Tracking intravenous adipose-derived mesenchymal stem cells in a model of elastase-induced emphysema.

BACKGROUND: Mesenchymal stem cells (MSCs) obtained from bone marrow or adipose tissue can successfully repair emphysematous animal lungs, which is a characteristic of chronic obstructive pulmonary disease. Here, we describe the cellular distribution of MSCs that were intravenously injected into mice with elastase-induced emphysema. The distributions were also compared to the distributions in control mice without emphysema.
METHODS: We used fluorescence optical imaging with quantum dots (QDs) to track intravenously injected MSCs. In addition, we used a human Alu sequence-based real-time polymerase chain reaction method to assess the lungs, liver, kidney, and spleen in mice with elastase-induced emphysema and control mice at 1, 4, 24, 72, and 168 hours after MSCs injection.
RESULTS: The injected MSCs were detected with QD fluorescence at 1- and 4-hour postinjection, and the human Alu sequence was detected at 1-, 4- and 24-hour postinjection in control mice (lungs only). Injected MSCs remained more in mice with elastase-induced emphysema at 1, 4, and 24 hours after MSCs injection than the control lungs without emphysema.
CONCLUSION: In conclusion, our results show that injected MSCs were observed at 1 and 4 hours post injection and more MSCs remain in lungs with emphysema.

Introduction

Stem cell-based cell therapy is relatively successful, demonstrating beneficial therapeutic effects across a wide range of disease such as heart disease, bone disease, cancer, hepatic disease, and lung disease1,2,3,4,5,6,7,8. Some preclinical trials report the therapeutic effects of using stem cells to treat chronic obstructive pulmonary disease (COPD), which is characterized by emphysema and chronic bronchitis9,10,11. Our recent research shows that bone marrow-derived mesenchymal stem cells (MSCs) are able to repair cigarette smoke-induced emphysema9. Other research also report that MSCs from bone marrow and adipose tissue demonstrate therapeutic effects against cigarette smoke- and elastase-induced emphysema11,12,13,14.

However, the role of engraftment in the mechanism of action of adult stem cells is controversial because engraftment rates of adults stem cells are low in recent studies15,16. In our previous study, only a few stem cells were observed from 1 day through 1month after intravenous injection9. Though many cells initially localize in the lungs as the major capillary bed encountered after systemic administration, the overall donor cell retention is minimal. It is important to know the distribution of systemically (intravenously) injected stem cells that need to be administered to COPD patients in clinical trials. Optical imaging demonstrates several advantages for tracking the distribution of injected cells: it is rapid, nontoxic, and provides the visible whole-body distribution. Detecting fluorescence is a general analytical method that uses a computer-controlled display detector. Quantum dot (QD) labeling kit is a commercially available for fluorescent labeling of cells and has strong intense, stable fluorescence that can be visible through several generations17,18.

Another method for tracking the distribution of injected human cells into mice is Alu sequencing. Alu sequences are present in 500,000-1,000,000 copies in the human genome and are observed primate specific19,20. Previous studies developed Alu-based real-time polymerase chain reaction (PCR) methods for quantifying human DNA in mixed samples obtained from various species21,22.

In our present study, we evaluate the distribution of intravenously injected MSCs by detecting fluorescence-labeled cells and the human Alu sequence in mice lung ex vivo. We also observed the distribution of intravenously injected MSCs in elastase-induced emphysematous mice.

Materials and Methods

1. Mice

C57BL/6 mice were purchased from Orient Bio (Seongnam, Korea). Mice were housed in a specific pathogen-free facility. All animal experiments were approved by the Institutional Animal Care and Use Committee of Asan Medical Center (Korea).

2. Induction of pulmonary emphysema and cell transplantation

To generate elastase induced model, 6-week-old female C57BL/6J mice were intratracheally instilled with porcine pancreatic elastase (Sigma-Aldrich, St. Louis, MO, USA). Each animal was intratracheally injected 0.4 U of elastase at day 0. The mice were intravenously injected with 5×105 of MSCs on day 7.

3. MSC culture and labeling

Human adipose-derived MSCs were purchased from Invitrogen (Carlsbad, CA, USA). MSCs were cultured in Mesen PRO RS Basal Medium supplemented with Mesen PRO RS growth supplement (Invitrogen) and incubated at 37℃ in a 5% CO2 atmosphere. Cells were used after 2-5 passages. MSCs were labeled using the QDs (Q-Tracker 800) Cell Labeling Kit (Invitrogen). MSCs (1×106) were suspended with 200 µL of complete growth medium and labeled with 10 nM of QDs labeling solution. After 60 minutes, labeled cells with QDs were washed twice with complete growth medium.

4. Ex vivo fluorescent imaging of QD-labeled MSCs

In total, 5×105 QDs 800-labeled MSCs were placed in 100 µL saline and injected into the mice through the tail vain. Mice were sacrificed after 1, 4, 24, 72, and 168 hours, and images of the major organs (e.g., lung, liver, and spleen kidneys) were taken using the IVIS Spectrum Pre-clinical In vivo Imaging System (PerkimElmer, Waltham, MA, USA).

5. DNA extraction and quantitative PCR (qPCR) analysis

Standard genomic DNA samples were prepared using human adipose-derived MSCs (Invitrogen). Genomic DNA samples were isolated from murine organs, including the lungs, liver, spleen and kidneys, using a genomic DNA extraction kit (Qiagen, Duesseldorf, Germany).

6. Primer design and PCR amplification

The primers for Alu were 5'-CGAGGCGGGTGGATCATGAGGT-3' and 5'-TCTGTCGCCCAGGCCGGACT-3'. To perform the Light Cycler reaction, a master mix of the following reaction components was prepared to the indicated end-concentration: 10 µL of 2× SYBR green buffer, 1 µL forward primer (10 pM), 1 µL reverse primer (10 pM), and 8 µL DNA template (10 ng). The following LightCycler experimental protocol was used: initial denaturation for 12 minutes at 95℃, followed by 40 amplification cycles of 95℃ for 15 seconds and 70℃ for 1 minute to anneal and extend, respectively. Quantitative PCR experiments were performed using LightCycler 480 Software (Roche Diagnostics, Basel, Switzerland).

7. Data analysis

Statistical analyses were performed using the Graphpad Prism ver. 5 (GraphPad software, La Jolla, CA, USA). The Mann-Whitney test was used to compare both groups, and statistical significance was set at p<0.05.

Results

1. MSC distribution after intravenous injection

To observe the intravenously injected MSCs, we injected 5×105 MSCs that had been labeled with 10 nM QDs. After injection, fluorescent signaling was detected in the lungs in 4 hours, but this signal gradually decreased (Figure 1A). The liver did not demonstrate any fluorescent signals (Figure 1B). There were no fluorescent signals in the kidneys or spleen in MSC-injected mice (data not shown). To determine fluorescent intensity, regions that contained only 1 organ were selected and region of interest (ROI) values were obtained. The intensity of the fluorescent signals in the MSC-injected mice was only detected in the lungs at 4 hours after intravenous injection (Figure 1C).

Figure 1

Ex vivo fluorescence imaging and intensity analysis various time after intravenously quantum dots (QDs)-labeled mesenchymal stem cell (MSC) injection. (A) The ex vivo fluorescence images of the lung from control mice or intravenously QDs labeled in MSC-injected mice. (B) The ex vivo fluorescence images of the liver from control mice or intravenously QDs labeled in MSC-injected mice. Representative images are shown (n=3). (C) Radiant efficiency of the lung from control mice or MSC-injected mice. The data had a significant difference (*p<0.05) between the control group and the MSC-injected group.

2. Quantitative PCR analysis using human Alu sequences

To assess the optical imaging data and quantify the relative amount of transplanted MSCs, we prepared genomic DNA obtained from the organs (including the lungs, liver, kidneys, and spleen) of MSC-injected and non-injected mice and performed quantitative PCR using human-specific Alu sequence primers. A previous study developed an assay that detects samples in real-time using human Alu-specific PCR and SYBR Green I. To validate assay using human Alu-specific sequence, the regression r2 value was calculated by performing quantitative PCR using 10 ng of genomic DNA from 10, 102, 103, 104, 105, and 106 numbers of MSCs (Figure 2A). The calculated r2 value was 0.998, and this was used as the standard curve to determine the number of transplanted MSCs. The number of MSCs in the lungs was about 3,500 after 1-hour postinjection and gradually decreased with time (Figure 2B). MSCs were undetectable at 72-hour postinjection and were undetectable in the liver at all times (Figure 2C). In addition, no MSCs were detectable in the kidneys or spleen (data not shown).

Figure 2

The amount of mesenchymal stem cells (MSCs) in the lung and liver at 1, 4, 24, 72 and 168 hours after MSC injection using real-time polymerase chain reaction for human Alu sequence. (A) Calibration curves for human Alu sequence to evaluate the amount of MSCs in some organs. The amount of MSC in the lung and liver (B, C) from MSC-injected mice or control mice were calculated based on panel (A).

3. MSC distribution after intravenous injection into elastase-induced emphysematous mice

To evaluate the distribution of intravenously injected MSCs in elastase-induced emphysema mice, the animals were intratracheally injected with 0.4 U porcine pancreas-derived elastase and intravenously injected with 5×105 QD-labeled MSCs at 7 days after elastase injection. More fluorescent signals were detected in the lungs of elastase-injected mice than non-elastase-injected mice (Figure 3A). MSCs in the lungs of elastase-induced emphysematous mice-but not non-elastase-injected mice-were detectable at 24 hours after injection. ROI analysis showed that larger amounts of MSCs had accumulated in the lungs of emphysematous mice in comparison with the lungs in non-elastase-injected mice at every documented time point (Figure 3B). The intravenously injected MSCs disappeared from the lungs of the emphysematous mice and control mice at 72 hours (Figure 4). There were no fluorescent signals in the liver, kidneys, or spleen in either the emphysematous or non-elastase-injected mice (Figure 5). MSCs were maintained for longer periods of time in emphysematous mice.

Figure 3

Ex vivo fluorescence imaging and intensity 1, 4, and 24 hours after intravenously quantum dots (QDs)-labeled mesenchymal stem cell (MSC) injection with elastase-induced emphysema. (A) The ex vivo fluorescence images of the lung from control mice or intravenously QDs-labeled MSC-injected mice with or without elastase-induced emphysema. Representative images are shown (n=7). (B) Radiant efficiency of the lung from control mice or MSC-injected mice with or without elastase-induced emphysema. The data had a significant difference (*p<0.05) between the control group and the MSC-injected group; **p<0.05 between MSC injected with and without elastase-induced emphysema group.

Figure 4

Ex vivo fluorescence imaging 72 hours after intravenously quantum dots (QDs)-labeled mesenchymal stem cell (MSC) injection with elastase-induced emphysema. (A) The ex vivo fluorescence images of the lung from control mice or intravenously QDs-labeled MSC-injected mice with or without elastase-induced emphysema. Representative images are shown (n=7). (B) Radiant efficiency of the lung from control mice or MSC-injected mice with or without elastase-induced emphysema. The data did not have a significant difference.

Figure 5

Ex vivo fluorescence imaging 1, 4, 24, and 72 hours after intravenously quantum dots (QDs)-labeled mesenchymal stem cell (MSC) injection with elastase-induced emphysema. The ex vivo fluorescence images of the liver, kidney or spleen (A-C) from control mice or intravenously QDs-labeled MSC-injected mice with or without elastase-induced emphysema. Representative images are shown (n=7).

Discussion

We here observed intravenously injected MSCs using optical imaging and Alu-based real-time PCR and compared MSCs distributions between emphysematous and control mice. QD-labeled MSCs were detected at 4-hour postintravenous injection in the lungs. No QD-labeled MSCs were observed in any other major organ, including the liver, kidneys, and spleen. These relative quantification methods in murine organs use human Alu-and xenograft-based real-time PCR. We were able to detect 10-106 MSCs per 10 ng genomic DNA. Intravenously injected MSCs were observed at 24 hours, but gradually decreased with time. Next, we intravenously injected MSCs into structure destructed emphysematous lung (on day 7 after elastase injection) because we focused on basic data to obtain for clinical application. Emphysematous lungs maintained more MSCs for longer periods of time in comparison with controls through 24 hours after injection.

The systemic injection of MSCs is relatively easy in clinical settings. Easy application allows intravenously injected MSCs to demonstrate high levels of initial accumulation for the treatment of pulmonary diseases. Our previous study has shown that that treatment with bone marrow cells (BMCs) can repair cigarette smoke-induced emphysema9,23. Moreover, intravenously injected BMCs from male animals are rarely detected in female recipients after 1 month9,14. 111In-oxine-labeled MSCs that were co-labeled with ferumoxides-poly-L-lysine were detected using combination single-photon emission computed tomography (SPECT), X-ray computed tomography (SPECT/CT), and magnetic resonance imaging in an animal model of acute myocardial infarction24. Enhanced green fluorescent protein (EGFP)-labeled MSCs were also tracked in a model of liver injury25. We used QDs with near-infrared region (NIR) emission to label MSCs. QDs are characterized by the deep penetration of excitation and emission light in the NIR region and low autogenetic fluorescence. QD-labeled stem cells are not affected in terms of viability, proliferation, or differentiation in comparison with non-labeled stem cells26. In our current study, we first observed intravenously injected QD-labeled MSCs at various times in elastase-induced emphysema model.

The detection for MSCs using molecular levels such as DNA and RNA were tired with stem cell from EGFP transgenic mice using techniques of qPCR for EGFP gene27. This assay quantifies the relative number of EGFP optimized by beta-actin genes, but low cell numbers, such as 10 EGFP+ cells per 105 total cells, were undetectable. The Alu-based quantification of human DNA requires real-time PCR and human Alu-specific primers21,22. We used Alu-based real-time PCR to relatively quantify some organs, such as the lungs, liver, and kidneys, and we successfully observed about 3,500 numbers of MSCs in the lungs without specifically genetically manipulating the mice, including EGFP transgenic mice.

Stem cell homing and engrafting to injury sites has been performed previously in some disease models, including those for traumatic brain injury, liver injury, and cardiac and brain injuries24,25,28. The recruitment of stem cells to the injury site relies on well-known mechanisms that release various chemokines and cytokines29. Representative signals include stromal-derived factor-1 (SDF-1) and the CXCR4 axis at the injury site and stem cell surface, respectively29,30,31. Although there are no data indicating that SDF-1 or the CXCR4 axis affect stem cell homing and mobilization into emphysematous lungs in present our results, the axis is related to the recruitment of stem cells to the injury sites of other diseases, such as inflammatory bowel disease and myocardiac infarction32,33. We have here observed for the first time that intravenously injected MSCs remain in greater numbers for longer periods of time in emphysematous lungs in comparison with controls. There are still very low numbers of intravenously injected MSCs although MSCs had therapeutic effects on emphysema and other diseases9. Some researchers explain one of action mechanism that is paracrine effects by some factors such as growth factors, anti-inflammatory cytokine and immune-modulator derived from MSCs34. Further studies are needed on the roles of SDF-1 and the CXCR4 axis in the recruitment of stem cells to emphysematous lung and the development of COPD.

In conclusion, our results show the cellular distribution of intravenously injected MSCs shortly after injection. The specific goal of this study was to observe the cellular distribution of MSCs using appropriate tools and cross-validate these results with fluorescent and genomic findings. This is the first study to cross-validate fluorescence-labeled MSCs (i.e., the cellular level) and human-specific Alu sequences (i.e., the molecular level) and observe the cellular distribution of intravenously injected MSCs in a mouse model of emphysema. The lungs of emphysematous mice contained intravenously injected MSCs for longer periods of time in comparison with controls. These results suggest that more MSCs migrate to and remain at the injury site and Alu-based assays for quantifying transplanted MSCs are suitable for tracking the distribution of human cells in disease models.