FND-labeled pcMSCs and HSA-FNDs into thepigs with a syringe pump. (b,c) Biodistribution of HSA-FND-labeled pcMSCs and HSA-FNDs in five different
organs at two different time points. (d) Distribution of HSA-FND-labeled pcMSCs in different sections of thelungs, including left lobes (LL), right lobes (RL), and the accessory lobe (AL) at 48 h after injection. (e) Amountsof HSA-FNDs taken up by 3 sets of pcMSCs (blue) and the corresponding percentages of HSA-FND-labeled
pcMSCs (red) found in the lungs 24 h after intravenous injection. The HSA-FND concentration used for thelabeling was 100 μ​g/mL. Experiments in (b–d) were repeated in triplicate and error bars represent one standard
deviation of uncertainty.
the same cells (Fig. 7c). In contrast to organic dyes, which suffer from the problem of photobleaching, FND is
non-photobleachable even under continuous, long-term high-power laser illumination. The easy identificationof FNDs lends strong support for the validity and reliability of this nanotechnology-based method in findingtransplanted human stem cells in the pig models.

Discussion

In recent years, carbon nanomaterials (including carbon nanotube, graphene, and nanodiamond) have attracted
considerable attention as promising fluorescent markers and drug delivery devices34. While some of the nanomaterials have found practical use in bioimaging and nanomedicinal therapy, absolute quantification of them in cells
and tissues remains a challenge. This is mainly because it is difficult, if not impossible, to distinguish them frombackground carbon atoms by inductively coupled plasma mass spectrometry as typically conducted for metal,
oxide, and semiconductor nanoparticles35. With the availability of FNDs, the quantification has become possible.
In this work, we have developed MMF into an innovative method for absolute and sensitive quantification of
FNDs in biological matrices. No extraction or other laborious separation steps prior to the fluorescence intensity
measurement are required. Although magnetic modulation is less common than optical modulation, which has
been successfully applied for probing photoswitchable fluorescent proteins36, it is more applicable to samples withstrong light scattering and complex chemical compositions37. The method is ideally suitable for selective imaging
of FNDs in tissues and also sensitive detection of FNDs in highly contaminated solution.
Lung failure is one of the leading causes of death worldwide. The lungs can be injured by trauma or affectedby a number of diseases, including pneumonia and lung cancer. Recent discoveries of the therapeutic effects ofhuman MSCs have made cell therapy an attractive option for lung injury treatment38,39. However, prior to theclinical trials, it is crucial to determine the ideal doses, injection speeds, biodistribution, physiological functionsof the stem cells migrating to the target organs, and the lifelong duration of these cells before they are metabolized in host tissues. Such analysis will provide important pharmacokinetics, pharmacodynamics, and toxicology
information for assessing the safety and efficacy of the MSC treatment. Although mice are the most commonlyused animal models, the marked differences in size and structure of the peripheral airways between humans androdents significantly limit their utility in preclinical settings.
We have chosen to use miniature pigs as the animal model in this study because they offer distinct advantages
in both size and availability over non-human primates9. Additionally, their intact respiratory systems display
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Figure 7. Fluorescence imaging of HSA-FND-labeled pcMSCs in pig tissues. (a) Fast screening of alung tissue section before deparaffinization to find HSA-FND-labeled pcMSCs: (1) bright-field image, (2)fluorescence image without time gating, (3) fluorescence image with time gating at >​ 8 ns, and (4) merged
images of (1) and (3). Scale bar: 50 μ​m. (b) Identification of HSA-FND-labeled pcMSCs in a lung tissue section
by confocal microscopy: (1) H&E staining image, (2) confocal fluorescence image without time gating, (3) timegated confocal fluorescence image, and (4) merged image of (1) and (3). White and black arrows denote HSAFND-labeled pcMSCs. Scale bar: 20 μ​m. (c) Immunostaining of pcMSCs in culture and in a lung tissue section:(1) nuclei of cultured cells stained with Hoechst 33342 in blue and FNDs in red, (2) cells in (1) additionallystained with FITC-tagged antibodies for human-specific nuclei in green, (3) a lung tissue section stained withthree fluorescent labels as in (2), and (4) photobleaching of the organic dyes (Hoechst 33342 and FITC) in (3) byrepeated laser illumination of the specimen. Scale bar: 20 μ​m.

human-like immune responses, similar to those of young adults40. Also, the anatomy and tissue structure of the
airways and lungs of these animals resemble those of humans. With pcMSCs introduced into the miniature pigs
by intravenous injection, our result shows that ~70% of the xenotransplanted cells are engrafted in the lungs formore than 48 h and these cells are predominantly located at the terminal bronchioles. It suggests a potential therapeutic use of pcMSCs to cure pulmonary disorders. Although the present study provides only the biodistribution
and pharmacokinetics data of human stem cells in healthy pigs, the developed methods can be readily applied todiseased or injured animal models. In our future experiments, we will conduct investigations on lung-injured pigs
and test the usefulness of the stem cells in repairing acute lung injury and treating other organ-specific diseases.
Furthermore, efforts will be made to conjugate FNDs with superparamagnetic iron oxide nanoparticles to form
dual-functional nanodevices for both magnetic resonance imaging and quantitative fluorescence tracking.
To conclude, we have demonstrated a viable application of FNDs for background-free imaging and quantitative tracking of human MSCs in animal models beyond rodents. The simplicity of this magneto-optical method,
together with the chemical robustness and biological inertness of the nanomaterials as well as the large quantityof the nanoparticles taken up by the cells, has permitted studies of the biodistribution and pharmacokinetics
of FND-labeled pcMSCs in miniature pigs for the first time. The technique is in excellent compatibility withFLIM and time-gated fluorescence imaging, which have been demonstrated to be a powerful means in acquiringhigh-contrast fluorescence images of FND-labeled cells in tissues. The ability to find single MSCs is particularly
valuable for ex vivo histological detection of these cells in clinical trials. This combined approach represents anappealing alternative to hazardous radioisotope labeling techniques in cell tracking applications.

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Methods

Chemicals.

Cell media MCDB201, SMEM, DMEM, and RPMI 1640 were obtained from Life Technologies,
human antibodies CD14-FITC, CD29-FITC, CD34-FITC, CD44-FITC, CD45-FITC, and CD90-FITC were from
GeneTex, CD73-FITC and HLA-DR-PE were from eBioscience, mouse anti-human IgG1-FITC, IgG2a-FITC, andIgG2a-PE were from GeneTex, anti-CD3 and anti-CD28 were from BD Biosciences, Hoechst 33342, Lysotracker
Green DND-265, and 6-carboxyfluorescein N-succinimidyl ester (CFSE) were from Invitrogen, Alizarin Red S,
Alcian Blue, Oil Red O, phosphate-buffered saline (PBS), human serum albumin (HSA), erythrocyte lysis buffer,and all other chemicals were from Sigma-Aldrich and used without further purification.

FND production.

FNDs (~100 nm in diameter) were produced by radiation-damage of synthetic diamond powders (Micro +​ MDA M0.10, Element Six) with a 40-keV He+ beam, followed by vacuum annealing at
800 °C, air oxidation at 450 °C, and acid washes in concentrated H2SO4-HNO3 (3:1, v/v) at 100 °C, as previously
described41,42.

Protein conjugation. HSA conjugation was made by first sonicating the FND particles in distilled deionizedwater (DDW) for 15 min. They were then mixed with the protein at a weight ratio of FND:HSA ≈​ 1:1 by gentle
shaking at room temperature for 2 h to allow physical adsorption. After removal of unbound HSA by centrifugation, the precipitate was extensively washed with PBS.
Cell culture and labelling.

A549 or HeLa cells were seeded at a density of 2 ×​  105 cells per 35-mm dish.
They were maintained in regular Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) and new born calf serum at 37 °C with 5% CO2 in a humidified incubator overnight for
attachment. Prior to cell labeling, HSA-coated FNDs were first dispersed in serum-free DMEM and then diluted
with the medium to have a concentration in the range of 10–200 μ​g/mL. After adding the labeling medium (1 mL)
to the dishes, cells were incubated at 37 °C for 4 h to facilitate uptake of the HSA-FND particles.

Magnetically modulated fluorescence (MMF).

Fluorescence spectra of FNDs suspended in aqueous
solution were acquired by using a MMF spectrometer built in house (Supplementary Fig. S1). The spectrometer
was equipped with a continuous-wave 532 nm laser (DPGL-2100F, Photop Suwtech), a dichroic beam splitter(Z532RDC, Chroma), a long-working distance microscope objective (50×​, NA 0.55, Mitutoyo), a long-pass edge
filter (E550LP, Chroma), and a multichannel analyzer (C7473, Hamamatsu). Backward fluorescence was collected
through the same objective to avoid spectral distortion due to strong light scattering by FNDs. To eliminate background noise, we magnetically modulated the FND fluorescence signal and then applied fast Fourier Transform(FFT) to extract concentration information from the measured fluorescence intensities. This was achieved byapplying a time-varying magnetic field of 20 mT from a round electromagnet (EM400-12-212, APW) to the sample solution held in a 10 mm cuvette with a frequency of 2 Hz. For every wavelength, we obtained a time-domain
spectrum with the magnetic field periodically switching on and off. A MATLAB program analyzed the time evolution of the spectra by performing FFT to yield intensities at the modulation frequency. The FND spectra were
finally restored by plotting the demodulated fluorescence intensities against wavelength.

pcMSC isolation. Human placentas were donated by women who had undergone cesarean sections with pro-cedures approved by the local ethics committees of Taipei Medical University Hospital (TMU-JIRB 201501063).
Written informed consent was obtained from all donors and experiments were performed in accordance with
relevant guidelines and regulations. To isolate pcMSCs, choriodecidual membrane was first physically separated
from the placentas and washed with Hank’s buffer to remove obvious blood clots. Clear choriodecidual tissues
were then shredded with a surgical knife in a digestion buffer (SMEM medium supplemented with 0.5 mg/mL
protease, 0.5 mg/mL collagenase B, and 1 mg/mL DNase I) and plated at 4 °C overnight. After pipetting and filtering the digestion buffer containing the tissue fractions through a 100-μ​m cell strainer, cells were collected bycentrifugation and washed with blank medium several times. The cells were subsequently re-suspended in culture
medium (MCDB201 supplemented with 1% insulin transferrin selenium, 10 ng/mL epidermal growth factor,
and 1% penicillin/streptomycin) and planted in culture dishes coated with human collagen type IV. After 24 h ofplantation, the dishes were shacked horizontally and washed with blank medium to remove non-adherent cells.
Finally, the adherent cells were kept in the culture medium changed every 3–4 days.

Immunophenotypic characterization.

pcMSCs after several passages were characterized by flow
cytometry with human antibodies against CD14-FITC, CD29-FITC, CD34-FITC, CD44-FITC, CD45-FITC,
CD73-FITC, CD90-FITC, HLA-DR-PE, mouse anti-human IgG1-FITC, mouse anti-human IgG2a-FITC, andmouse anti-human IgG2a-PE for their immunophenotypes.

Immunostaining and fluorescence in situ hybridization (FISH).

Immunostaining for human cell
nuclei was carried out by using anti-nuclei antibodies (MAB1281, Merck Millipore) and FISH analysis by using
the CEP X SpectrumOrange/Y SpectrumGreen Direct Labeled Fluorescent DNA Probe Kit (Abbott Molecular),both following the manufacturer’s protocols.

In vitro differentiation assays. Three differentiation assays were performed as follows. (1) Osteogenesis:
pcMSCs were plated at a density of 1 ×​  104 cells/well in a 24-well plate and supplied with osteogenic induction
medium (DMEM-high glucose with 10% FBS, 10 mM β​-glycerosphate, 50 μ​g/mL ascorbic acid, and 0.1 μ​M dexamethasone). After 28 days of incubation, cells were fixed in 4% paraformaldehyde (PFA), washed with H2O, andstained with 40 mM Alizarin Red S. (2) Chondrogenesis: pcMSCs were plated at a density of 5 ×​  105 cells/well ina 24-well plate and supplied with chondrogenic induction medium (DMEM-high glucose with 10% FBS, 10 ng/
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ml TGFβ​-1, 1% ITS, and 50 μ​g/mL ascorbate). After 21 days of incubation, cells were fixed in 4% PFA, rinsed with
0.1 N HCl for 5 min, and stained with Alcian Blue. (3) Adipogenesis: pcMSCs were plated at a density of 6 ×​  104cells/well in a 24-well plate and supplied with adipogenic induction medium (DMEM-low glucose with 10% FBS,
1 μ​M dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 μ​g/mL insulin, and 100 μ​M indomethacin). After21 days of incubation, cells were fixed and the lipid droplets were stained with Oil Red O using the mesenchymal
adipogenesis kit (SCR020, Millipore).

pcMSC labeling. pcMSCs after attachment were labeled with HSA-coated FNDs (10–200 μ​g/mL) throughendocytosis at 37 °C with 5% CO2 for 4 h. The HSA-FND-labeled pcMSCs were then thoroughly washed with PBS
to remove untaken FNDs. This was followed by trypsinization and analysis using a flow cytometer (FACSAria
II, BD Bioscience) equipped with a 532 nm laser for the cellular uptake. To enable fluorescence imaging, thecells were additionally labeled with Hoechst 33342 and Lysotracker Green DND-26 for nuclei and lysosomes,
respectively.
Cell viability assay. A cell counting kit (CCK-8, Sigma) was used to evaluate the cell viability after FND
labeling. First, pcMSCs (5 ×​  103 cells) were plated in a 48-well plate for 24 h and then labeled with 10–200 μ​g/mL
HSA-coated FND for 4 h. After PBS wash to remove untaken FNDs, WST-8 (a water-soluble tetrazolium salt) was
added to the wells and incubated for 3 h. Absorbance was measured at 450 nm using a microplate reader (BioTek)
and converted into cell numbers according to the calibration curve made with the known numbers of pcMSCs(1 ×​  103–1 ×​  105 cells/well) in the plate.
Cell proliferation assay. pcMSCs were labeled with 100 μ​g/mL HSA-coated FND for 4 h. Next, the cells
were trypsinized and replated at a density of 3 ×​  103 cells/well in a 48-well plate. The numbers of live cells were
measured by using the cell counting kit (CCK-8, Sigma) every day from day 1 to day 7.
Immunomodulation assay. Spleens isolated from C57BL/6 mice were mechanically shredded in RPMI
1640 medium supplemented with 10% FBS, 1% L-Glutamine, 100 U/mL penicillin and 100 μ​g/mL streptomycin
to release splenocytes. After standing for 10 min, the upper portion of the medium was collected and centrifuged
to obtain the primary splenocytes in the pellets. This was followed by the addition of erythrocyte lysis buffer to thepellets for 1 min, stopping of the reaction with the RPMI 1640 medium, and isolation of the splenocytes by centrifugation. After an additional wash with the same medium to remove traces of the lysis buffer, the splenocytes
were re-suspended in culture medium and incubated in a humidified chamber with 5% CO2/air mixture at 37 °C.
Immunomodulation assays were performed by labeling splenocytes with 5 μ​M CFSE at room temperature, following the manufacturer’s protocol. The CFSE-labeled splenocytes (1 ×​  106 cells/well) were then co-cultured witheither unlabeled pcMSCs or FND-labeled pcMSCs (1 ×​  104–2 ×​  105 cells/well) in a 24-well plate. The splenocyte
proliferation rates were finally assessed by flow cytometry to determine the numbers of CFSE-positive cells at 48 h
after activation with 0.1 μ​g/mL anti-CD3 and anti-CD28.
Animal experiments. Miniature pigs (weighing 18–24 kg each) were obtained from the Experimental Farmof National Taiwan University or the Taitung Animal Propagation Station, Taiwan. They were pair-housed on
a 12-h light/dark schedule (lights on at 0700) with controlled temperature and humidity. Food and water wereavailable ad libitum. The study was approved by the Institutional Animal Care and Use Committee (with IACUC
Approval No: 20140478) of College of Medicine and College of Public Health, National Taiwan University. All
experiments were performed in accordance with the guidelines and regulations established in the Guide for theCare and Use of Laboratory Animals.
Twelve miniature pigs were used in this study. They were randomized into 4 groups, each consisting of 3 pigs.
Prior to the experiments, the pigs were fasted overnight and premedicated intramuscularly with atropine (0.1 mg/
kg BW), zoletil 50 (10 mg/kg BW), and xylazine (10 mg/kg BW) to allow placement of an intravenous catheter
and continuous electrocardiogram and pulse oximetry monitoring. After mask preoxygenation, anesthesia was
induced with propofol (2–3 mg/kg BW) and succinylcholine (1 mg/kg BW) by intravenous bolus via an ear vein,and the trachea was orally intubated with a 6.0–8.0 mm inner diameter endotracheal tube. The pigs were provided
mechanical ventilation until breathing spontaneously following initial anesthesia at a volume-controlled settingof 20 mL/kg BW (body weight). During preparation, the respiratory frequency was adjusted at 15–20 breaths/
min to maintain the mean end-tidal CO2 pressure at 35–40 mmHg and the inspiratory oxygen concentration
was titrated to maintain saturation (>​96%), measured by pulse oximetry. Titrated anesthesia was maintained bypropofol infusion with a rate of 10 mg/kg/h, guided by the prospectively set target parameters for heart rate, bloodpressure, tail and hoof pinch response, and spontaneous breathing during the entire course of the experiment.
A central venous catheter was placed in the left or right internal jugular vein of the pig being experimented
on under sterile conditions. The animal was administered with a dose of 500 U heparin to prevent venous catheter clot formation and a dose of 1.5 million U penicillin to prevent infection. Each pig received an intravenous
injection of HSA-FNDs (served as the control group) or HSA-FND-labeled pcMSCs (served as the treatmentgroup) infused at a dose of 1 ×​  106 cells/kg BW in 60 mL by using a syringe pump at a rate of 150 mL/h. Afterinjection of the particles or cells for 24 h or 48 h, the pigs were sacrificed and five major organs (including bilateral lungs, spleen, bilateral kidneys, heart, and liver) were collected for fluorescence imaging and biodistribution
measurements.

Fluorescence imaging. Confocal fluorescence imaging was conducted by using an inverted confocal micro-

scope (Leica, SP8) equipped with a white-light continuum laser operating at 405, 488, and 561 nm for the excitation of Hoechst 33342, Lysotracker Green DND-26 (or FITC), and FND, respectively. Fluorescence was collected

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through an oil-immersion objective (63 ×​ , NA 1.4) and detected with either a photomultiplier (PMT) for Hoechst
33342 and Lysotracker Green DND-26 (or FITC) or a hybrid detector (HyD) for FND at emission wavelengths of
420–450 nm, 500–550 nm, and 700–800 nm, respectively. The use of HyD allowed effective removal of unwanted
background signals by time gating.

Biodistribution measurement.

Organs collected from sacrificed pigs were weighted and dried in oven.
After being cut into small pieces and mixed together, they were divided into several parts and each part (typically 20 g) was placed in a Florence flask (50 mL) containing aqua regia (24 mL) and 30% H2O2 (5 mL) heated to
85–100 °C for digestion until the solution became transparent. Indigestible portions were then collected by centrifugation and dissolved in acetone to remove lipid components. The remaining indigestible and undissolvabletissues were collected by centrifugation, placed in a Teflon jar containing 70% HNO3 (5 mL) in an autoclave reactor (BO-300, Hondwen), and heated to 90–200 °C under high pressure for 2 h. The residues were finally digested
in 70% HNO3 at 140 °C in a microwave reactor (Discover BenchMate, CEM) for 1.5 h and then added with 30%
H2O2 for decoloration. The reaction continued until the solution became clear. All fractions were finally pooled
together for fluorescence intensity measurements.

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Acknowledgements

We thank C.-Y. Fang, C.-K. Huang, W.-C. Ho, and C.-J. Lu for technical assistance. This work was supported byAcademia Sinica, Taiwan Lung Foundation, and the Ministry of Science and Technology, Taiwan, with Grant Nos
104-2119-M-001-001 and 104-2314-B-002-186-MY2.

Author Contributions

L.J.S., M.S.W., Y.Y.H., T.Y.L., H.H.H., and H.C.C. conceived and designed the experiments, Y.T.C., H.N.H.,
and Y.H.H. supplied materials and equipments, L.J.S., M.S.W., Y.Y.H., B.M.C., L.P., and P.C.H. performed theexperiments, L.J.S., M.S.W., Y.Y.H., T.Y.L., H.H.H., and H.C.C. analysed the data and co-wrote the paper.

Additional Information

Supplementary information accompanies this paper at http://www.nature.com/srep
Competing Interests: The authors declare no competing financial interests.
How to cite this article: Su, L.-J. et al. Fluorescent Nanodiamonds Enable Quantitative Tracking of Human
Mesenchymal Stem Cells in Miniature Pigs. Sci. Rep. 7, 45607; doi: 10.1038/srep45607 (2017).
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