Quantum dot imaging for embryonic stem cells
- Shuan Lin†1,
- Xiaoyan Xie†1,
- Manishkumar R Patel1,
- Yao-Hung Yang1,
- Zongjin Li1,
- Feng Cao1,
- Oliver Gheysens1,
- Yan Zhang1,
- Sanjiv S Gambhir1, 2,
- Jiang Hong Rao1 and
- Joseph C Wu1, 3Email author
© Lin et al.; licensee BioMed Central Ltd. 2007
Received: 14 April 2007
Accepted: 09 October 2007
Published: 09 October 2007
Semiconductor quantum dots (QDs) hold increasing potential for cellular imaging both in vitro and in vivo. In this report, we aimed to evaluate in vivo multiplex imaging of mouse embryonic stem (ES) cells labeled with Qtracker delivered quantum dots (QDs).
Murine embryonic stem (ES) cells were labeled with six different QDs using Qtracker. ES cell viability, proliferation, and differentiation were not adversely affected by QDs compared with non-labeled control cells (P = NS). Afterward, labeled ES cells were injected subcutaneously onto the backs of athymic nude mice. These labeled ES cells could be imaged with good contrast with one single excitation wavelength. With the same excitation wavelength, the signal intensity, defined as (total signal-background)/exposure time in millisecond was 11 ± 2 for cells labeled with QD 525, 12 ± 9 for QD 565, 176 ± 81 for QD 605, 176 ± 136 for QD 655, 167 ± 104 for QD 705, and 1,713 ± 482 for QD 800. Finally, we have shown that QD 800 offers greater fluorescent intensity than the other QDs tested.
In summary, this is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements, QDs will have a greater potential for tracking stem cells within deep tissues. These results provide a promising tool for imaging stem cell therapy non-invasively in vivo.
QDs' photophysical properties have broadened their application and shown great promise as imaging probes in bioimaging, drug discovery, and diagnosis. To keep up with their burgeoning utility, current QD technology has rapidly evolved. QDs have been used for tumor targeting and imaging , lymph node  and vascular mapping , and cellular trafficking [8, 10]. QDs can be delivered in a targeted fashion by conjugating them with ligands and antibodies. QDs can also be introduced into cells non-specifically, which serves as a potential tracking marker for cellular imaging.
Stem cell therapy holds promise for treatment of intractable conditions such as Parkinson's disease, ischemic heart disease, diabetes, and degenerative joint diseases [11–14]. There are two types of cells used in stem cell therapy, adult stem cells and embryonic stem (ES) cells. Of the two, ES cells are the ultimate source for use in cell-based therapy because they posses a virtually unlimited capacity for self-renewal and pluripotency, which is defined as the ability to differentiate into all cell types, including neurons, cardiomyocytes, hepatocytes, islet cells, skeletal muscle cells, and endothelial cells . In stem cell therapy, monitoring of cell survival and location after transplantation is important for determining their efficacy. Because the absorption and scattering of light in biological tissue can be considerable, any optical signal transmitted from deep tissues to the surface tends to diminish in strength (reviewed by Choy et al. ). With QDs' many advantages over traditional organic dyes, QDs may provide an excellent tool for imaging stem cell therapy.
In this study, we use the peptide-based reagent QTracker to label mouse ES cells with QDs and evaluate the utility of QDs for imaging stem cell therapy. We next show that labeling mouse ES cells with QDs does not adversely affect ES cell viability, proliferation, and differentiation. Finally, we examine QDs' potential for imaging ES cells in vitro and in vivo.
Qtracker intracellular QD delivery
QDs do not affect ES cell viability and proliferation
QDs have no profound effects on ES cell differentiation in vitro
Having demonstrated that QD labeling had no detectable effect on ES cell growth, we next tested its effect on cellular development and differentiation. Dubertret et al. showed that at high concentrations, QDs injected into an individual blastomere of Xenopus during very early cleavage stages can cause apparent abnormalities in late stage embryos . Therefore, we examined the pluripotency of QD labeled mouse ES cells to ascertain if any developmental interference would occur. In the literature, both human and murine ES cells have well-documented differentiation and replication capacities [19, 20]. Mouse ES cells were differentiated in vitro by hanging drop assay. We then isolated RNA samples from undifferentiated mouse ES cells and embryoid body at day 14 and analyzed them by RT-PCR. Both labeled and unlabeled undifferentiated ES cells (day 0) expressed ES cell specific marker Oct4. Likewise, both labeled and unlabeled differentiated ES cells (day 14) expressed specific markers for endoderm (alpha-1-fetoprotein, AFP), mesoderm (fetal liver kinase-1, Flk1), and ectoderm (neural cell adhesion molecule, Ncam) germ layers  (Figure 3c).
In vivomultiplex imaging using QDs
Detection sensitivity for in vivoimaging using QD800
Postmortem histologic analysis of QD labeled ES cells
Stem cells offer an exciting new branch of therapy to treat a variety of conditions and diseases. It is therefore important to develop methods to monitor cell survival and location after transplantation. Due to its many advantages over conventional organic dyes, QDs serve as good candidates to monitor these parameters. In order to evaluate their in vivo ability, we delivered them by using commercially available QTracker. Strategies for ex vivo cell labeling by QDs include non-specific endocytosis, microinjection, liposome mediated uptake, electroporation, and peptide-based reagents. Previous studies have shown that the liposome-based reagent Lipofectamin 2000 had the highest delivery efficiency, but the QDs were delivered in aggregates . Electroporation also delivered QDs in aggregates , and may even cause cell death. Peptide-based QTracker  reagents (Invitrogen, CA) deliver QDs into the live cells, and have been shown to be an excellent and easy tool for studying live cell mobility  and cell fusion .
In this report, we evaluated ES cells labeled with QDs using commercially available Qtracker for non invasive in vivo imaging in living mice. Twenty-four hours after labeling ES cells with QDs, 72% of the cells were positive. However, by day 4 the percentage of positive cells dropped to 4%. This dramatic decrease could be due to the rapid division of ES cells (doubling time of 12 – 15 hours) or QD diffusion out of dividing cells over time thus causing a dilution of QD signal. The dramatic decrease in signal is consistent with a previous study that used QDs to label human cervical adenocarcinoma cells .
Another important question is whether QDs affect ES cell properties (i.e., pluripotency and self-renewal) that make them an attractive choice for regenerative therapy. Previous studies have shown that QD toxicity is dose dependent with increasing concentrations affecting cell growth and viability . However, we were interested in any toxicity caused at concentrations used for labeling cells for in vivo applications. Therefore, we examined ES cell proliferation and viability at one QD concentration (10 nM) and observed no significant changes between QD labeled ES cells and control unlabeled ES cells. This was true for all QDs tested: QD 525, 565, 605, 655, 705, and 800. These results concur with the study by Jaiswal et al. that also showed no adverse effects by QDs on the viability, morphology, function, and development of various other cells . Likewise, we confirmed that QDs also had no adverse affect on ES cell differentiation based on RT-PCR analysis of germ layer specific genes. Implanted ES cells are known to form teratoma tumors with a variety of differentiated tissues . In Figure 6, we found that the teratoma consisted of a variety of tissues including respiratory epithelium, osteochondroid, squamous cell, and immature brain-like neural cell based on histology. This confirmed that QD labeling did not affect in vivo differentiation as well. However, although ES cell-derived teratomas were retrieved from the animals, they were not shown to be QD labelled. We believe that the in vivo signal could be due to uptake of QD by neighboring host cells. Thus, the poor retention of QDs in targets cells may be a problem for long-term tracking, and more detailed analysis are needed to address this issue in the future.
Another advantage of QDs is their ability to do multiplex imaging of different QDs at the same time. However, in our study, ES cells labeled with different QDs were only capable of being imaged up to day 2 after subcutaneous implantation. A likely cause for this could be the loss of signal due to rapid cell division. Another possible cause could be serum instability of the QDs. Cai et al. reported that QD 705 lost 14% of its original intensity after 24 hours of incubation in mouse serum . Any loss of signal could hamper detection of QD labeled cells at later time points, especially those that are not within the near-infrared region since signals from these QDs will also be mostly absorbed by the skin. For those QDs that are in near-infrared region, QD 705 and QD 800, the difference in intensity could be due to transfection efficiency since these two QDs have similar extinction coefficients and quantum yield according to the manufacturer. However, extinction coefficients and quantum yield data were obtained in vitro and not in an animal setting. Moreover, the transfection efficiency was similar across all QDs. Therefore, we believe transfection efficiency is unlikely to be the cause of the difference in intensity observed in vivo. Due to its higher extinction coefficient and wider emission spectra within near-infrared region, only QD 800 signals were capable to be imaged in the animals for up to 14 days. We observed an increase in signal intensity when using a red shifted excitation laser (640 nm) to image QD 800 labeled ES cells. The normal excitation wavelength is 465 nm. This was somewhat surprising since the excitation coefficient of QD 800 is lower at 640 nm than it is at 465 nm. That is at 640 nm, QD 800 absorbs light with less efficiency than at 465 nm, so less QDs become excited and thus give off lower signal intensities. However, the tissue penetration is much greater at 640 nm. Therefore, labeled cells that would not have been excited at 465 nm could be excited at 640 nm. Thus, these newly excited cells could contribute to the greater signal intensity seen at the detection wavelength of 800 nm.
In summary, we report the successful demonstration of labeling ES cells with QDs and imaging these labeled cells in vivo. We have shown that it is feasible to label ES cells with QDs by Q-Tracker with high efficiency. After labeling, QDs did not affect the viability, and proliferation of ES cells, and have no profound effect on differentiation capacity of ES cells within the sensitivities of the screening assays used. We tested multiplex imaging in vivo using the Maestro system and showed that QD 525, QD 565, QD605, QD 655, QD 705, and QD 800 labeled ES cells can be detected in vivo using a single excitation wavelength (465 nm). This versatility makes them good candidates for tumor targeting , lymph node  and vascular mapping , and cell trafficking [8, 10] in small animal imaging. Nevertheless, the use of QD in stem cells is only beginning to be explored. To our knowledge, this is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements (e.g., near-infrared QDs, better serum stability, and improved cell retention), QDs will have greater potential for tracking of stem cells within deep tissues.
Culture of undifferentiated ES cells
The murine ES-D3 cell line (CRL-1934) was obtained from the American Type Culture Collection (ATCC; Manassas, VA). ES cells were kept in an undifferentiated, pluripotent state with 1000 IU/ml leukemia inhibitory factor (LIF; Chemicon. ESGRO, ESG1107) and grown on top of the murine embryonic fibroblasts feeder layer inactivated by 10 ug/ml mitomycin C (Sigma). ES cells were cultured on 0.1% gelatin-coated plastic dishes in ES medium containing Dulbecco Modified Eagle Medium supplemented with 15% fetal calf serum, 0.1 mmol/l β-mercaptoethanol, 2 mmol/l glutamine, and 0.1 mmol/l nonessential amino acids as described previously [27, 28].
Flow cytometry and fluorescent microscopy
Trypsinized mouse ES cells were labeled with QD 655 (10 nM) using Qtracker according to the manufacturer's protocol. Briefly, 10 nM of labeling solution was prepared according to the kit direction. Trypsinized mouse ES cells (1 × 106) were added to the 0.2 ml of labeling solution. After incubating at 37°C for 60 minutes with intermittent mixing, the ES cells were washed twice with PBS to remove any free QDs and plated on 0.01% gelatin coated plates. Fluorescence microscopy (Carl Zeiss Axiovert 200M) was used to image the cells on day 1. Labeled ES cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA) using the FL3 channel to detect QD 655 labelled cells on days 1, 4, and 7 post-labeling. Acquisition data were analyzed by the FlowJo software.
Effect of QDs on ES cell viability and proliferation
ES cells labeled with six different QDs (10 nM each) and control unlabeled ES cells were plated uniformly in 96-well plates at a density of 5,000 cells per well. Cells were treated according to the manufacturer's protocol and read out on a fluorescence microplate reader (SpectraMax Gemini EM, Molecular Devices Corporation, Sunnyvale, CA) at 24, 48, and 72 hours post labeling. For Trypan blue exclusion assay (indicative of cell death), aliquots of labeled cells were removed at specific time points and mixed with Trypan blue. The number of dead cells was determined by counting blue cells under a light microscope.
Embryoid body formation and differentiation
ES cells were differentiated in vitro by the "hanging drop" method as described previously [28–30]. Briefly, the main steps included withdrawal of LIF and cultivation of 400 cells in 18 μl hanging drops to produce embryoid bodies for 3 days, followed by cultivation as suspension in ultra-low-cluster 96-well flat-bottom plates for 2 days. Next, the embryoid bodies were seeded onto 48-well plates.
RT-PCR analysis of embryonic and germ layer specific transcripts
Reverse-transcription polymerase chain reaction (RT-PCR) was used to compare the expression of embryonic marker (Oct4), endoderm (alpha-1-fetoprotein, AFP), mesoderm (fetal liver kinase-1, Flk1), and ectoderm (neural cell adhesion molecule, Ncam) germ layer markers  between control unlabeled ES and QD-labeled ES cells. Total RNA was prepared from cells with Trizol reagent (Invitrogen) according to the manufacturer's protocol. The primer sets used in the amplification reaction were as follow: Oct4 forward primer GGCGTTCTCTTTGCAAAGGTGTTC, reverse primer CTCGAACCACATCCTTCTCT; AFP forward primer TATCAGCCACTGCTGCAACT, reverse primer GTTCAGGCTTTTGCTTCACC; Flk1 forward primer CACCTGGCACTCTCCACCTTC, reverse primer GATTTCATCCCACTACCGAAAG; Ncam forward primer GGAAGGGAACCAAGTGAACA, reverse primer ACGGTGTGTCTGCTTGAACA. PCR products were separated on 1% agarose gel electrophoresis and quantified with Labworks 4.6 Image Acquisition and analysis software (UVP Bio-Imaging Systems).
In vivofluorescence imaging of QD-labeled ES cells
Right after labeling ES cells with QDs by QTracker, the labeled cells were subcutaneously injected with Matrigel (50 μl, vol. 1:1, BD Biosciences, San Jose, CA) into various locations on the back of athymic nude mice (n = 6). Images were taken with an excitation filter of 465 nm and an emission filter of 510 nm long-pass using the Maestro Optical imaging (CRI Inc, Woburn, MA) as shown in Figure 1b. Detection was set to capture images automatically at 10 nm increments from 500 to 850 nm. The vendor's software (Nuance 2p12_beta) determined the correct exposure time for each QD labeled cells. The resulting TIFF image was loaded into the software and analyzed. Spectral unmixing was done using a user-defined library according to manufacturer's direction for each QD. Briefly, images of six different QD labeled ES cells in 1.5 ml micro-centrifuge tubes were taken separately. Each QD library spectra was decided and set by unmixing autofluorescence spectra and QD spectra manually selected from the image using the computer mouse to select appropriate regions. Images for QD800 sensitivity experiment was taken with an excitation filter of 640 nm and an emission filter of 700 nm long-pass.
Postmortem immunohistochemical stainings
After imaging, all animals were euthanized by protocol approved by the Stanford Animal Research Committee. Explanted subcutaneous teratomas were routinely processed for hematoxylin-and-eosin staining. Slides were interpreted by an expert pathologist blinded to the study (AJC).
Data were presented as mean ± SD. For Statistical analysis, the 2-tailed Student t test was used. Differences were considered significant at P < 0.05.
This work was supported in part by grants from the NIH HL089027 (JCW), NIH HL074883 (JCW), CIRM RS1-00322-1 (JCW), ACCF-GE (JCW) and SNM Bradley-Alavi Fellowship (SL).
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