Intravenously injected mesenchymal stem cells home to viable myocardium after coronary occlusion and preserve systolic function without altering infarct size

International Journal of Cardiology 122 (2007) 17 – 28 www.elsevier.com/locate/ijcard

Intravenously injected mesenchymal stem cells home to viable myocardium after coronary occlusion and preserve systolic function without altering infarct size Robert A. Boomsma a , Paari Dominic Swaminathan b , David L. Geenen c,⁎ a

b

Department of Biology, Trinity Christian College, Palos Heights, IL, 60463, United States Department of Medicine, Rosalind Franklin University of Medicine and Science, North Chicago, IL, 60064, United States c Center for Cardiovascular Research, University of Illinois at Chicago, Cardiology Section, Department of Medicine, 840 South Wood Street, Chicago, IL 60612, United States Received 7 July 2006; received in revised form 28 September 2006; accepted 2 November 2006 Available online 21 December 2006

Abstract Background: The purpose of this study was to determine whether murine mesenchymal stem cells (MSC) are able to home to the viable myocardium when injected intravenously and attenuate cardiac dysfunction and ventricular remodeling associated with myocardial infarction. Methods and results: Murine bone marrow cells were negatively selected for lineage markers and adherent MSC differentiated into adipocytes and osteocytes following treatment in culture. Two weeks after coronary occlusion that resulted in a permanent transmural infarct we observed a significant drop in LV systolic pressure, dP/dtmax, dP/dtmin, ESPVR and Emax and a significant increase in end-diastolic volume in vivo. Femoral vein injection of MSC 1 h after occlusion attenuated the cardiac dysfunction without altering infarct size, or enddiastolic volume. Injected MSC pre-labeled with fluorescent paramagnetic microspheres were observed scattered in noninfarcted regions of the myocardium. Flow cytometry of whole heart digests after intravenous injection of MSC labeled with either fluorescent microspheres or fluorescent PKH26 dye demonstrated that infarcted hearts from mice that received MSC injections contained significantly more cells that integrated into the heart (20×) than those from uninfarcted controls. Conclusion: We conclude that intravenously injected MSC were able to home to viable myocardium and preserve systolic function by 2 weeks following ligation. The preserved contractility is likely an MSC-mediated paracrine response since infarct morphology was unchanged and labeled cells observed at two weeks exhibited the same characteristics as the injected MSC. These data underscore the importance of using MSC as a potential therapeutic intervention in preserving cardiac function following infarction. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Heart; Transdifferentiation; Myocyte; Bone marrow

1. Introduction Since the heart is generally considered a terminally differentiated organ, various strategies have been attempted to replace scar tissue with functional myocardium following infarction [1]. There is considerable interest in using stem cells as agents of myocardial regeneration, and this topic has been extensively reviewed [2–5]. Results from various studies have been mixed, due in part to methodological ⁎ Corresponding author. Tel.: +1 312 355 1623; fax: +1 312 996 5062. E-mail address: [email protected] (D.L. Geenen). 0167-5273/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2006.11.022

variations in the ischemic model used, the type of cell studied, and the mode of cellular administration. Stem cell administration appears to improve cardiac function after myocardial infarction, but it is unclear whether this effect is due to the production of new cardiomyocytes, angiogenesis, or the secretion of factors that improve the function of existing cells. There have also been reports of resident cardiac stem cells [6–8], and the role that these cells play in cardiac regeneration must be delineated. There is evidence that ischemic injury to the heart can lead to increased homing of bone marrow stem cells to the injured tissue [3], and cytokine mobilization of endogenous

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bone marrow stem cells may improve cardiac function following myocardial injury [9,10]. Recent studies in our lab demonstrated that mobilization of bone marrow cells in rats by intraperitoneal injection of granulocyte colony stimulating factor and stem cell factor caused an improvement in the functional deficits associated with myocardial infarction [11]. We were interested in understanding the type of bone marrow stem cells that might be mobilized and attracted to the damaged myocardium. Mesenchymal stem cells (MSC) are non-hematopoietic cells from the bone marrow and other tissues that have been shown to differentiate into various cell types including adipocytes, osteocytes and cardiomyocytes [12–14] and have shown promise in treating myocardial infarctions [15–17]. Therefore, we isolated MSC from the bone marrow of mice and injected them intravenously into animals with myocardial infarctions in order to study whether these cells would integrate in the heart and improve myocardial function. 2. Methods 2.1. Animals Mice were housed in the Biological Resources Laboratory at UIC (AAALAC accredited) and maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, revised 1996). Experimental protocols were approved by the Animal Care Policies and Procedures Committee at UIC (IACUC accredited). 2.2. Mesenchymal stem cell (MSC) culture Bone marrow was isolated from FVB.Cg-Tg(GFPu) 5Nagy/J mice (Jackson Laboratory) that expressed green fluorescent protein according to a modification of the technique described by Peister and colleagues [18]. Tibia and femur bones from six mice were stripped of muscle and placed in ice cold PBS + 2% FBS. The epiphyseal ends were removed and the bones were centrifuged at 4000 ×g for 1 min in a microfuge tube containing the cut end of a 1 ml pipette tip inside the cut end of a 5 ml tip. The tips provided support for the bones during centrifugation and allowed the marrow to collect at the bottom of the tube. The bone marrow cells were suspended in ice cold PBS + 2% FBS, passed through a 70 μm filter and counted with a hemocytometer. Filtered bone marrow cells were suspended in PBS + 2% FBS + 0.1 g/L phenol red and enriched for lineage negative (Lin−) cells using the SpinSep system (Stem Cell Technologies). Briefly, the cells were incubated with Murine Progenitor Enrichment Cocktail (anti-CD5, anti-CD45R, anti-CD11b, anti-Gr-1, anti-TER119, and anti-7/4; Stem Cell Technologies) on ice for 30 min and, after washing, incubated with dense particles on ice for 20 min. The cells were layered on density medium, centrifuged at 1200 ×g for 10 min, and the layer of cells at the density medium/PBS interface was collected, washed and counted.

Enriched bone marrow cells were plated on tissue culture treated plates at a density of 0.1 × 106 cells/cm2 in murine Mesencult media (Stem Cell Technologies) with 100 units/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B added. The media was changed after 48 h and adherent cells were maintained in culture with twice weekly media changes. After 4 weeks the confluent cells were detached with trypsin and one dish was split into three dishes. Some MSC were labeled with Dragon Green 0.96 μm diameter paramagnetic microspheres (Cat# ME02F, Bang Laboratories, Inc. Fishers, IN) by incubating cultures with 5 μl of microsphere mixture/ml of Mesencult media at 48 and 24 h prior to use. This resulted in over 80% of the cells containing at least one microsphere. Other MSC were labeled with the PKH26GL Red Fluorescent Cell Membrane Labeling Kit (Sigma-Aldrich) 24 h prior to use resulting in greater than 95% labeling of the cells. Cell labeling with either the paramagnetic microspheres or the PKH26 label did not influence cell morphology, viability or proliferation in culture. Cell labeling was necessary since the endogenous GFP fluorescence of the MSC proved insufficient for cell identification. 2.3. Characterization of MSC Lin− MSC were characterized for surface antigens using flow cytometry. Antibodies were obtained from BD Pharmigen unless otherwise noted. Cultured cells were detached with trypsin and incubated with 1 μl mouse Fc block (1:50 dilution; clone 2.4G2, rat anti-mouse CD16/ CD32) for 5 min on ice. Cells were then incubated with 1 μl fluorescent-conjugated antibodies (1:50 dilution) for 1 h on ice, washed and analyzed. The following mouse monoclonal antibodies were used: anti-CD45-Cy5 (clone 30-F11), anti-Sca1-PE (clone E13– 161.7), anti-Sca1-Cy5 (clone D7; eBioscience), anti-CD34-PE (clone RAM34), anti-CD90.1-PE (clone HIS51), anti-CD117 (cKit)-PE (clone 2B8), anti-CD105-biotin (clone MJ7/18; eBioscience) and anti-CD184-biotin (clone 2B11/CXCR4). Those treated with biotinylated antibodies were washed and incubated 20 min at room temperature with 1 μl streptavidinPE or streptavidin-Cy5. Control samples were treated with immunoglobulin of the appropriate isotype. Cells were analyzed in a (FACS) Calibur flow cytometer or in a Beckman Coulter FC500 equipped with one (Calibur) or two lasers (Beckman) for multiparametric and multicolor analysis, including a 488 nm argon laser for measurement of forward light scatter (FSC) and orthogonal scatter (SSC), and three to five colors. The cells were acquired by FSC and SSC and analyzed in density plots displaying dragon green (FITC) in Fluorescence 1 (green channel) and PKH26 in Fluorescence 2 and 3 (red channel). Because previous experiments have shown that the green fluorescence is very strong, compensation was performed in order to eliminate overlapping signals. The data were analyzed with Cell Quest (Becton Dickinson) and Excel (Microsoft) on a G4 Macintosh computer.

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Adipogenic differentiation was induced by treating 50% confluent cultures twice weekly with media change for 14 days with 10 nM dexamethasone and 5 μg/ml insulin [19], and osteocyte differentiation was induced by treating 50% confluent cultures twice weekly with media change for 4 weeks with 10 nM dexamethasone, 50 μg/ml ascorbic acid and 10 mM β-glycerol phosphate [19,20]. Cells were fixed for 20 min in 10% buffered formalin. Lipid droplets in adipocytes were stained with oil red O (0.5% in isopropropanol stock diluted 3:2 in H2O) for 10 min, and bone matrix was stained for 20 min in 2% alizarin red. Cell nuclei were counterstained in Mayer's hematoxylin (no ethanol; Sigma-Aldrich).

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MgSO4, 0.032 phenol red, 12 mM NaHCO3, 10 mM KHCO3, 10 mM HEPES, 30 mM taurine, 10 mM 2,3butanedione monoxime, 5.5 mM glucose, pH 7.4. The hearts were cannulated and perfused retrograde at 37 °C with perfusion buffer for 4 min followed by perfusion buffer, 0.25 mg/ml liberase blendzyme 1 (collagenase and dispase;

2.4. Coronary ligation, injection of MSC, and hemodynamic measurements Mice (SJL/BL6; Jackson Laboratories) were anesthetized with methoxyflurane and maintained with 1.0% isoflurane through a ventilator. The left anterior descending coronary artery was ligated with an 8–0 monofilament nylon suture as previously described [21]. A suture was passed under the coronary artery without ligation in sham-operated mice. Mice were randomly placed into the following groups: unoperated control (Con), sham-operated control (Sham), sham-operated control + MSC injection (Sham + MSC), coronary ligation (MI), coronary ligation + vehicle injection (MI + vehicle), and coronary ligation + MSC injection (MI + MSC). Lin− MSC were washed in injection buffer (Ca++/Mg++ free PBS + 2 mM EDTA + 0.25% BSA), passed through a 70 μm filter and suspended in a final concentration of 0.5 × 106 cells/100 μl. 100 μl of MSC (0.5 × 106 cells) or vehicle alone was injected into the femoral vein 1 h after coronary ligation while the mice remained on isoflurane anesthesia from the thoracotomy. Immediately prior to all injections (MSC or vehicle), a rapid, transient vasodilatory response was induced by injecting the animals with 50 μl of 0.5 mg/ml sodium nitroprusside (1 mg/kg body weight) allowing the cells to bypass the lung capillaries [22,23]. Two weeks following cell injections, hemodynamic measurements were taken using an ultra-miniature pressure–volume catheter (SPR-839; 1.4 French; Millar Instruments) as previously described [24]. We analyzed hemodynamic data from four different groups: Shamoperated mice (n = 7), sham-operated mice injected with MSC (n = 5), mice with myocardial infarctions (n = 8) and mice with myocardial infarctions injected with MSC (n = 5). 2.5. Whole heart digests To estimate the total number of MSC present in hearts following intravenous cell administration, animals were injected with cells labeled with fluorescent paramagnetic microspheres or PKH26 dye followed two weeks later with enzymatic digestion of the whole heart [25] and analysis by flow cytometry. Perfusion buffer contained 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2H2PO4, 1.2 mM

Fig. 1. Photomicrographs of MSC cultures. A, Untreated passage four MSC visualized by phase-contrast microscopy (200×). B, MSC differentiated in vitro into adipocytes (300×). Cells were stained with oil red O and counterstained with hematoxylin. C, MSC differentiated in vitro into osteocytes (150×). The tissue was stained with alizarin red. Scale bars are equal to 50 μm.

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Table 1 Flow cytometric analysis of MSC surface antigens Cells

CD34

CD45

CD90.1

CD105

CD117

Sca-1

BM before BM after MSC P4 MSC P17

0.4% 3% 1% 81%

36% 80% 54% 24%

3% 34% 8% 3%

6% 23% 60% 1%

4% 43% 0.2% 0.5%

2% 21% 91% 95%

Data presented as % cells expressing the antigen. BM before: bone marrow cells before negative selection; BM after: bone marrow cells after negative selection; MSC P4: stem cells after passage four; MSC P17: stem cells after passage seventeen.

Roche Applied Science) and 0.14 mg/ml trypsin + 12.5 μM CaCl2 for 7–10 min. The hearts were removed from the cannula and minced in the same buffer at 37 °C until completely digested. Digestion was terminated by adding an equal volume of stop buffer (perfusion buffer + 10% Bovine Calf Serum (BCS) + 12.5 μM CaCl2). Cells were resuspended in stop buffer containing 5% BCS and the CaCl2 was slowly raised to a final concentration of 1 mM in a final volume of 10 ml. The cells were washed in PBS + 0.2% BSA, passed through a 100 μm filter, and analyzed by flow cytometry for the presence of either the fluorescent

paramagnetic microspheres or the red fluorescent PKH26 label. The cardiac digest was examined prior to flow cytometry to identify viable labeled cells and to determine whether any microspheres were present outside of cell membranes. Cells were gated on the basis of size and fluorescence, and counting continued until 10,000 events of medium size or larger were counted. Final analysis was based on ungated data, however, since most MSC were in the smaller size range. The volume utilized for analysis was determined, and the number of MSC present per heart was determined. We analyzed data from four different groups: unoperated and sham-operated mice (n = 6), unoperated mice injected with MSC (n = 6), sham-operated mice injected with MSC (n = 6) and mice with infarcted hearts injected with MSC (n = 8). Heart digests from unoperated and noninjected control animals were spiked with known quantities of microsphere (n = 3) or PKH26 (n = 3) labeled cells to determine the detection sensitivity of labeled MSC in the digests. Labeled MSC were suspended in injection buffer in a final concentration of 5000, 1000, 500 and 100 cells per 10 μl. Whole heart digests were separated into 500 μl aliquots, and then 10 μl of labeled cells were added to each aliquot. During flow

Fig. 2. Representative pressure–volume loops from one mouse in each of the four groups during inferior vena cava occlusion 2 weeks after coronary artery occlusion. Aggregate data derived from the pressure–volume loops for all mice are presented in Table 3. The end-systolic pressure–volume relationship (ESPVR) is shown by the solid line. A, Sham operated. B, Sham operated injected with MSC. C, Coronary artery occlusion injected with vehicle. D, Coronary artery occlusion injected with MSC.

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Table 2 Hemodynamic measurements: steady state Treatment

LVsys (mm Hg)

LVed (mm Hg)

Ved (μl)

dP/dtmax (mm Hg/s)

dP/dtmin (mm Hg/s)

Tau (ms)

Sham (7) Sham + MSC (5) MI (8) MI + MSC (5)

90 ± 3 91 ± 4 70 ± 4† 84 ± 9

4±0 4±0 5±1 4±1

52.9 ± 2.8 52.5 ± 5 103.7 ± 5.6† 93.5 ± 11.1†

8512 ± 573 9254 ± 626 5234 ± 6509 8738 ± 1415‡

7529 ± 531 8947 ± 698 4585 ± 453† 7391 ± 1206‡

5.60 ± 0.22 4.80 ± 0.25 11.20 ± 1.45† 8.42 ± 0.98

Hemodynamic measurements were taken prior to inferior vena cava occlusion using an intraventricular pressure–volume catheter. Data are presented as mean ± standard error with number of animals per group (n). LVsys = peak left ventricular systolic pressure; LVed = left ventricular end-diastolic pressure; Ved = enddiastolic volume; dP/dtmax = maximum rate of increase in left ventricular pressure; dP/dtmin = maximum rate of decrease in left ventricular pressure; Tau = time of isovolumic relaxation (Weiss). †P b 0.05 vs. sham groups; ‡P b 0.05 vs. MI.

cytometry, approximately 21,000 cells (100 μl) were counted, and the number of MSC detected per 500 μl was determined. 2.6. Microscopy Following hemodynamic measurements, the hearts were removed and perfused retrograde through the aorta with cold saline followed by Methacarn fixation. The hearts were divided into four equal transverse blocks (5 mm) and immersion fixed for an additional 30 min. The tissue was dehydrated in ethanol, cleared in xylene and embedded in paraffin. Sections (7 μm) were placed on slides, deparaffinized and rehydrated in water. Some sections were stained with Masson's trichrome or hematoxylin and eosin while others were analyzed for specific proteins by immunofluorescence. For immunofluorescence, antigens were unmasked with Retrievagen (Becton Dickinson). For cTnI staining, biotin activity was blocked with an avidin/biotin blocking kit (Vector Labs). Sections were then blocked in 3% normal horse serum and incubated overnight with a 1:250 dilution of mouse monoclonal anti-cardiac troponin I (Research Diagnostics; clone C5) at 4 °C, followed by room temperature incubation with biotinylated horse anti-mouse IgG and then streptavidinconjugated Alexa Fluor 594 (Molecular Probes) diluted 1:500. For von Willebrand factor (vWF) staining, slides were blocked in 3% normal goat serum and incubated overnight with a 1:50 dilution of rabbit polyclonal anti-human factor VIII-related antigen (vWF; DAKO; A0082) at 4 °C, followed by room temperature incubation with Cy3-conjugated goat anti-rabbit IgG diluted 1:600. Following staining, the slides were treated with Sudan black B to reduce autofluorescence and mounted with Vectashield containing DAPI (Vector Labs). Images for immunofluorescence were obtained using a Zeiss LSM510 confocal microscope equipped with a 63× water immersion objective. The 488 nm and 568 nm beams from an argon-krypton laser and 354 nm beam from an argon UV laser were used for excitation and emission. FITC, Alexa Fluor 594 and DAPI were detected through an LP505, LP585 and BP385–470 filter respectively. Sections stained with Masson's trichrome were used to estimate the size of the myocardial infarction. The epicardial circumference was measured, and infarct size was expressed as the percent of the left ventricular epicardial surface that contained scar tissue. Sections from all four blocks of each

heart were analyzed and averaged for each infarct group. The authors acknowledge that Masson's trichrome can lead to underestimation of scar quantification and that picosirius red or triphenyltetrazolium staining may have provided more accurate determination of infarct size. 2.7. Statistical analysis The hemodynamic data were analyzed using a one-way ANOVA (Sigma-Stat) with group being the main factor. The alpha level was set at 0.05. If there was a significant group effect, multiple comparisons were made for all pairwise comparisons between the four groups using the Duncan multiple comparisons test. Student's T-test was used to make comparisons between the Con/Sham groups and all other groups for the flow cytometry data. The alpha level was set at 0.05. 3. Results 3.1. Characterization of MSC Following negative selection of bone marrow for lineage markers, adherent MSC cultures were established that contained cells with a spindle-shaped morphology typical of MSC (Fig. 1A). The cells grew slowly at first, with initial confluence being reached after 4 weeks. Cells grew more rapidly as they were passaged so that by the 4th passage they were replated weekly and one dish was split into four dishes. To demonstrate the ability of our MSC to differentiate into other cell types, we treated the cells with dexamethasone Table 3 Hemodynamic measurements: inferior vena cava occlusion Treatment

ESPVR (mm Hg/μl)

PRSW (mm Hg)

dP/dt-EDV (mm Hg/s/μl)

Emax (mm Hg/μl)

Sham (7) Sham + MSC (5) MI (8) MI + MSC (5)

7.40 ± 1.43 7.23 ± 1.37 2.69 ± 0.53† 6.96 ± 1.63‡

62 ± 11 60 ± 13 32 ± 8 44 ± 19

301 ± 17 292 ± 49 131 ± 33† 362 ± 74‡

13.49 ± 1.81 13.77 ± 3.35 5.55 ± 1.13† 13.90 ± 2.84‡

Hemodynamic parameters calculated after inferior vena cava occlusion. Data presented as mean ± standard error with number of animals per group (n). ESPVR = end-systolic pressure–volume relationship; PRSW = preload recruitable stroke work; dP/dt-EDV = slope of +dP/dt max end-diastolic volume relation; Emax = maximum chamber elasticity. †P b 0.05 vs. sham groups; ‡P b 0.05 vs. MI.

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or insulin to induce adipocyte differentiation or dexamethasone, ascorbic acid and β-glycerol phosphate to induce osteocyte differentiation. Adipocytes (Fig. 1B) and bone matrix deposition (Fig. 1C), respectively, were observed after 2–4 weeks of treatment. Bone marrow cells and MSC were analyzed by flow cytometry for the presence of various surface antigens before and after negative lineage selection and following the fourth and seventeenth passage after the establishment of MSC cultures. As seen in Table 1, negative selection for the lineage markers CD5, CD45R, CD11b, Gr-1, TER119, and 7/4 enriched the bone marrow cells for CD45, CD 90.1, CD105, CD117 (cKit) and Sca-1. Passage 4 MSC derived from these Lin− bone marrow cells were Sca-1 positive whereas they were CD34 and CD117 negative. A small number of MSC contained CD90.1 while approximately half of the cells contained CD45 and CD105. Passage 17 MSC continued to be strongly Sca-1 positive and CD117 negative. They showed a reduction in CD45, CD90.1 and CD 105 such that they were essentially CD90.1 and CD105 negative and only 24% of the cells were CD45 positive. There was a dramatic increase in CD34 by passage 17 so that 80% of the cells were positive for this antigen. In addition, 5% of passage 17 cells were positive for CD184 (CXCR4). Expression of GFP fluorescence in MSC was generally weak when studied by flow cytometry (data not shown). GFP expression characteristics changed over time such that approximately 10% of passage 4 cells displayed moderate to high levels of GFP while passage 17 cultures were homogenously weak for GFP expression. Passage 20 MSC were analyzed for CXCR4 (CD184), the receptor for SDF-1, in addition to the surface antigens mentioned above. Their surface characteristics were similar to passage 17 cells, and 8% of the cells displayed CXCR4. Interestingly, 92% of the CXCR4 positive cells were negative for CD45. 3.2. Hemodynamic measurements Mice underwent coronary artery occlusion (MI) or sham surgery and some were then injected intravenously with MSC or vehicle 1 h later. Hemodynamic measurements were taken 2 weeks later. Representative pressure–volume loops are shown in Fig. 2, aggregate steady state hemodynamic data in Table 2, and contractile parameters from pressure–volume loops collected during inferior vena cava occlusion, in Table 3. No differences were observed between the sham and sham + vehicle groups or between the MI and MI + vehicle groups, thus the data were combined in Tables 2 and 3. Fig. 3. Confocal images of paraffin-embedded heart sections from mice two weeks after coronary artery occlusion and treatment with MSC labeled with fluorescent paramagnetic microspheres. Arrows point to green fluorescent microspheres. All sections were stained with DAPI (blue fluorescence); sections A and B were stained for TnI (red fluorescence) while section C was stained for von Willebrand factor (red fluorescence). Scale bars are equal to 10 μm. (For interpretation of the references to colour of this figure legend, the reader is referred to the web version of this article.)

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Cardiac function was significantly altered two weeks after coronary artery occlusion when compared to sham-operated controls. End-diastolic volume and tau were significantly increased while LV systolic pressure, dP/dtmax, dP/dtmin, ESPVR, dP/dtmax-EDV, and Emax were all significantly decreased. MSC injection 1 h after coronary artery occlusion significantly attenuated the deleterious effects as measured two weeks later such that the contractile parameters were not different from control animals. The primary exception was that the diastolic volume remained elevated, consistent with the morphological analysis of scar size presented below. In addition, tau and preload recruitable stroke work were not improved. MSC injection in sham-operated mice had no effect on cardiac function. 3.3. Microscopic observations Tissue sections stained with Masson's trichrome were used to assess myocardial infarct size in animals without (n = 4) and with (n = 4) MSC injections. Coronary artery occlusion resulted in an infarct that covered 30.7 ± 2.5% of the left ventricular area, and MSC injection did not alter the size of the infarct (29.5 ± 3.6%). The morphology of the infarct was similar in all animals, with significant wall thinning, loss of cardiomyocytes, collagen deposition and decreased cellularity. Our initial intention was to use the inherent fluorescence of the GFP MSC to identify stem cells and their progeny in heart sections. However, due to the high level of autofluorescence in the heart and the low level of GFP fluorescence of the injected MSC, we were unable to unequivocally identify them. Therefore, MSC labeled with fluorescent paramagnetic microspheres were used to assess incorporation into the myocardium two weeks after coronary artery occlusion by observing tissue sections with confocal microscopy. Cells containing green fluorescent microspheres were observed in healthy myocardium of animals with myocardial infarctions (Fig. 3) but were not observed in the scar tissue of the infarct. Fluorescent immunostaining demonstrated that fluorescent microsphere positive cells were found in the interstitial space (Fig. 3A and B) or the endothelium (Fig. 3C) and colocalized with either troponin I or vWF. 3.4. Quantification of MSC incorporation Flow cytometry of whole heart digests was used to determine the quantity of MSC present in the heart following intravenous injection two weeks earlier. The fluorescent pattern of MSC suspensions alone labeled with either fluorescent paramagnetic microspheres (Fig. 4A) or PKH26 (Fig. 4B) was compared to the fluorescent pattern of cardiac cell suspensions alone from mice without MSC injections (Fig. 4C and D). A consistent pattern of green (Fig. 4C) and red (Fig. 4D) autofluorescence was detected in heart digests that was distinct from the fluorescence pattern detected in fluorescent microsphere (Fig. 4A) and PHK26

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(Fig. 4B) labeled MSC alone. This allowed us to identify regions of non-overlap, shown as regions 1, 2 and 3 (Fig. 4), that could be used to identify labeled MSC in heart digests. Subsequent analysis led us to use only regions 1 and 2 since region 3 often represented cells in the border area between autofluorescent cardiocytes and weakly fluorescing labeled MSC. Although this would tend to underestimate the number of cells present, regions 1 and 2 still contained over 90% of the MSC detected in Fig. 4A and B. To determine the sensitivity of detection of labeled MSC by flow cytometry, heart digests from normal uninjected SJL mice were spiked with either iron-oxide or PKH26 labeled MSC. An increase in the number of fluorescent cells was clearly observed in regions 1 and 2 after 5000 fluorescent microsphere labeled MSC were added to 500 μl of heart digest (compare Fig. 4E with C). A similar increase in MSC was observed when 5000 PKH26 labeled MSC were added to the same amount of heart digest (compare Fig. 4F with D). Note that fluorescent microspheres labeled cells had a higher fluorescence than did their PKH26 labeled counterparts so that the majority of fluorescent microsphere labeled MSC were detected in region 1 while the majority of PKH26 MSC were detected in region 2. A concentration dependent effect was observed when different concentrations of fluorescent microsphere labeled (Fig. 5A) or PKH26 labeled (Fig. 5B) MSC were added to heart digests. The detection of MSC in heart digests was similar when either label was utilized, and approximately 50% of the cells added to the digest could be detected by this methodology. Fluorescent microsphere or PKH26 labeled cells were intravenously injected into animals followed by whole heart digestion two weeks later. Small fluorescent microsphere positive cells (Fig. 6A) or PKH26 positive cells (Fig. 6B) were observed in heart digests with the fluorescence microscope. The number of labeled cells present in the digests was determined by flow cytometry as discussed above and the number of labeled cells per heart was calculated. Fig. 4G is a representative flow cytometry dotplot identifying cells with green fluorescence (regions 1 and 2) that were considered positive for fluorescent microsphere. Fig. 4H is a representative plot for PKH26 cells. Consistent with results discussed above, hearts from animals injected with fluorescent microsphere labeled cells contained more highly fluorescent cells in region 1 than those injected with PKH26 labeled cells. Since the number of cells detected with the fluorescent microsphere label was similar to the number detected with PKH26, the data was combined for each treatment group and presented in Fig. 7. In unoperated and sham-operated control mice that did not receive an MSC injection, 492 ± 86 (n = 6) cells/heart were identified in regions 1 and 2 and represent autofluorescent background. No significant differences were observed between this group and un-operated MSC-injected controls (589 ± 92 cells/heart; n = 6) or sham-operated MSC injected controls (1045 ± 274; n = 6). In contrast, infarcted hearts from mice that received MSC injections exhibited a significant

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Fig. 5. Summary of flow cytometric analysis of heart digests from normal mice without MSC injection that were spiked with increasing amounts of fluorescent microsphere (A) or PKH26 (B) labeled MSC. The number of cells added to an aliquot of heart digest was compared to the mean ± SE (n = 3) number of cells detected by flow cytometry in regions 1 and 2 (see Fig. 4).

(P b 0.01) increase in the number of cells that integrated into the heart (12,237 ± 3,278 cells/heart; n = 8). 4. Discussion Our study is the first to demonstrate, to our knowledge, that MSC administered in the femoral vein 1 h after infarction home to the viable myocardium and improve load-independent systolic performance without affecting

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ventricular remodeling. Permanent coronary artery ligation resulted in an infarct that covered 30% of the left ventricle that was unaltered by MSC injection. Additionally, intraventricular end-diastolic volumes measured by the indwelling catheter provided further evidence that MSC did not improve the dilatation associated with MI. Despite the lack of an effect on remodeling, contractility was almost completely restored in infarcted hearts with MSC compared to the untreated MI. One implication of these results is that the remaining viable myocytes exhibited enhanced contractility as a result of MSC engraftment. Our data are in contrast to other studies that reported stem cell migration into the ischemic region and remodeling associated with functional improvement. Several groups have documented improvement in cardiac function after intravenous administration of MSC. Tail vein injection (48 h postMI) of cytokine-mobilized human CD34 + bone marrow cells increased fractional shortening (22%) and decreased left ventricular end-systolic area (26%) compared to noninjected infarcted hearts that was sustained for up to 15 weeks. These improvements were attributed to vasculogenesis, angiogenesis, decreased apoptosis of hypertrophied myocytes and reduction in collagen deposition [26]. Jugular vein injection of rats with MSC 3 h after coronary ligation resulted in reduced infarct size and left ventricular end-diastolic pressure along with engrafted MSC that were positive for von Willebrand factor and formed vascular structures [15]. The authors reported that MSC were preferentially attracted to the infarcted, but not the noninfarcted myocardium, although this was not quantified. Delivery of MSC by tail vein injection within 24 h after ligation of the coronary artery promoted angiogenesis and vascularization and improved cardiac function 28 days later [27]. These differences may be due, in part, to the length of follow-up after infarction. Initial dilation of the ventricle following acute coronary artery ligation is primarily due to thinning of the ventricular wall in the infarcted region and replacement by scar tissue. Further dilation occurs by loss of myocytes and connective tissue replacement in the noninfarcted regions of ventricle. We may have observed a more pronounced dilation of the ventricle in the untreated infarct group and preserved ventricular dilation in the treated group if the endpoint of our experiments had been extended beyond 2 weeks. It is also possible that the differences observed in cardiac remodeling and ventricular function compared to earlier reports may also be due to characteristics of the stem cells used in other studies. We were careful to characterize the MSC used for injection and noted significant cell surface changes with later passages. Cells

Fig. 4. Flow cytometric analysis of MSC and heart digests for the quantification of MSC analyzed for either green (A, C, E, G) or red (B, D, F, H) fluorescence. MSC were labeled with either green fluorescent paramagnetic microspheres (A, E, G) or red fluorescent PKH26 (B, F, H). MSC alone were subjected to flow cytometry after fluorescent microsphere (A) or PKH26 (B) labeling to assess the fluorescent characteristics of labeled cells. Heart digests from normal animals without MSC injection were analyzed for green (C) or red (D) fluorescence to assess the autofluorescence of heart tissue. Aliquots of heart digests from normal animals without MSC injection were spiked with 5000 fluorescent microspheres (E) or PKH26 (F) labeled cells to determine the ability of flow cytometry to identify MSC. Digests from infarcted hearts two weeks after fluorescent microsphere (G) or PKH26 (H) labeled MSC injection were analyzed to assess the number of MSC present. Regions 1, 2 and 3 represent areas of fluorescent non-overlap between MSC labeled cells and autofluorescent heart tissue. Quantification of MSC was done using only regions 1 and 2 to minimize the inclusion of autofluorescent cells.

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a means to detect regional integration within the heart but this method does not yet possess single cell resolution capabilities [31]. Quantifying the number of labeled cells that integrate in the heart after intravenous injection or mobilization has obvious limitations. In hearts spiked with labeled cells we were able to detect approximately half of the added MSC in a dose-dependent manner suggesting that we underestimated the total number of integrated cells after 2 weeks by approximately 50%, most likely related to the efficiency of labeling. In addition, the enriched MSC in our study were highly proliferating cells yielding a dilution of the fluorescent signal over time, and the number of MSC that integrated and subsequently died during the ensuing 2 weeks could not be quantified. Interestingly, we found little evidence of cell death after integration and cell disruption from enzyme digestions since extracellular fluorescent microspheres were found in only two heart digests. Because of these factors it is likely that at least twice as many MSC (N 25,000) were present in the viable myocardium and contributed to the improved systolic function. It is not clear, however, how many of the cells detected were the result of cellular proliferation once the cells initially integrated into the myocardium. One potential mechanism for the homing of stem cells to the heart is the chemokine stromal cell-derived factor-1α (SDF)–CXCR4 axis. SDF is highly expressed in the bone marrow and also during tissue injury. The SDF released by cells from the injured tissue form a complex with its receptor CXCR4 present on bone marrow cells including the enriched MSC used in the present study. Other investigators have shown that the amount of SDF in the heart peaks within the

Fig. 6. Photomicrographs of heart digests from mice two weeks after injection with MSC (500×). Cells were observed using phase contrast (main image) and fluorescence (inset) microscopy. The arrows point to the cells shown in the insets. A, MSC were labeled with green fluorescent microspheres prior to injection. B, MSC were labeled with red fluorescent PKH26 prior to injection. Scale bars are equal to 30 μm.

passaged for different lengths of time or derived from human bone marrow [26] likely effect the engraftment and cell differentiation potential. We were able to quantify stem cell integration in whole hearts in a novel way by enzymatic digestion of whole hearts and then counting labeled cells using flow cytometry. Using this approach, we found approximately 12,000 labeled cells in cardiac digests 2 weeks after femoral injection of 0.5 × 106 total cells. Quantification was dose dependent and reproducible, and was comparable in independent experiments when MSC were labeled with either fluorescent microspheres or PKH26 dye. Other investigators have used histochemical detection of labeled cells in cardiac sections to assess integration but quantification errors are introduced when this data is extrapolated to the entire heart [28–31]. More recently, magnetic resonance imaging has been used as

Fig. 7. Summary of flow cytometric analysis of heart digests from mice injected with MSC labeled with either fluorescent microspheres or PKH26. The data were calculated as the total number of cells found per heart and reported as mean ± SE. Co = unoperated and sham-operated animals (n = 6); Co + M = unoperated animals injected with MSC (n = 6); Sh + M = shamoperated animals injected with MSCs (n = 6); MI + M = animals with myocardial infarctions injected with MSC (n = 8), ⁎P b 0.01 vs. Co.

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first 24 h after infarction and returns to normal levels by 8 days post-infarction [27]. We injected MSC 1 h after tissue injury which coincides with increased expression of SDF, and the number of cells found in the hearts two weeks after infarction closely parallels the percentage of CXCR4 positive cells (5%) of the total MSC we injected. The inflammatory response may also be critical to stem cell homing since other investigators have shown that an SDF gradient in normal hearts does not lead to the same level of stem cell homing as observed during SDF expression in the injured myocardium [32]. Although we cannot rule out the potential role of other chemokines as homing factors in MSC engraftment, our data are consistent with the important role the SDF–CXCR4 axis plays in recruitment associated with injury and inflammation. The systolic performance of the noninfarcted myocardium observed in our study at 2 weeks post-MI was capable of overcoming the deficit caused by infarction and dilation of the ventricle, and our data are consistent with a paracrine effect since little if any transdifferentiation of MSC into cardiomyocytes was observed. Evidence suggests that paracrine mechanisms, such as growth factor expression, may lead to increased vascularization, enhanced myofiber performance and/or reduced apoptosis. MSC injected into the ischemic area at the time of myocardial infarction [33] or into the peri-infarct region one week after infarction [34] resulted in elevated levels of bFGF and VEGF and was associated with a 40% increase in capillary density and improved systolic pressure and fractional shortening between 1 and 4 weeks. Similarly, local delivery of MSC into ischemic skeletal muscle resulted in increased hindlimb perfusion and increased expression of bFGF, VEGF, and monocyte chemoattractant protein-1, although labeled cells did not incorporate into mature collateral vessels [35]. Ablation of VEGF interferes with cardiac contractility through the phospholipase C gamma 1 pathway controlling calcium transients [36] and injection of plasmid DNA encoding for VEGF and FGF-2 improves myocardial function in chronic ischemia [37]. Recent evidence also suggests that MSC transplantation may improve cardiac function 28 days post-MI through stabilization of the infarct by overexpression of stem cell factor [38]. Finally, anoxic preconditioning of bone marrow stem cells enhanced their ability to inhibit cardiomyocyte apoptosis by paracrine mechanisms in ischemic hearts [39]. Investigators have observed elevated apoptosis in noninfarcted regions of hearts undergoing decompensation and heart failure [40]. It may be that our MSC inhibited the apoptosis that occurs in the remote (viable) myocardium following myocardial infarction. Future studies need to address the mechanism by which MSC improved systolic performance in our system. In conclusion, our data show that MSC integrate within the viable myocardium and enhance systolic performance 2 weeks post-MI without altering infarct size or ventricular dilation. The primary mechanism by which MSC exert their effect is not by transdifferentiation into cardiomyocytes but

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likely by a paracrine mechanism that may involve a direct effect of growth factors on angiogenesis and/or viable muscle contractility or viability. Acknowledgements We thank Trinity Christian College, Palos Heights, IL for providing sabbatical support for R.A.B. while working on this project at the University of Illinois at Chicago. We also acknowledge Dr. Mei Ling Chen from the UIC Research Resources Center for her expert technical assistance with the confocal microscope and Mr. Raphael Nunez for assistance with the flow cytometry measurements. D.L.G. is the recipient of a seed grant from the University of Illinois at Chicago Central Campus Research Board. This study was funded by The Illinois Regenerative Medicine Institute (D.L.G., Project Director). References [1] Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J Clin Invest 2005;115:572–83. [2] Forrester JS, Price M, Makkar RR. Mending the broken heart. Clin Cardiol 2003;26:449–50. [3] Orlic D, Hill JM, Arai AE. Stem cells for myocardial regeneration. Circ Res 2002;91:1092–102. [4] Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 2004;95:9–20. [5] Timmermans F, De Sutter J, Gillebert TC. Stem cells for the heart, are we there yet? Cardiology 2003;100:176–85. [6] Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA 2003;100:12313–8. [7] Matsuura K, Nagai T, Nishigaki N, et al. Adult cardiac Sca-1positive cells differentiate into beating cardiomyocytes. J Biol Chem 2004;279:11384–91. [8] Messina E, De Angelis L, Frati G, et al. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res 2004;95:911–21. [9] Norol F, Merlet P, Isnard R, et al. Influence of mobilized stem cells on myocardial infarct repair in a nonhuman primate model. Blood 2003;102:4361–8. [10] Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA 2001;98:10344–9. [11] Sareh SS, Gandhi S, Pop A, et al. Mobilization of hematopoietic stem cells following infarction improves ventricular performance. Circulation 2003;108:137. [12] Gregory CA, Prockop DJ, Spees JL. Non-hematopoietic bone marrow stem cells: Molecular control of expansion and differentiation. Exp Cell Res 2005;306:330–5. [13] Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103:697–705. [14] Phinney DG. Building a consensus regarding the nature and origin of mesenchymal stem cells. J Cell Biochem Suppl 2002;38:7–12. [15] Nagaya N, Fujii T, Iwase T, et al. Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis. Am J Physiol Heart Circ Physiol 2004;287:H2670–6. [16] Tang YL, Zhao Q, Zhang YC, et al. Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium. Regul Pept 2004;117:3–10. [17] Tomita S, Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100: II247–56.

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