PRE-CLINICAL RESEARCH
Imaging Survival and Function of Transplanted Cardiac Resident Stem Cells
Zongjin Li, MD, PhD*,
Andrew Lee, BS*,
Mei Huang, PhD*,
Hyung Chun, MD ,
Jaehoon Chung, MD,
Pauline Chu, MS ,
Grant Hoyt, BS ,
Phillip Yang, MD, PhD,
Jarrett Rosenberg, PhD*,
Robert C. Robbins, MD and
Joseph C. Wu, MD, PhD*,,*
* Department of Radiology and Molecular Imaging Program (MIPS) Stanford University School of Medicine, Stanford, California
Department of Medicine, Division of Cardiology Stanford University School of Medicine, Stanford, California
Department of Cardiothoracic Surgery Stanford University School of Medicine, Stanford, California
Department of Comparative Medicine, Stanford University School of Medicine, Stanford, California
Manuscript received September 26, 2008;
revised manuscript received December 1, 2008,
accepted December 23, 2008.
* Reprint requests and correspondence: Dr. Joseph C. Wu, Stanford University School of Medicine, Edwards Building, R354, Stanford, California 94305-5344. (Email: joewu{at}stanford.edu).
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Abstract
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Objectives: The goal of this study is to characterize resident cardiac stem cells (CSCs) and investigate their therapeutic efficacy in myocardial infarction by molecular imaging methods.
Background: CSCs have been isolated and characterized in vitro. These cells offer a provocative method to regenerate the damaged myocardium. However, the survival kinetics and function of transplanted CSCs have not been fully elucidated.
Methods: CSCs were isolated from L2G85 transgenic mice (FVB strain background) that constitutively express both firefly luciferase and enhanced green fluorescence protein reporter gene. CSCs were characterized in vitro and transplanted in vivo into murine infarction models. Multimodality noninvasive imaging techniques were used to assess CSC survival and therapeutic efficacy for restoration of cardiac function.
Results: CSCs can be isolated from L2G85 mice, and fluorescence-activated cell sorting analysis showed expression of resident CSC markers (Sca-1, c-Kit) and mesenchymal stem cell markers (CD90, CD106). Afterwards, 5 x 105 CSCs (n = 30) or phosphate-buffered saline control (n = 15) was injected into the hearts of syngeneic FVB mice undergoing left anterior descending artery ligation. Bioluminescence imaging showed poor donor cell survival by week 8. Echocardiogram, invasive hemodynamic pressure-volume analysis, positron emission tomography imaging with fluorine-18-fluorodeoxyglucose, and cardiac magnetic resonance imaging demonstrated no significant difference in cardiac contractility and viability between the CSC and control group. Finally, postmortem analysis confirmed transplanted CSCs integrated with host cardiomyocytes by immunohistology.
Conclusions: In a mouse myocardial infarction model, Sca-1–positive CSCs provide no long-term engraftment and benefit to cardiac function as determined by multimodality imaging.
Key Words: molecular imaging • cardiac stem cells • ischemic heart disease • differentiation • cell fate
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Abbreviations and Acronyms
| | BLI = bioluminescence imaging | | BM-MSC = bone marrow-derived mesenchymal stem cell | | CSC = cardiac stem cell(s) | | DAPI = 4'6-diamidino-2-phenylindole | | DiI-ac-LDL = DiI acetylated low-density lipoprotein | | EF = ejection fraction | | eGFP = enhanced green fluorescence protein | | FBS = fetal bovine serum | | Fluc = firefly luciferase | | FS = fractional shortening | | %ID/g = injected dose per gram of heart | | LAD = left anterior descending artery | | LVEDd = left ventricular end-diastolic diameter | | LVESd = left ventricular end-systolic diameter | | MI = myocardial infarction | | MRI = magnetic resonance imaging | | PBS = phosphate-buffered saline | | PET = positron emission tomography | | PV = pressure-volume | | ROI = region of interest | | RT-PCR = reverse transcription-polymerase chain reaction | | [18F]-FDG = fluorine-18-fluorodeoxyglucose |
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Recent years have brought stem cell therapy for heart disease to the forefront. Several clinical trials have shown beneficial effects of stem cell transplantation to improve cardiac function after myocardial infarction (MI) (1–3). However, the mechanism(s) behind this benefit is not clearly understood. Although numerous studies have suggested the cardioprotective benefits from various stem cell types, they also carry limitations including the potential of tumorigenicity with embryonic stem cells (4), arrhythmogenicity with skeletal myoblasts (5), as well as existing controversies surrounding transdifferentiation of bone marrow-derived stem cells (6,7).
The identification of undifferentiated cells with stem cell-like properties derived from the myocardium has renewed the excitement in this field. A number of recent studies have confirmed the presence of cells bearing stem cell markers in the myocardium, which were successfully terminally differentiated into cell lineages that can benefit myocardial function (8–12). A recent study even used resident cardiac stem cells (CSCs) derived from human myocardial biopsy specimens and injected them into mice, showing engraftment and improved myocardial function (12).
We have previously described the L2G85 transgenic mice, which constitutively express both the firefly luciferase (Fluc) and enhanced green fluorescence protein (eGFP) genes (13,14). Cells derived from these mice provide the advantage of being "trackable" over time. Using the previously described methods for CSC isolation and proliferation (10), we now provide in detail the survival kinetics of transplanted CSCs in infarcted murine hearts. In contrast to previous studies (9,10,12,15,16), our results suggest that these CSCs do not constitute long-term improvement in myocardial function. We also show definitive evidence via bioluminescence imaging (BLI) that the majority of these cells are not viable by 8 weeks post-transplantation.
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Methods
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Isolation and culture of CSCs..
Animal protocols were approved by the Stanford University Animal Care and Use Committee. The L2G85 transgenic mice of FVB background with beta-actin promoter driving Fluc-eGFP have been described previously (14,17). Normal adult female FVB mice were used as recipient controls. CSCs were isolated from 6- to 12-week-old L2G85 mice as described with some modifications (10,11). In brief, myocardial tissue was cut into a 1- to 2-mm piece, washed with Hanks' balanced salt solution (Invitrogen, Carlsbad, California), and incubated with 0.1% collagenase II for 30 min at 37°C with frequent shaking. Cells were then filtered through 100-µm mesh. The cells obtained were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, Utah), 0.1 mmol/l nonessential amino acids, 100 U/ml penicillin G, 100 µg/ml streptomycin, 2 mmol/l glutamine, and 0.1 mmol/l beta-mercaptoethanol. After 2 to 3 weeks, a population of phase-bright cells appeared over the adhered fibroblast-like cells. These phase-bright cells were collected by 2 washes with phosphate-buffered saline (PBS), and 1 wash with cell dissolution buffer (Gibco, Grand Island, New York) at room temperature under microscopic monitoring, and subcultured in poly-lysine-coated plates (BD Pharmingen, San Jose, California) with the same medium (Online Videos 1 and 2).
Immunofluorescence staining of cultured CSCs.
The cells were fixed with 4% paraformaldehyde and processed for immunofluorescence with antibodies to Sca-1 (BD Pharmingen) in situ. Briefly, cells were incubated with rat antimouse Sca-1 for 1 h at room temperature and incubated with goat antirat Alexa Fluor 594 (Invitrogen). 4'6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining.
Flow cytometry analysis
Fluorescence-activated cell sorting (FACS) analysis of the bright cells and the cells subcultured on poly-lysine-coated plates were carried out, and the third passage of bone marrow-derived mesenchymal stem cells (BM-MSCs) from L2G85 mice was used as control cells. The isolation and culturing techniques of BM-MSCs were similar to a previous report (18). Antibodies used in this study were phycoerythrin-conjugated anti-CD34, CD29, CD90, CD44, CD54, Flk1, Sca-1, allophycocyanin-conjugated anti-CD31 and c-Kit, allophycocyanin-conjugated antirat IgG2a, and rat antimouse CD62E, CD62P, Tie-2, CD144 (all from BD Pharmingen). The stained cells were analyzed using BD LSR (BD Pharmingen). Dead cells stained by propidium-iodide were excluded from the analysis. Isotype-identical antibodies served as controls (BD Pharmingen). FlowJo software (Tree Star Inc., Ashland, Oregon) was used for followed data analysis.
In vitro differentiation of CSC
For cardiac and smooth muscle differentiation, CSCs were cultured in poly-lysine-coated plates in differentiation medium containing 35% Iscove's modified Dulbecco's medium with 10% FBS/65% Dulbecco's modified Eagle medium-Ham F-12 mix containing 2% B27, 0.1 mmol/l 2-mercaptoethanol, 10 ng/ml epidermal growth factor (R&D Systems Inc., Minneapolis, Minnesota), 20 ng/ml basic fibroblast growth factor (R&D Systems Inc.), 40 nmol/l cardiotrophin-1 (R&D Systems Inc.), 40 nmol/l thrombin (Sigma-Aldrich, St. Louis, Missouri), 100 U/ml penicillin G, 100 µg/ml streptomycin, and 2 mmol/l glutamine (10). After 1 week, the cells were fixed with 4% paraformaldehyde and processed for immunofluorescence with antibodies specific to cardiac troponin T, myocyte enhancer factor 2C, connexin 43, and  -smooth muscle actin (all from Santa Cruz Biotech, Santa Cruz, California) in situ. Briefly, cells were incubated with first antibody for 1 h at room temperature and incubated with goat antirabbit or donkey antimouse Alexa Fluor 594. DAPI was used for nuclear counterstaining. For endothelial differentiation, the phase-bright cells were cultured on fibronectin-coated plates with EGM-2 (Cambrex, Walkersville, Maryland) with an extra 20 ng/ml of vascular endothelial growth factor. DiI acetylated low-density lipoprotein (DiI-ac-LDL) uptake assay and Matrigel assay were used to confirm endothelial cell phenotype differentiation. For DiI-ac-LDL uptake assay, cells were incubated with 10 µg/ml of DiI-ac-LDL (Molecular Probes, Eugene, Oregon) at 37°C for 6 h. After washing with PBS twice, the slides were fixed and detected with fluorescence microscopy as described (19,20). The formation of endothelial tubes was assessed by seeding cells in 24-well plates coated with Matrigel (BD Pharmingen) and incubating them at 37°C for 12 h as described (19,20). For adipogenic differentiation, the phase-bright cells were cultured in DMEM with 10% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin, 2 mmol/l glutamine, 1 µmol/l dexamethasone, 10 µg/ml insulin, 0.5 mmol/l isobulyl-1-methylxanthione, and 200 µmol/l indomethacin for 2 weeks and medium was changed every 3 days. The differentiated adipocytes were confirmed by oil red staining and reverse transcription-polymerase chain reaction (RT-PCR) analysis for peroxisome proliferator-activated receptors gamma and lipoprotein lipase expression. For osteogenic differentiation, the phase-bright cells were cultured in osteogenic differentiation medium (Cambrex, Walkersville, Maryland; PT-3002) for 2 to 3 weeks and medium was changed every 3 days. The osteocyte differentiation was confirmed by Alizarin red S staining and RT-PCR analysis for osteocalcin expression as described (21).
MI and cell delivery
All surgical procedures were performed on 8- to 10-week-old female FVB mice (Charles River Laboratories, Inc., Davis, California) by a single experienced micro-surgeon (G.H.). FVB mice were anesthetized with inhaled isoflurane (2% to 3%). Mice were intubated, ventilated, and anesthesia was maintained with inhaled isoflurane (1% to 2.5%). A left thoracotomy was performed, and the pericardium was opened. The left anterior descending artery (LAD) was permanently ligated with a 9-0 Ethilon suture (Ethicon Inc., Somerville, New Jersey) just distal to the level of the left atrium. Infarction was visually confirmed by blanching of the anterior region of the left ventricle along with dyskinesis. After 30 min, 5 x 105 CSCs were injected intramyocardially into the peri-infarct zone at 2 different sites with a total volume of 20 µl in each animal (n = 30). Control animals received a PBS injection instead (n = 15). For sham-operated animals (n = 5), open thoracotomy was performed without suturing of the LAD and without injection of CSCs or PBS. A thoracotomy tube was placed and lungs were re-expanded using positive pressure ventilation. The chest cavity was closed in layers with 5-0 Vicryl suture (Ethicon Inc.), and the animal was gradually weaned from the respirator. Once spontaneous respiration resumed, the endotracheal and thoracotomy tubes were removed, and the animals were placed in a temperature-controlled chamber until they resumed full alertness and mobility.
Optical BLI of transplanted cell survival
BLI was performed on all animals (n = 30 CSC, n = 15 PBS, and n = 5 sham) using the IVIS 200 system (Xenogen, Alameda, California) by a blinded investigator (M.H.). A majority of the animals were scanned on days 2, 7, 14, 21, 28, 49, and 56. After intraperitoneal injection of the reporter probe D-Luciferin (150 mg/kg), animals were imaged for 1 to 10 min. The same mice were imaged longitudinally for up to 8 weeks. Bioluminescence signals were quantified in units of maximum photons per second per centimeter square per steridian (photons/s/cm2/sr) as described (19).
Cardiac viability with fluorine-18-fluorodeoxyglucose ([18F]-FDG) positron emission tomography (PET) imaging
Small animal microPET imaging (Vista system, GE Healthcare, Chalfont St. Giles, United Kingdom) was performed in a subset of the animals (n = 14 CSC and n = 8 PBS) on baseline (day –7) and days 2, 28, and 56 post-operatively. Mice were fasted for 3 h before radioisotope injection. Animals were then injected with 139 ± 23 µCi [18F]-FDG via the tail vein. At 45 to 60 min after injection, animals were anesthetized with inhaled 2% isoflurane. Images were performed, reconstructed by filtered back projection, and analyzed using image software Amide (Amide's Medical Image Data Examiner, SourceForge, Inc., Mountain View, California) by a blinded investigator (A.L.). Three-dimensional regions of interest (ROIs) were drawn encompassing the heart. For each ROI, counts/ml/min were then converted to counts/g/min and divided by the injected dose to obtain the image ROI derived [18F]-FDG percentage injected dose per gram of heart (% ID/g) as described (22). To measure the myocardium infarction size, [18F]-FDG PET images were assembled into polar maps (23). The size of perfusion defect was measured as the myocardium with 50% of maximum activity and expressed as percent total myocardium polar map by GE Healthcare Advantage Workstation.
Left ventricular functional analysis with echocardiogram and pressure-volume (PV) acquisition
Echocardiography was performed in all animals (n = 30 CSC, n = 15 PBS, and n = 5 sham). A majority of the animals were scanned on baseline (day –7) and days 2, 28, and 56 post-operatively by a blinded investigator (M.H.) using the Siemens-Acuson Sequoia C512 system (Siemens Healthcare, Malvern, Pennsylvania) equipped with a multifrequency (8 to 14 MHz) 15L8 transducer. Animals were anesthetized with inhaled 2% isoflurane. Analysis of M-mode images was performed. Left ventricular end-diastolic diameter (LVEDd) and end-systolic diameter (LVESd) were measured and used to calculate fractional shortening (FS) by the following formula: FS = [LVEDd – LVESd]/LVEDd. At the end of the study (week 8), invasive hemodynamic measurements were performed in a subset of the animals (n = 5/group for CSC, PBS, and sham) as described (24). Briefly, after midline neck incision, a conductance 1.4 conductance catheter (Millar Instruments, Houston, Texas) was introduced into the left ventricle through the right carotid artery. After stabilization, the signals were continuously recorded at a sampling rate of 1,000/s using PV conductance system coupled to a PowerLab/4SP analog to digital converter (AD Instruments, Colorado Springs, Colorado). Data were analyzed by using a cardiac PV analysis program (PVAN 3.4, Millar Instruments) and Chart/Scope Software (AD Instruments).
Assessment of cardiac contractility with magnetic resonance imaging (MRI)
Please see the Online Supplemental Methods section.
Histological analysis
After completion of the imaging protocols, all animals were sacrificed at week 8. For immunostaining, explanted hearts (n = 10 for CSC, n = 5 for PBS, and n = 2 for sham) were embedded into Tissue-Tek O.C.T. compound (Sakura Finetek USA Inc., Torrance, California), snap frozen in liquid nitrogen, and cut into transverse sections at 5-µm thickness. To track GFP-positive CSC-derived cells and cardiomyocytes differentiation in hearts, anti-GFP antibody (Invitrogen) and anti--sarcomeric actin antibody (Sigma) were used. Alexa Fluor 488- and Fluor 594-conjugated secondary antibodies were applied appropriately. DAPI was used for nuclear counterstaining. To detect microvascular density in the peri-infarct area, a rat antimouse CD31 (BD Pharmingen) and Alexa Fluor 647-conjugated secondary antibody were used. The number of capillary vessels was counted in 10 randomly selected areas using a fluorescence microscope (200x magnification). To detect fibrosis in cardiac muscles, explanted hearts (n = 10 for CSC, n = 5 for PBS, and n = 2 for sham) were fixed in 4% paraformaldehyde, cut transversely, embedded in paraffin, and stained with Masson's trichome (25). Five to 6 sections from apex to base were subjected to staining. Images of each section were taken to calculate the fibrotic and nonfibrotic areas as well as ventricular and septal wall thickness. The measurement of area of fibrosis was determined by Image J software (National Institutes of Health, Bethesda, Maryland) performed on 3 separate sections, and the averages were used for statistical analysis.
Statistical analysis
Comparison of FS, ejection fraction (EF), cardiac viability, and infarction size between CSC and sham/PBS groups over time was done by 2-way repeated measures analysis of variance (ANOVA) with group and day as factors, using a Greenhouse-Geisser correction for lack of sphericity. Comparison of end-systolic and -diastolic volume and pressure between CSC and sham/PBS groups was done by 2-way repeated measures ANOVA with group and cardiac phase as factors, using a Greenhouse-Geisser correction for lack of sphericity. Comparison of histological infarct size and microvascular density between PBS and CSC groups was done by 2-tailed t test. Statistics were calculated using SPSS 14.0 (SPSS Inc., Chicago, Illinois). Descriptive statistics included mean and standard error. Differences were considered significant at p values of <0.05.
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Results
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Isolation and culturing of CSCs.
Explanted hearts were subjected to enzymatic digestion, cultured, and are composed of Sca-1–positive cells (Fig. 1A). Phase bright spherical cells that spontaneously separated from the myocardium samples were identified after 2 to 3 weeks. These cells demonstrated a high nucleus-to-cytoplasm ratio and expressed robust GFP (Fig. 1B). The cells also demonstrated linear growth with population doubling time of approximately 5 days (Fig. 1C), similar to previous studies (10,12,16). Importantly, assessment of Fluc expression showed a linear correlation (r2 = 0.98) between the total cell numbers and the bioluminescence signals (Figs. 1D and 1E), which is needed for accurate tracking of cell fate in subsequent in vivo imaging studies.
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Figure 1 Isolation and Culturing of CSCs
(A) Ventricular cells from L2G85 transgenic mice stained for Sca-1 (red) (I). After a period ranging from 1 to 3 weeks, phase-bright cells migrated over a layer of fibroblast-like cells (II). The phase-bright cells were collected, and Giemsa stain showed the cell with a large nucleus (III). Scale bar = 50 µm (I, II), = 2 µm (III). (B) Subculture and in vitro differentiation of phase-bright cells. Morphology of cardiac stem cells (CSCs) (I) cultured in poly-D-lysine-coated plates and expressing green fluorescence protein (II). With cardiac differentiation medium, some CSCs can form cardiac sphere (arrow) with green fluorescence protein expression (III, IV). (C) Proliferation curves show linear growth of cultured CSCs over a 1-month period. (D and E) Ex vivo imaging analysis of CSCs shows increasing bioluminescence signals with cell numbers (r2 = 0.98). Also see Online Videos 1 and 2.
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Characterization of surface markers in CSCs.
Previous studies have shown that Sca-1–expressing cells possess the potential to differentiate into cardiomyocytes (10,11). Our results showed that  40.0 ± 5.5% of the phase-bright cells were Sca-1 positive. After subculturing for 1 to 2 weeks, Sca-1 expression on CSCs was up-regulated close to 95% (Fig. 2A). In order to further characterize their cellular phenotypes, detailed flow cytometry analysis was performed. Overall, the subcultured group had higher expression of mesenchymal stem cell markers (CD29, CD90, CD44, CD106), but endothelial (CD31, CD34, CD144) and c-Kit markers were both down-regulated (Fig. 2B). Interestingly, compared with BM-MSCs, subcultured CSCs have a similar expression pattern, but express higher CD29 and CD90 and lower CD54. CSCs isolated from normal syngeneic FVB mice also showed similar results (data not shown), suggesting no significant effects on cellular characteristics from the stable expression of the Fluc-eGFP reporter gene. Overall, the expression of these markers in our CSCs is similar to previously published data (12,26).
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Figure 2 Characterization of Surface Markers in CSCs
Quantification by FACS analysis of cardiac stem cells (CSCs), phase-bright cells, and their derived cells. Bone marrow mononuclear cell-derived mesenchymal stem cells (BM-MSCs) were used as a control. (A) CSCs express robust green fluorescence protein (GFP) both on phase-bright CSCs and their subcultures. After 2 to 3 passages, there was up-regulation of Sca-1. Left panels show Sca-1 antibody isotype control. (B) Quantitative analysis of cell markers expression by FACS. CSCs express high-level mesenchymal stem cells markers, CD29, CD90, CD44, and CD106. After subculturing, Sca-1 up-regulated, but c-Kit, CD34, CD31, and CD144 markers all down-regulated. Compared with BM-MSCs, CSCs express less CD54, but higher CD29 and CD90. All fluorescence-activated cell sorting experiments were performed in triplicate.
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Multipotent differentiation of CSCs.
We next examined the differentiation of CSCs into various cell types. Similar to other studies (10,21,27,28), these cells can differentiate into cardiomyocytes and smooth muscle cells, as documented by positive staining for cardiac troponin T, connexin 43, myocyte enhancer factor 2C, and -smooth muscle actin (Fig. 3A). After 7 days in differentiation medium, the cells developed into clusters, and after 2 weeks they formed into spherical collection of cells, some of which showed spontaneous contractility (Online Videos 1 and 2). In addition, these cells were capable of differentiating into endothelial cells as demonstrated by DiI-ac-LDL uptake and Matrigel angiogenesis assays (Fig. 3B), into adipocytes as demonstrated by oil red staining and RT-PCR analysis (Fig. 3C), and into osteoblasts as demonstrated by Alizarin red S staining and RT-PCR analysis (Fig. 3D).
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Figure 3 Multipotent Capacity of CSCs
(A) Cardiac and smooth muscle differentiation of cardiac stem cells (CSCs) in vitro. Immunostaining of green fluorescence protein-positive CSCs with cardiac troponin T (cTnT), myocyte enhancer factor 2C (MEF-2C), connexin-43, and -smooth muscle actin (SMA). (B) Endothelial differentiation of CSCs in vitro. The cells were cultured in EGM-2 medium with 10 ng/ml vascular endothelial growth factor and showed endothelial differentiation by morphology (I) and uptake of DiI acetylated low-density lipoprotein (II). Endothelial tube formation by differentiated CSCs after 12 h of plating on Matrigel (III) whereas undifferentiated CSCs cells do not form cord-like structures (IV). (C) Oil red staining and reverse transcription-polymerase chain reaction analysis of peroxisome proliferator-activated receptors gamma (PPAR ) and lipoprotein lipase (LPL) expression shows adipogenic differentiation of the CSCs induced for 2 weeks. (D) Alizarin red S staining of calcium and reverse transcription-polymerase chain reaction analysis of osteocalcin expression show CSCs induced to differentiate into osteoblasts.
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Injection of CSCs into infarcted myocardium.
Several groups have reported that CSCs can proliferate indefinitely in vitro (10,21,27,28). This begs the question whether CSCs can have similar growth properties in vivo, and if so, whether this could lead to potential tumorigenesis. In order to address this question, adult FVB mice were subjected to LAD artery ligation followed by injection with either 5 x 105 cultured CSCs (n = 30) or PBS (n = 15). Injection of CSCs into infarcted myocardium resulted in a robust bioluminescence signal at day 2. However, serial imaging of the same animals out to 8 weeks demonstrated a significant decay in the bioluminescence signal. When normalized to results from day 2, the percent bioluminescence signals were 6.7 ± 0.5 at day 7, 3.9 ± 0.8 at day 14, 1.7 ± 0.2 at day 21, 1.4 ± 0.1 at day 28, and 0.4 ± 0.1 at day 56 (Fig. 4). Control animals injected with PBS showed no imaging signals as expected.
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Figure 4 Reporter Gene Imaging of CSC Fate After Transplantation
(A) A representative animal injected with 5 x 105 cardiac stem cells (CSCs) shows significant bioluminescence activity at day 2, which decreases progressively over the following 8 weeks. (B) Detailed quantitative analysis of signals from all animals transplanted with CSCs. Signal activity is expressed as photons/s/cm2/sr. (C) Estimation of percent donor cell survival plotted as % signal activity (normalized to day 2) over the 8-week period after transplantation. PBS = phosphate-buffered saline.
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Assessment of cardiac contractility and hemodynamic status after CSC therapy.
To date, only a few studies have demonstrated that CSCs can improve cardiac function (10,21,27,28). In order to confirm these results, echocardiograms were performed at baseline, day 2, day 28, and day 56 post-surgery (Figs. 5A and 5B). At day 2 after infarction, there was a significant decrease in the FS in both the CSC group and the PBS group compared with that seen in baseline, consistent with successful induction of MI. At days 28 and 56, there was a trend toward slight improvement in the FS for both groups, but the difference was not statistically significant. These results were also confirmed in a subset of the animals that underwent cardiac MRI, which showed no functional change in EF (Online Supplemental Fig. 1). In addition, invasive hemodynamic parameters showed a decrease in EF for both CSC and control groups compared with that seen in the sham group (Online Supplemental Fig. 2). This was associated with higher left ventricular end-diastolic volume and left ventricular end-systolic volume in both groups compared with sham. Finally, as highlighted in the Online Supplemental Table 2, there was no significant difference in other functional parameters (e.g., cardiac output, stroke volume) between CSC transplanted mice and PBS control mice.
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Figure 5 Echocardiographic Evaluation of Cardiac Contractility
(A) Representative M-mode echocardiographic data of infarcted hearts receiving PBS versus CSCs at day 2, week 4, and week 8. (B and C) Comparison of fractional shortening (FS) and ejection fraction (EF) between the 2 groups 7 days before (baseline), 2 days, 28 days, and 56 days after left anterior descending coronary artery ligation. Abbreviations as in Figure 4.
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Assessment of cardiac viability after CSC therapy.
In addition, we performed [18F]-FDG PET scans of the mice at baseline, day 2, day 28, and day 56 post-surgery. This technique can be used to detect changes in cardiac viability after interventions in small animal models (29). At day 2 after infarction, there was a significant reduction in cardiac viability for both groups (20.2 ± 4.9% ID/g for PBS and 20.8 ± 4.2% ID/g for CSCs; p < 0.001), consistent with successful induction of MI. However, at day 28 and day 56, there was no significant difference between the treatment arm and the control group (Figs. 6A and 6B). Moreover, analysis of the [18F]-FDG PET images using polar-map reconstruction revealed no significant difference of infarction size between the CSC transplantation group and the PBS group (Figs. 6C and 6D).
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Figure 6 [18F]-FDG PET Imaging of Cardiac Viability
(A) Representative image in a normal mouse heart and at days 2, 28, and 56 in infarcted hearts receiving PBS versus CSCs. *p = 0.46 and p = 0.38 versus PBS group at days 28 and 56, respectively. (B) Detailed quantitative analysis of signals from all animals transplanted with CSCs and PBS group. There is no significant difference of the FDG uptake (injected dose per gram of heart [% ID/g]) between the 2 groups. *p = 0.31 and p = 0.40 versus PBS group at days 28 and 56, respectively. (C and D) Representative polar map of the micropositron emission tomography (PET) images obtained from mice treated with PBS versus CSC. Measurements are based on 50% thresholds. [18F]-FDG = fluorine-18-fluorodeoxyglucose; other abbreviations as in Figure 4.
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Histological assessment of infarct size and CSC engraftment.
Histological analysis of the myocardium was performed by both looking at thin sections of the gross specimen and via immunofluorescent microscopic examination. Microscopic examination showed the presence of GFP-positive cells within myocardium at day 3 (Figs. 7A and 7B). At day 14, CSCs could differentiate into cardiomyocytes as confirmed by -smooth muscle actin and GFP double stainings (Fig. 7C). However, this population became significantly decreased when the tissues were examined at day 28 (Fig. 7D), which is also consistent with the decrease in bioluminescence signals over this period of time (Fig. 4). Finally, examination of the explanted hearts showed no significant difference in the infarct size and microvascular density between the 2 groups (Online Supplemental Fig. 3).
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Figure 7 Tracking of Grafted CSCs by Immunofluorescence
(A and B) Cardiac stem cells (CSCs) within the recipient myocardium 3 days after injection shown at low and high magnification. (C and D) Transplanted CSCs can differentiate and integrate with host myocardium as confirmed by green fluorescence protein (GFP) and -sarcomeric actin (SA) double staining. At day 14, CSCs could differentiate into cardiomyocytes as confirmed by -SA and GFP double stainings (C). However, this population became significantly decreased when the tissues were examined at day 28 (D), which is also consistent with the decrease in bioluminescence signals over this period of time. Scale bar = 100 µm (A), 20 µm (B to D).
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Discussion
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In this study, we isolated cardiac resident stem cells from L2G85 transgenic mice that constitutively expressed Fluc-eGFP, enabling us to track stem cells by both noninvasive imaging and invasive histopathology. Upon culturing, we demonstrated that these phase-bright cells up-regulated Sca-1 and expressed surface markers resembling mesenchymal stem cells. We also present evidence that molecular imaging can be used to track CSCs survival in a murine model of MI. When injected directly into the infarcted heart, these cells initially demonstrated viability by BLI. But after a period of 8 weeks, the majority of the imaging signals were no longer present, suggesting elimination from the myocardium. Consequently, we found no significant physiologic benefit from CSC therapy as determined from multiple parameters, including FS by echocardiogram, myocardial glucose uptake by [18F]-FDG PET scan, EF by MRI, invasive PV loop analysis, and infarct size by histopathological assessment.
Recent works have documented the presence of a reservoir of stem and progenitor cells in the myocardium (8,10,16,21,30–32). These cells have been successfully isolated and expanded ex vivo, and have been differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells (10,16,21,26,30–32). Previous works using CSCs have also demonstrated significant improvement in cardiac function after CSC injection, which was, in part, attributed to the persistent engraftment of the injected CSCs into the infarct zone (9,12,16,33). A higher percentage of viable myocardium within the infarct zone and improved EF were demonstrated up to 5 weeks after cell transplantation (9). In contrast, BLI data from our study suggest that by week 8, <0.5% of the transplanted CSCs are still alive. By echocardiography and PET imaging, no significant changes were observed at day 28 and day 56 in cardiac contractility and viability.
At present, we can only speculate that the discrepancy in findings may be attributed to differences in isolation techniques (cardiosphere, side population), stem cell marker expression (c-Kit, Sca-1), donor cell sources (human, mice), particular strains of mice used, or host animal models (nonreperfused LAD ligation, ischemia-reperfusion) (34). From the procedural standpoint, we cannot exclude that the possibility that our mice may have been oversedated (2% isoflurane) given the lower baseline FS values (35% to 40%) observed compared with those in previous studies (35). This could have undermined the chances of detecting a small but significant change between the 2 groups. From the transgenic model standpoint, it is possible that Fluc reporter gene silencing may have occurred and limited our ability to detect surviving CSCs at late time points. However, a previous study using hematopoietic stem cells isolated from these mice was able to demonstrate complete reconstitution of the irradiated bone marrow at late time points as detected by BLI, which argues against significant transgene silencing (17). From the technical standpoint, BLI is limited to small animal imaging as the low-energy photons (2 to 3 eV) can become attenuated within deeper tissues (e.g., heart) compared with more superficial locations (e.g., skeletal muscles). At present, a conservative estimate of minimal detectable cell number by BLI is 1,000 in the heart versus 100 in the leg (14). Finally, from the instrumentation standpoint, it is plausible that the image resolution (1.5 mm3) and detection sensitivity (10–9 to 10–12 mol/l) of the small animal microPET scanner is still insufficient to distinguish subtle but significant differences in [18F]-FDG uptake between the 2 groups. Thus, additional studies will be needed in the future to further examine each of these variables separately.
Just as the mechanism of stem cells exerting benefit on myocardial function remains to be fully elucidated, the mechanism of their elimination over time also remains poorly understood. The pattern of acute donor cell death seen in our CSC transplantation is also consistent with previous studies showing poor donor cell survival after transplantation of neonatal cardiomyocytes (36), mesenchymal stem cells (37), bone marrow mononuclear cells (14), and human embryonic stem cell-derived cardiomyocytes (38). A number of culprits may be involved, including inflammation, ischemia, apoptosis, anoikis, and autophagy, and may all play contributory roles. Furthermore, the myocardial milieu in vivo is in stark contrast to the rich nutrients and oxygen concentration that have been optimized for their cell culturing, growth, and expansions in vitro. It is interesting to note historically that myoblast transplantation as a potential cell-based therapy for patients with Duchenne muscular dystrophy met with disappointing failures in the 1990s (39). One of the main reasons was acute donor cell death after transplantation. Future clinical studies aimed at transplanting CSCs in patients will likely need to first understand the mechanism of acute donor cell death.
Despite the limitation of acute donor stem cell death, both animal and clinical studies have suggested beneficial effects after transplantation of various stem cell types (40), perhaps acting through paracrine pathways (41). Thus, strategies aimed at prolonging cell survival (and, hence, prolonging paracrine activation) may lead to even more favorable results. To that extent, the application of bioengineering methods (42), pro-survival cocktails (43), or genetic modification (44), rather than direct stem cell injection, may prove to be a more viable approach for achieving long-term engraftment in the future. Encouragingly, a recent study by Tillmanns et al. (45) showed that CSCs activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection into infarcted sites had significantly improved cell survival rate and were able to form conductive arterioles and capillaries in the host myocardium.
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Conclusions
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In summary, our study demonstrates that imaging can be used to monitor CSC fate noninvasively in living animals and that CSC transplantation provides no significant benefits to cardiac function in a mouse MI model. As the field of CSC therapy continues to mature, it is critical to better understand the underlying mechanism of the treatment modality, and to identify the potential pitfalls of the therapy before its incorporation into the clinical realm (1). Although a stem cell source from the heart is of tremendous potential, cautious optimism is necessary before full clinical use of these cells.
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Appendix
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For the Supplemental Methods section, figures, tables, and videos, please see the online version of this article.
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Footnotes
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This work was supported, in part, by grants from the National Institutes of Health HL089027 (to Dr. Wu), the Burroughs Wellcome Foundation Career Award for Medical Scientists (to Dr. Wu), Stanford Cardiovascular Institute (to Dr. Wu), and the Society of Nuclear Medicine Pilot Research Award (to Dr. Li).
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References
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