Author + information
- Received May 11, 2011
- Revision received July 25, 2011
- Accepted July 28, 2011
- Published online January 1, 2012.
- Koen E.A. van der Bogt, MD, PhD⁎,†,⁎ (, )
- Alwine A. Hellingman, MD, PhD†,
- Maarten A. Lijkwan, MD⁎,†,
- Ernst-Jan Bos, MD⁎,†,
- Margreet R. de Vries, BSc‡,
- Juliaan R.M. van Rappard, MS⁎,
- Michael P. Fischbein, MD⁎,
- Paul H. Quax, PhD†,‡,
- Robert C. Robbins, MD⁎,
- Jaap F. Hamming, MD, PhD† and
- Joseph C. Wu, MD, PhD§∥,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Koen E. A. van der Bogt, Leiden University Medical Center, K6-41, P.O. Box 9600, 2300 RC Leiden, the Netherlands. OR Dr. Joseph C. Wu, Stanford University School of Medicine, 265 Campus Drive, G1120B, Stanford, California 94305-5454
Objectives This study aims to provide insight into cellular kinetics using molecular imaging after different transplantation methods of bone marrow–derived mononuclear cells (MNCs) in a mouse model of peripheral artery disease (PAD).
Background MNC therapy is a promising treatment for PAD. Although clinical translation has already been established, there is a lack of knowledge about cell behavior after transplantation and about the mechanism whereby MNC therapy might ameliorate complaints of PAD.
Methods MNCs were isolated from F6 transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). Male FVB and C57Bl6 mice (n = 50) underwent femoral artery ligation and were randomized into 4 groups receiving the following: 1) single intramuscular (IM) injection of 2 × 106 MNCs; 2) 4 weekly IM injections of 5 × 105 MNCs; 3) 2 × 106 MNCs intravenously; and 4) phosphate-buffered saline as control. Cells were characterized by flow cytometry and in vitro bioluminescence imaging (BLI). Cell survival, proliferation, and migration were monitored by in vivo BLI, which was validated by ex vivo BLI, post-mortem immunohistochemistry, and flow cytometry. Paw perfusion and neovascularization was measured with laser Doppler perfusion imaging (LDPI) and histology, respectively.
Results In vivo BLI revealed near-complete donor cell death 4 weeks after IM transplantation. After intravenous transplantation, BLI revealed that cells migrated to the injured area in the limb, as well as to the liver, spleen, and bone marrow. Ex vivo BLI showed presence of MNCs in the scar tissue and adductor muscle. However, no significant effects on neovascularization were observed, as monitored by LDPI and histology.
Conclusions This is one of the first studies to assess kinetics of transplanted MNCs in PAD using in vivo molecular imaging. MNC survival is short-lived, MNCs do not preferentially home to injured areas, and MNCs do not significantly stimulate perfusion in this particular model.
Peripheral artery disease (PAD) currently affects more than 27 million North Americans and Europeans and is associated with impaired leg function and decreased quality of life, leading to significant morbidity and mortality (1,2). Despite a variety of treatment options, including percutaneous transluminal angioplasty, stenting, and bypass surgery, a cluster of patients remain unresponsive to therapy, a third of whom have no option other than amputation (3).
Recently, stem cell therapy has emerged from bench to bedside as a treatment for end-stage PAD, potentially offering a last option for revascularization of the ischemic limb. Although results from pre-clinical experiments using bone marrow–derived mononuclear cells (MNCs) appear hopeful, outcomes from clinical studies are divergent (4), raising questions about cell behavior and mechanisms of action involved in the benefits of stem cell transplantation. Regarding cell behavior, 2 major questions to be addressed are the potential lack of donor cell survival after introduction into ischemic target tissue and inadequate cell homing to the injured area after systemic administration (5). Three mechanisms have been proposed as explanations for the results from cell transplantation thus far: donor cell death might hamper a lasting scaffolding effect, impair cell-induced neovascularization, and limit the secretion of protective paracrine factors by the transplanted cells.
To study stem cell behavior, one must be able to monitor cell location, migration, proliferation, and death. Recent proof-of-principle studies have demonstrated the ability to track cell fate after cardiac injections (5,6). In the present study, we monitor by bioluminescence imaging (BLI) the presence of MNCs after transplantation in mice with induced hind limb ischemia. These experiments are designed to answer critical questions regarding cell survival and homing patterns to the affected leg, as well as functional consequences of different transplantation strategies.
Animal study protocols were approved by the Animal Research Committees from both institutions (Stanford University and Leiden University Medical Center). The donor group for imaging experiments consisted of 8-week old male F6 mice, which were bred on FVB background and ubiquitously express green fluorescent protein (GFP) and firefly luciferase (Fluc) reporter genes driven by a β-actin promoter, as previously described (7). Recipient animals for these experiments consisted of syngeneic, male FVB mice (10 to 12 weeks old, Jackson Laboratories, Bar Harbor, Maine). Additionally, C57Bl6 mice were used (10 to 12 weeks old, Jackson Laboratories).
Preparation and characterization of MNCs
Please refer to the Online Appendix.
Characterization of MNCs by flow cytometry
Please refer to the Online Appendix.
In vivo optical BLI
BLI was performed using the IVIS 200 (Xenogen, Alameda, California) system. For in vitro characterization of luciferase expression, cells were suspended in different quantities in 1 ml of phosphate-buffered saline (PBS). After administration of 10 μl of D-Luciferin (43.5 μg/ml), peak signals (photons/s/cm2/sr) from a fixed region of interest were evaluated and plotted versus cell number. For in vivo experiments, recipient mice were anesthetized with isoflurane, shaved, and placed in the imaging chamber. After acquisition of a baseline image, mice were intraperitoneally injected with D-Luciferin (400 mg/kg body weight). Mice were imaged on post-injection days 1, 3, 6, 9, 13, 20, and 27. Peak signals (photons/s/cm2/sr) from a region of interest were evaluated as described (7). For ex vivo experiments, animals were euthanized immediately after the moment when peak signals were achieved. The organs were rapidly explanted and imaged according to the protocol described previously.
Laser Doppler perfusion imaging
Neovascularization was monitored by measurements of perfusion of the mouse hind limbs at the level of the paws and was performed before, directly after, and weekly over a period of 4 weeks after the surgical procedure with laser Doppler perfusion imaging (LDPI) (Moor Instruments, Axminster, United Kingdom) (8). To control for temperature variability during measurements, all animals were kept in a double-glassed jar filled with 37°C water, keeping environment temperature at a constant level during the LDPI measurements. Each animal served as its own control. Eventually, perfusion was expressed as a ratio of the flow in the left (ischemic) to right (nonischemic) paw. Before each LDPI measurement, mice were anesthetized with an intraperitoneal injection of midazolam (5 mg/kg, Roche, Woerden, the Netherlands) and medetomidine (0.5 mg/kg, Orion Corp., Espoo, Finland).
Surgical model for hind limb ischemia and cell injections
Before surgery, mice were anesthetized with either isoflurane (2%) or an intraperitoneal injection of a combination of midazolam (5 mg/kg), medetomidine (0.5 mg/kg), and fentanyl (0.05 mg/kg, Janssen, Tilburg, the Netherlands). The effect of MNC injections was tested in 2 models of hind limb ischemia: a single electrocoagulation model and a double electrocoagulation model (8). For unilateral single electrocoagulation of the femoral artery, ischemia was created by an electrocoagulation of the femoral artery just proximally to the superficial epigastric artery. Moreover, a double electrocoagulation was performed to create a larger therapeutic window for assessment of possible arteriogenesis (8). For this model, first an electrocoagulation of the common iliac artery was performed, followed by an electrocoagulation of the femoral artery. Subsequently, the skin was closed using 6-0 silk sutures. One day after operation, 40 μl cell/PBS injections were given into the adductor muscle, or 100 μl cell/PBS solution was injected into the tail vein. To compare the efficacy of a single versus repeated injection with cells, FVB mice were randomized into 3 groups: 1) single intramuscular (IM) injection of 2 × 106 MNCs; 2) 4 repeated IM injections of 5 × 105 MNCs; and 3) IM injection of PBS injection as control (n = 10 per group). The reason for using FVB mice in the first experiment was to establish a clinically resembling model of autologous cell transplantation as our F6 transgenic donor mice, used for in vivo BLI, were bred on FVB background. In addition, to investigate the functional effects of intravenous (IV) MNC injection, C57Bl6 mice were randomized into 2 groups: 1) single IV injection of 2 × 106 MNCs; and 2) IV injection of PBS (n = 10 per group). Additionally, in order to investigate the impact of the surgical model, 8 FVB mice were randomized to undergo either electrocoagulation or suture ligation of the femoral artery just proximally to the superficial epigastric artery. Both groups received IM injections of 2 × 106 MNCs into the adductor muscle. Subsequently, cell survival was monitored by BLI, and paw perfusion was followed by LDPI for 4 weeks or until full restoration was achieved in either group.
Ex vivo enzyme-linked immunosorbent assay for apoptosis on digested muscle
Please refer to the Online Appendix.
Ex vivo assays of reporter gene expression
To validate in vivo BLI findings, the bone marrow was collected as described earlier and assayed for GFP expression by flow cytometry, as described previously.
Please refer to the Online Appendix.
Please refer to the Online Appendix.
After isolation and Ficoll selection, the MNC population showed subpopulations of CD31+, CD34+, CD45+, and Sca-1+, but minimal Flk-1− cells, representing hematopoietic but not early endothelial progenitors cells. Moreover, the strong expression of CD11b, Gr-1, and NK 1.1, representative of macrophages, granulocytes, and natural killer cells, respectively, indicated the largely inflammatory character of this donor cell population (Fig. 1A).
Reporter gene characterization
For tracing the cells in an in vivo fashion by BLI, we first set out to characterize the expression of the reporter gene Fluc in vitro. As suggested in Figure 1B, luciferase expression intensity increased concordantly with increasing cell number. When maximal expression per well was plotted versus the amount of cells, a robust correlation was observed with an r2 value equaling 0.97 (Fig. 1C). Thus BLI signal intensity is closely representative of the amount of living cells carrying the luciferase reporter gene. Moreover, the activity of GFP in the donor-specific Fluc-GFP double-fusion reporter gene construct was confirmed by in vitro fluorescence microscopy (Fig. 1D).
Monitoring kinetics of transplanted MNCs by in vivo BLI
After single transplantation of 2 × 106 MNCs, a short-term post-transplant increase in BLI signal from 6.6 ± 1.5×104 at day 1 to 8.9 ± 2.5×104 p/s/cm2/sr at day 3 (p = NS) indicated an increase in cells in the adductor muscle region during the first days. Thereafter, however, cell death resulted in a rapid decrease in signal intensity down to background level after 4 weeks (Fig. 2). A similar cumulative dose of MNCs, divided in 4 weekly doses of 5 × 105 MNCs, led to a relatively stable presence of donor cells. No statistically significant differences after 4 weeks (5.1 ± 0.8 × 103 in single vs. 5.7 ± 0.3×103 in multiple dose group; p = NS) were detected.
Ex vivo postmortem localization of GFP+ -MNCs in the ischemic adductor muscles
Skeletal muscles of mice treated with a single injection of 2 × 106 MNCs and weekly injections of 5 × 105 MNCs were harvested 28 days after hind limb ischemia induction. Dismal numbers of GFP+ MNCs were only observed in the adductor muscle of mice that received weekly injections of MNCs (Fig. 3). These GFP+-MNCs surrounded vessels within the muscle tissue, suggesting a role for these cells in neovascularization. By contrast, GFP+-MNCs were not observed in the adductor muscles of mice receiving a single injection of MNCs.
LDPI of blood flow restoration after MNC transplantation in FVB mice
Single femoral artery electrocoagulation resulted in a significant decrease in paw perfusion when compared with the healthy right hind limb (p < 0.001 for all groups) (Fig. 4). Three days after MNC transplantation, a trend toward better flow recovery with increased MNC number was observed, as the ischemic/nonischemic paw perfusion ratios in the single 2 × 106 MNC and 4-weekly 5 × 105 MNC injection groups were 0.75 ± 0.07 and 0.67 ± 0.07, respectively, as compared with 0.62 ± 0.07 in the PBS group (p = NS). However, no significant differences were observed, as robust recovery of paw perfusion was seen in all groups by week 4.
Histological analyses of collateral formation
Figure 5 shows no significant differences in collateral density and collateral size in the post-ischemic adductor muscle after a single 2 × 106 MNC injection, 4 repeated 5 × 105 MNC injections, and PBS control, further confirming the lack of functional improvement in LDPI results. As shown in Online Figure 1, treatment with both single 2 × 106 MNCs and 4 weekly 5 × 105 MNCs led to significantly (p = 0.03 and p = 0.02, respectively) decreased amounts of fragmented DNA (mirroring apoptosis) as compared with the PBS group, which had an almost 3-fold higher expression than its healthy contralateral counterparts.
Monitoring cell homing in vivo after systemic MNC injection
The initial BLI signals on day 0 (1 h after IV transplantation of MNCs) equaled background, confirming that the cells were spread throughout the circulatory system (Fig. 6). No signs of retention in the pulmonary capillaries were observed, in contrast to previous studies using larger size cell types such as mesenchymal stem cells (9). Over time, however, signal intensity increased due to homing and migration to the injured area. In addition, signals arose from the bone marrow, spleen, and liver, suggesting homing patterns that mimic endogenous myelomonocytic pathways (10).
Ex vivo confirmation of in vivo patterns of cellular kinetics
To validate and further specify the observed in vivo findings after systemic MNC injection, organs were procured immediately after mice were euthanized. As shown in Online Figure 2A, BLI after dissection of the skin showed in situ signals from liver, spleen, and the long bones similar to in vivo results. However, the signals that were previously observed from the injured area in vivo were now largely concentrated in the subcutaneous fat pad as well as in the femoral bone. Indeed, when the different tissues were explanted, low signal was seen from the adductor muscle, whereas equally strong signals were observed from the scarred skin, the subcutaneous fat pad, and the bone marrow in the femoral bone. Thus the ex vivo imaging results confirmed the robust in vivo signals from liver and spleen. Moreover, the presence of donor GFP+-MNCs in the bone marrow was validated with flow cytometry (Online Fig. 2B). Taken together, these experiments showed that BLI is a reliable method to monitor MNC trafficking in an in vivo environment and that homing to the injured area was not limited to the adductor muscle, but also occurred in more natural biological niches such as marrow, liver, and spleen.
Monitoring effects of intravenously injected MNC therapy
For the previous experiments, FVB mice were used in order to establish a clinically resembling model of autologous cell transplantation in which the donor group consisted of transgenic FVB mice expressing GFP-Fluc. However, we observed a robust endogenous restoration of paw perfusion in these FVB mice. Therefore, to investigate the functional effects of IV MNC injection, another strain of mice (C57Bl6) was used in combination with a double electrocoagulation to ensure a larger therapeutic window (8). After double electrocoagulation of both the femoral and iliac arteries, the ischemic/nonischemic paw perfusion ratio decreased dramatically from an overall mean of 1.04 pre-operatively to 0.04 post-operatively (p < 0.0001). IV injection of MNCs was incapable of restoring paw perfusion significantly, with a ratio of 0.60 in the MNC group compared with 0.57 in the PBS group (p = NS) at 4 weeks post-operatively (Fig. 7).
Effect of electrocoagulation on paw perfusion and MNC survival
Considerable differences exist between different murine models of hind limb ischemia (8). We next sought to determine whether the electrocoagulation or suture ligation method would better fit the purposes of investigating the therapeutic effect of cell therapy. As outlined in Online Figure 3, the suture ligation method did not result in a prolongation of the therapeutic window, as there were no significant differences in LDPI, with fast recovery of paw perfusion within 1 week (0.97 ± 0.10 vs. 0.62 ± 0.08; p = NS) (Online Figs. 3A and 3B). Moreover, cell survival was not significantly different in both groups, with a trend toward an increased early cell survival in the suture ligation group (p = NS) (Online Fig. 3C).
This is one of the first studies to evaluate post-transplant MNC behavior in a murine model of PAD using in vivo molecular imaging techniques. The main findings can be summarized as follows: 1) MNC survival after a single IM injection is short-lived; 2) repeated MNC injections do not provide a significantly prolonged cell survival; 3) homing of MNCs after IV injection is not limited to the area of injury but other natural niches; and 4) neither IM nor IV injection of MNCs results in an improved blood flow recovery after hind limb ischemia induction.
The clinical relevance of these findings is significant. Since the pioneering work of Tateishi-Yuyama et al. (11), more than 25 clinical trials have been registered on www.clinicaltrials.gov, using either IM or systemic injections into the ischemic leg. Although the findings from this first study raised early hopes, the initial positive outcomes have not since been confirmed by large randomized clinical trials. The original hope for using progenitor cells in regenerative medicine was to regenerate the damaged tissue by forming new blood vessels (12), skeletal muscle (13), or even myocardium (14). However, as the true regenerative capacity has been questioned (15), and considering the dismal survival capacity of MNCs and other progenitor cells in this and other studies (5), a more plausible explanation for a possible beneficial effect would be the secretion of protective cytokines, as suggested before (16). Indeed, it has recently been shown that a more profound angiogenic response can be achieved in ischemic muscle by transplanting progenitor cells overexpressing both vascular endothelial growth factor and stromal-derived factor-1 (17). Alternatively, and in order to achieve true regeneration, one could switch to more specialized cell types rather than whole MNCs. To this end, it has recently been shown that embryonic stem cell–derived endothelial cells can improve perfusion due to the favorable effect of engraftment and biological activity (18). Thus, in the future, it might be a feasible approach to use a set of growth factors or specialized cells for gene therapy or to use a combination of these 2 approaches.
Previous studies have assessed MNC function and mechanism after transplantation into the ischemic leg largely by using post-mortem histological techniques (19). However, this approach requires that animals be euthanized, which increases inter-animal variance and hampers longitudinal studies of the same subject. Moreover, the search for scant donor cells on histological slides from all organs is extremely difficult and time-consuming. As a consequence, these techniques are less suitable for studying the kinetics of cells through the body over time. To overcome these limitations, here we were able to take advantage of the double-fusion reporter construct carrying Fluc and GFP to yield valuable insight into longitudinal cell fate. By doing so, we were able to track the spatiotemporal kinetics of MNC homing, retention, and survival in a murine model of PAD.
Interestingly, we observed a relatively limited cell survival after IM injection in the adductor muscle that was independent upon the vascular occlusion method. After a short-term increase in BLI signal up to day 3, a rapid decrease in BLI signal intensity relative to background after 4 weeks was observed. The limited cell survival was confirmed by the immunohistochemical staining, against GFP+ cells. No MNCs were detected 28 days after a single 2 × 106 MNC injection with an anti-GFP immunohistochemical staining, whereas 1 week after the fourth transplantation of 5 × 105 MNCs, a small proportion of these cells could be found. These GFP+-MNCs were present near blood vessels, suggesting either a role in neovascularization or that these cells preferred being adjacent to oxygenated blood. The dismal survival in the adductor muscle is interesting because femoral artery ligation results in less profound ischemia in the adductor muscle as compared with the gastrocnemius muscle. This suggests that even in a normoxic niche, MNCs require more biologically attractive environments to be capable of robust survival. This once again stresses the need for development of cell survival–augmenting approaches such as scaffolds or transduction of cells with pro-survival factors.
Results from this study show that, after systemic injection, MNCs migrated extensively to the bone marrow, spleen, and liver. This pattern indicates that MNCs traveled to their natural biological niches, as all of these organs play a role in intramedullary and extramedullary hematopoiesis. Confirming this observation, our BLI findings are concordant with previous leukocyte scans showing retention in the liver and spleen (20). For future experiments, it will be important to improve homing to the ischemic muscles, which may increase angiogenic response as measured by LDPI. This could be realized in 2 ways: 1) improving the attractiveness of the target environment with stem cell mobilizer such as short hairpin knockdown of prolyl hydroxylase and factor-inhibiting hypoxia inducible factor (21); or 2) manipulating the cells to be more specifically guided. In this respect, it might be a better approach to isolate a subset of the mononuclear fraction such as the CD14+ expressing cells that are expected to play a more active role in the restorative process after ischemia (22).
First, GFP+-MNCs were used in the hind limb ischemia mouse model in order to study post-ischemic neovascularization. It has been suggested that GFP can elicit an immune response (23), which may influence collateral artery formation because this is an inflammatory driven process. However, the lack of any effect on post-ischemic perfusion recovery or collateral artery formation at the tissue level makes the interference of immunogenic GFP+ cells on arteriogenesis unlikely. Second, the present report studied MNC behavior in an acute model of hind limb ischemia. Clearly, this model is not truly reflective of PAD, which is a chronic disease. Unfortunately, a superior model with more chronically occluded arteries is not available yet in mice.
Taken together, to our knowledge, this is one of the first studies to monitor the kinetics of MNCs in PAD in an in vivo fashion using molecular imaging techniques. Results from this study highlight the caution that should be taken when interpreting results from experimental as well as clinical studies. The poor survival and homing patterns warrant further research that should aim for better retention and increased biological activity of the cells in the injured area. Additional research may make cell therapy a more valuable option for treating end-stage PAD, which is in urgent need of better treatment alternatives. To this end, molecular imaging should be fully exploited to provide more insight into the mechanisms of action for cell therapy.
For an expanded Methods section and supplementary figures, please see the online version of this article.
Dr. van der Bogt is supported by the Michaël van Vloten fund. Dr. Hellingman is supported by the TeRM Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science. Dr. Robbins is supported by National Institutes of Health Grant No. U01HL099776. Dr. Wu is supported by the Burroughs Wellcome Foundation Career Award for Medical Scientists and National Institutes of Health Grants No. RC1HL099117, R01HL093172, and R01EB009689. All authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. van der Bogt and Hellingman contributed equally to this study.
- Abbreviations And Acronyms
- bioluminescence imaging
- firefly luciferase
- green fluorescent protein
- laser Doppler perfusion imaging
- bone marrow–derived mononuclear cell
- peripheral artery disease
- phosphate-buffered saline
- Received May 11, 2011.
- Revision received July 25, 2011.
- Accepted July 28, 2011.
- American College of Cardiology Foundation
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