Almost 80 million Americans suffer from cardiovascular disease, and with an average of one death every 36 sec, cardiovascular disease is the number one killer of Americans. Despite a wide range of therapeutic options to prevent progression of heart failure, end-stage disease can only be treated by heart transplantation, which is, in turn, hampered by a lack of suitable donor organs (1). Bone marrow mononuclear stem cells (BMSC) have raised hope as a new therapeutic modality as they were believed to differentiate into cardiomyocytes when transplanted into the infarcted murine myocardium (2). Although this observation is subject to controversy (3), BMSC-mediated cardiac repair has recently been introduced into clinical medicine (4). A small stromal subset of BMSC, called mesenchymal stem cells (MSC), is capable of proliferation in vitro (5) and therefore gains popularity as a candidate to replace the infarcted myocardium. MSC have been proposed to improve cardiac function after myocardial infarction (MI) in both animals (6) and humans (7), and might even have an immunomodulating effect (8). However, the process of bone marrow harvesting can be painful and is limited in the quantity of aspirate.
Recently, stromal cells have been isolated from the adipose tissue (9), which would be an ideal source regarding procurement procedure (e.g., elective abdominoplasty) and yield. These adipose stromal cells (ASC) largely express the same surface markers as MSC (10) and have shown to preserve cardiac function after infarction (11). Although the in vitro properties of ASC and MSC have been compared before (12–14), there are no reports evaluating the cellular behavior and functional effects of either cell type when transplanted into the ischemic myocardium. Here, we present the first report using a molecular imaging technique to unveil and compare the in vivo behaviors and functional effects of ASC and MSC after transplantation into the infarcted heart.
All animal study protocols were approved by the Stanford Animal Research Committee. The donor group consisted of male L2G mice (n=4, 8-weeks old), 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 (15). Recipient animals (n=37) consisted of syngeneic, female FVB mice (8-weeks old, Jackson Laboratories, Bar Harbor, ME). Animals were randomized into four recipient groups (n=8 per group): (1) adipose tissue-derived stromal cells (ASC), (2) bone marrow-derived mesenchymal cells (MSC), (3) fibroblasts (Fibro) as cellular control group, and (4) phosphate-buffered saline (PBS) as noncellular control group.
Cell Culture of Fibro, ASC, and MSC
Donor mice were sacrificed cervical dislocation after ample anesthesia with isoflurane and were placed in 70% ethanol for 5 min.
- For the isolation of Fibro, skin biopsies were taken from the tail and ears, minced and incubated overnight in collagenase type II (400 U/mL, Gibco-Invitrogen, Carlsbad, CA), dissolved in DMEM (Gibco, NY) supplemented with 20% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT), 1% antibiotics/antimycotic solution (Penicillin/Streptomycin, Gibco-Invitrogen, Carlsbad, CA) at 37°C and 5% CO2 in air as described (16). The next day, cells were dislodged from digested tissue by repeated pipetting and were passed through 70 μm sterile netting into sterile 15-mL centrifuge tubes. The samples were centrifuged for 5 min at 1200 rounds per minute (rpm), and the cell pellet was resuspended in DMEM/20% FBS/1%Penicillin- Streptomycin to be plated in a 25 cm2 tissue flask at 37°C/5%CO2.
- For the isolation of ASC, the adipose tissue was isolated from the inguinal and abdominal region as described before (17). In brief, the adipose tissue was washed in PBS and digested using 5 mL 0.075% collagenase (type I, Gibco-Invitrogen, Carlsbad, CA) in PBS for 30 min, followed by deactivation by DMEM/20% FBS/1%Penicillin-Streptomycin. After centrifuging for 5 min at 1200 rpm, the cell pellet was resuspended and incubated for 10 min in ACK lysing buffer to eliminate red blood cells. The suspension was centrifuged, resuspended in DMEM/20% FBS/1%Penicillin-Streptomycin, filtered through a 70-μm mesh, and plated in a 25 cm2 tissue flask at 37°C/5%CO2 to grow ASC.
- For the isolation of MSC, the long bones were explanted, washed and flushed with PBS using a 25-gauge needle to collect bone marrow. After passing through a 70 μm strainer, the isolate was centrifuged at 1200 rpm for 5 min, washed and resuspended into DMEM/20%FBS/1%Penicillin/Streptomycin medium to grow MSC as described (5).
At passage 8 to 10, the cells were labeled using specific FITC-conjugated antibodies against CD34, CD45, C-kit, Sca-1, CD90, and CD106 and processed through a FACSCalibur system (BD, San Jose, CA) according to the manufacturer’s protocol. Results were compared with appropriate isotype controls.
In vitro Firefly Luciferase (Fluc) Assays
Cells were dislodged from culture flasks to be resuspended in PBS. Cell suspensions were divided into a 6-well plate in known concentrations. After administration of D-Luciferin (Xenogen, Alameda, CA, 4.5 μg/mL), peak signal (photons/second/square centimeter/steridian or p/s/cm2/sr) was measured using a charged coupled device camera (IVIS200, Xenogen, Alameda, CA). Same amounts of dislodged cells were lysed using 200 μL of 10× Passive Lysis Buffer (Promega, Madison, WI) and centrifuged at maximum speed for 2 min at 4°C. For every sample, 20 μL of supernatant was added to 100 μL of Luciferase Assay Reagent (LAR-II, Promega, Madison, WI) and luminosity in relative light units was measured on a 20/20n luminometer (Turner Biosystems, Sunnyvale, CA). All samples were conducted in triplets.
Female FVB mice (8-weeks-old) were intubated with a 20-gauge angiocath (Ethicon Endo-Surgery, Inc., Cincinnati, OH) and were placed under general anesthesia with isoflurane (2%). MI was created by ligation of the mid-left anterior descending artery with 8–0 Ethilon suture through a left anterolateral thoracotomy. After approximately 10 min, the infarct region was injected with 5×105 cells or PBS respective of group randomization using a Hamilton syringe with a 29-gauge needle. The chest was closed in four layers with 5–0 vicryl suture. All surgical procedures were performed in a blinded fashion by a micro-surgeon (G.H.) with several years of experience with this model.
Echocardiography studies were performed 2, 4, and 6 weeks postoperatively. Three independent two-dimensional transversal-targeted M-mode traces were obtained at the level of the papillary muscles using a 14.7-MHz transducer on a Sequoia C512 Echocardiography system (Siemens, Malvern, PA). Using the enclosed software, left ventricular end- diastolic and -systolic posterior and anterior dimensions were measured by a blinded member of our group (A.Y.S.) and processed to calculate left ventricular fractional shortening (LVFS).
In vivo Optical Bioluminescence Imaging
Bioluminescence imaging (BLI) was performed using the IVIS200 (Xenogen, Alameda, CA) system. Recipient mice were anesthetized with isoflurane and then 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; Xenogen, CA). Mice were imaged on postoperative day 2, 4, 7, 10, and at week 2, 4, 5, and 6. Peak signal (p/s/cm2/sr) from a fixed region of interest was evaluated using Living Image 2.50.1 software (Xenogen, CA).
Before euthanizing the animal, invasive hemodynamic measurements were conducted by closed-chest pressure-volume (PV) loop analysis at week 6. The animal was placed under general anesthesia as described earlier. After midline neck incision, a 1.4F conductance catheter (Millar Instruments, Houston, TX) was retrogradely advanced through the right carotid artery into the left ventricle. The measurements of segmental conductance were recorded that allowed extrapolation of the left ventricular volume, which was coupled with pressure. These data were analyzed in a blinded fashion using PVAN 3.4 Software (Millar Instruments, Houston, TX) and Chart/Scope Software (AD Instruments, Colorado Springs, CO).
Hearts (n=3 per group at week 6) were flushed with saline and placed in 2% paraformaldehyde for 2 hr at room temperature followed by 12 to 24 hr in 30% sucrose at 4°C. The tissue was embedded in optical cutting temperature compound (Tissue-Tek Sakura Finetek USA Inc., Torrance, CA) and snap-frozen on dry ice. Five-micrometer sections were cut in both the proximal and apical regions of the infarct zone. Slides were stained for hematoxylin-eosin. Stained tissue was examined by Leica DMRB fluorescent microscope.
Ex vivo TaqMan Polymerase Chain Reaction
Animals were killed and hearts were explanted, followed by mincing and homogenization in 2 mL DNAzol (Invitrogen, Carlsbad, CA). DNA was isolated according to the manufacturer’s protocol. The DNA was quantified on a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and 500 ng DNA was processed for TaqMan polymerase chain reaction (PCR) using primers specific for the Sry locus selectively found on the Y chromosome in the male donor cells. Reverse-transcriptase PCR reactions were conducted in iCylcer IQ Real-Time Detection Systems (Bio-Rad, Hercules, CA). Lower cycle numbers represent higher donor cell counts (18). Samples were conducted in triplets.
Effects of Gender Mismatch, Myocardial Ischemia, and GFP on Cell Survival
To investigate the effect of our gender mismatch model, we injected 5×105 male and female cells into the hindlimbs of male FVB mice (n=5). To assess the effects of myocardial milieus (ischemia vs. non-ischemia) on cell survival, 5×105 MSC were injected into noninfarcted hearts from female FVB recipients (n=3). To evaluate whether GFP could have an effect on transplanted cell survival, 5×105 GFP+-Fluc+ cells were injected into the hindlimbs of female FVB mice that previously received intramyocardial GFP-Fluc plasmid injection 3 months prior (n=3). As control, 5×105 GFP+-Fluc+ were similarly injected in nonmanipulated FVB mice (n=3). In these experiments, cell survival was measured using in vivo optical BLI as described earlier.
Statistics were calculated using SPSS 15.0 (SPSS Inc., Chicago, IL). Descriptive statistics included mean and standard error. Comparison between groups was performed using a one-way between groups ANOVA, or, when compared over time, one-way repeated measures ANOVA, both with Bonferroni correction. A logarithmic transformation of values was performed when needed to ensure normal distribution within each group and significance was assumed when P is less than 0.05.
Characterization of ASC, MSC, and Fibro
After culturing for approximately eight passages, hematopoietic cells were eliminated from both ASC and MSC cultures. This was confirmed by flow cytometry showing absent CD34, CD45, and C-kit markers. Moreover, both populations were Sca-1+, CD90+, and CD106+ (MSC) or CD106− (ASC) (Fig. 1), consistent with prior literature comparing expression patterns of ASC and MSC (13). On microscopy, both cell types showed spindle-shaped morphology (Fig. 2a). Following isolation, MSC have slower population doubling time compared with ASC and Fibro before eventually growing like ASC and Fibro with an average population doubling time of approximately 2 days at passage 7 (Fig. 2b). All populations were furthermore tested for the expression of the reporter gene firefly luciferase (Fluc) (Fig. 2c). In all groups, cell number and Fluc signal correlated robustly with r2 values of 0.95 (ASC), 0.80 (MSC), and 0.97 (Fibro). Moreover, the assay of Fluc enzyme activity by luminometry also showed good correlation with cell number (ASC: r2=0.95; MSC: r2=0.85; Fibro: r2=0.98; Fig. 2d). Importantly, Fluc enzyme activity correlated well with the previous BLI findings (ASC: r2=0.86; MSC: r2=0.75; Fibro: r2=0.95). Taken together, these data suggest that BLI is a reliable tool for measuring viable cell numbers and can be used instead of luminometry. Moreover, we found a robust correlation between in vivo BLI signals and ex vivo TaqMan PCR cycle counts of Sry expression, indicating that cardiac BLI signal is representative of the presence of male donor cells in the female hearts (see Figure, Supplemental Digital Content 1, https://links.lww.com/A785).
Kinetics of Cell Survival by Longitudinal BLI
Previously, several groups have used reverse-transcriptase PCR or histological techniques and observed radical cell death after MSC transplantation into the ischemic myocardium (18, 19). However, these techniques do not allow for longitudinal imaging of cellular kinetics. Our BLI data showed that 2 days after intramyocardial transplantation, all cell types exhibited robust signals from the cardiac region, thereby confirming successful transplantation (20). However, in the following days, all three cell types experienced significant donor cell death (Fig. 3a). Quantitative analysis shows decreased signals at day 7 to 10 as compared with day 2, which reached background levels by week 4 to 5 (Fig. 3b–d). When normalized to the signal of day 2, there were no significant differences between ASC, MSC, and the Fibro control group (Fig. 3e, P=NS, repeated measurements ANOVA).
Influence of Gender Mismatch, Myocardial Milieus, and GFP Expression on In vivo Transplanted Cell Survival
To enable ex vivo validation of our in vivo BLI study, we performed a gender mismatch model with male donors and female recipients followed by TaqMan PCR of the male Sry gene. From studies with organ transplant patients, it has been noticed that male patients receiving female grafts have decreased graft survival and require more immunosuppressant drugs (21). Although this effect was generally less apparent in male-female transplants (21), we set out to investigate the role of gender mismatch by transplanting equal numbers of male and female ASC into hindlimbs of male mice. As shown in Supplemental Figure 2 (see Figure, Supplemental Digital Content 2, https://links.lww.com/A786), there were no significant differences in cell survival between the cell types from both genders. After two weeks, BLI signals were 6.3×104±0.8×104 p/s/cm2/sr for male ASC and 5.7×104±1.3×104 p/s/cm2/sr for female ASC (P=NS). In our study, it is also possible that GFP can elicit an immune response (22), which may have led to a decreased survival in our cardiac experiments. To differentiate between the effects of ischemic versus normal cardiac tissue on stem-cell survival, MSC were also transplanted into noninfarcted hearts. While acute survival was observed in the nonischemia group, the cells were still not capable of surviving for a prolonged period (see Figure, Supplemental Digital Content 3, https://links.lww.com/A787). To explore the influence of GFP-expression on cell survival, we transplanted GFP+-Fluc+ into the hindlimbs of naïve FVB mice or FVB mice that were presensitized by means of previous Fluc-GFP plasmid injection. By comparison of cell survival pattern, again there were no significant differences between BLI signals from both animals (see Figure, Supplemental Digital Content 4, https://links.lww.com/A788). In summary, while both gene mismatch and GFP immunogenicity could have affected cell survival, our direct comparison studies suggest that they were not the main contributing factors for the loss of imaging signal, and thus cell survival, in our experiments.
Assessment of Cardiac Contractility by Echocardiography
Previously, it has been observed that ASC (11) and MSC (23) preserved left ventricular dimensions and fractional shortening after infarction. However, there is no comparative functional data available between both cell types, after prolonged time in culture. In the current study, measurements of left ventricular dimensions revealed a gradual increase in diameters in both diastole and systole, suggesting negative remodeling in all groups without any significant benefits of cell transplantation when evaluated at weeks 2, 4, and 6 (Fig. 4a–c). For the cell groups (ASC, MSC, Fibro), after an initial nonsignificant increase in LVFS compared with the PBS group at week 2, the increasing ventricular dilatation resulted in a declining LVFS over time (Fig. 4d). By week 6 after cell transplantation, there was a trend toward improved LVFS in the ASC (33.1±1.0%) and MSC (33.0±3.5%) groups compared with the Fibro (31.3±2.2%) and PBS (32.0±1.8%) (Fig. 4d, P=NS, repeated measurements ANOVA) control groups, of which the latter had similar values compared with the literature (24). For complete echocardiography results, please refer to Supplemental Table 1 (see Table, Supplemental Digital Content 5, https://links.lww.com/A789).
Hemodynamic Measurements Using PV Loops
Recently, it has been shown that MSC were capable of preserving myocardial compliance, as measured by invasive hemodynamic measurements (25). Thus, to validate the echocardiography measurements of ventricular dimensions, invasive steady-state hemodynamic measurements of left ventricular diastolic and systolic volumes were conducted immediately after echocardiography at week 6. When plotted versus each other, mean diastolic and systolic volumes and diameters from each group correlated with r2 values of 0.88 and 0.70, respectively (Fig. 5a–c). Stroke work and cardiac output, important parameters of ventricular performance (26), did not differ among cellular groups but was slightly better than the PBS control group (Fig. 5d,e, P=NS, ANOVA). Ventricular contraction was not different between groups, but there was a trend toward an improved ventricular relaxation, as measured by the minimum ΔP/Δt, in the ASC and MSC groups (Fig. 5f,g, P=NS, ANOVA). Furthermore, an increase in arterial elastance, suggestive of higher afterload caused by arterial stiffening or increased peripheral resistance (27), was seen in the PBS group but this was not significantly different from the ASC and MSC group and the Fibro controls (Fig. 5d, P=NS, ANOVA).
Immediately after PV-analysis, the animals were euthanized after which the hearts were explanted. Gross morphology showed that ventricular dilatation and fibrous scar formation had not been prevented by cell transplantation (Fig. 6). As suggested by the BLI, echocardiography, and PV findings, transplantation of both ASC and MSC did not result in either repopulation (data not shown), or endogenous preservation of myocardial tissue.
This study has evaluated for the first time the in vivo behavior and functional effects of both ASC and MSC after injection into the ischemic myocardium, as compared with a cellular (Fibro) and noncellular (PBS) control group. The major findings are as follows: (1) molecular imaging using the Fluc reporter gene is a reliable tool for repetitively monitoring donor cell survival in vivo; (2) similar to the Fibro control group, ASC and MSC rapidly die off after injection into the infarcted heart; and (3) ASC and MSC were not capable of significantly preventing left ventricular remodeling and subsequent loss of cardiac function.
Until now, ASC and MSC studies have been largely based on in vitro studies (10) or investigations that monitored cell location and quantity by postmortem histological analysis (28) or PCR techniques (18). To study the true in vivo behavior of stem cells, one needs to be able to repetitively image cell location and count in a noninvasive fashion. One such approach would be the labeling of stem cells with iron particles, which would enable imaging by MRI (29). However, this technique does not provide insight into cell number because the same amount of iron particles are divided among daughter cells during cell proliferation or ingested by macrophages in case of cell death (30). By contrast, the current study demonstrates that molecular imaging of the Fluc reporter gene with the D-Luciferin reporter probe can provide repetitive, longitudinal in vivo imaging of donor cell survival in the infarcted heart of the same animal, thereby preventing sampling biases that can occur with the use of multiple animals that need to be killed on different time points to perform conventional histological staining (31).
The clinical relevance of this study is significant. MSC have been suggested as potential treatments for a variety of diseases including graft versus host disease, osteogenesis imperfecta, rheumatoid arthritis, multiple sclerosis, and MI (32). Because the ASC and MSC seem to be comparable cells with similar in vivo behavior, it would be possible to yield stromal cells from fat instead of bone marrow. Not only would this provide a less restricting source regarding yield, but it would also be a more patient friendly isolation procedure. In fact, it has even been proposed that human ASC are superior to MSC with regard to their paracrine and angiogenic potential in response to ischemia (33). Despite these reported advantages, an important finding from our study is that both MSC and ASC do not survive for longer term following transplantation into the infarcted heart. On the basis of our quantitative measurements of BLI signals, all cells have died by week 6. This is in concordance with earlier PCR findings from Muller-Ehmsen et al. (18), who noticed robust cell death after transplantation with less than 2% of the initially transplanted MSC being present at 6 weeks after cardiac transplantation. Similarly, Nakamura et al. (19) were only able to find 4.4% engraftment of MSC 1 week after intramyocardial transplantation, whereas Amsalem et al. (23) were unable to find any MSC 4 weeks after transplantation. Moreover, Mangi et al. (34) observed robust cell death early after transplantation, which was mitigated by transfecting the MSC with the pro-survival gene Akt-1. Thus, although prolonged survival of MSC and ASC has been proven possible in different models (35, 36), the environment in the heart proves hostile to these cells, resulting in decreased cell survival.
There have been several studies reporting preservation of cardiac function after MI and subsequent ASC (11) and MSC (37) transplantation. On the contrary, in this study we did not observe any functional benefit from ASC or MSC after transplantation into the infarcted heart. In fact, after an initial preservation, we have observed decreasing fractional shortening in both ASC and MSC groups from week 2 to week 6. Although these differences could be a consequence of differences in the experimental animal and surgical models, the observed short-term effect of cell transplantation is in concordance with findings from a growing body of experimental studies. Ultra-sensitive small animal MRI has recently been used to show that MSC fail to repair the ischemic heart and do not have any beneficial effect on cardiac function after infarction (38). This non-beneficial effect corresponds to other studies, which show that nontransduced MSC do not ameliorate functional heart failure after transplantation (39). In addition, Dai et al. (28) observed no long-term functional effect after an initial significant benefit 1 month after transplantation of MSC in infarcted rat hearts. In combination with the BLI data of the current study and as suggested in a recent review (40), the dismal cell survival of ASC and MSC might underlie this short-term effect. This poor survival pattern makes robust repopulation impossible and furthermore limits the protective paracrine action of the cells. This paracrine effect has been shown to be of crucial importance in MSC function (34, 41). By transfecting MSC with Akt-1, researchers have observed not only a better cell survival under hypoxic conditions, but also a robust increase in paracrine factors that resulted in a preservation of morphology and function of the infarcted heart (34, 41), effects that were also shown using MSC transfected with the pro-survival gene Bcl-2 (39). Thus, further research is needed to identify the factors responsible for the acute donor cell death in the heart (both infarcted and noninfarcted). Improving stem-cell survival may offer a sustained paracrine effect that could lead to protective effects on resident cardiomyocytes from apoptosis and a subsequent preservation of cardiac function.
Several limitations of this study can be raised. First, we used Fluc- and GFP-expressing cells to be able to investigate cellular fate in case of robust cell survival after 6 weeks. It has been suggested that GFP could impair actin-myosin interactions in muscle cells (42) that might have influenced cardiac function. However, the lack of functional benefit of transplantation of either cell type in this study is also in concordance with other studies using non-GFP-labeled MSC (28, 38). Second, we chose to use longer-term cultured cells because this generally increases purity and also because the ability to culture these cells underlies a crucial advantage which is important for its off-the-shelf clinical potential. However, it has been shown that higher passage MSC had decreased growth factor release under hypoxic conditions and that passage number of MSC was inversely correlated to the protective effect on infarcted hearts (43). Thus, it is possible that the lack of functional benefit is a consequence of dismal cell survival, diminished paracrine signaling, and high passage of the cells.
In conclusion, we have reported that the stromal population from the adipose tissue has close in vitro resemblance with its counterpart from the bone marrow. Importantly, using noninvasive molecular imaging techniques, this study has shown for the first time that these cells also have similar in vivo behavior in the infarcted heart. Finally, we did not observe a clear functional benefit after transplantation of both cell types. These results should be a stimulus for further research regarding improvement of cellular behavior to ultimately be able to restore cardiac function after MI by transplanting long-term cultured, off-the-shelf adipose tissue-derived stromal cells.
The authors thank V. Mariano for animal care and Ms. P. Chu for assistance with histology.
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