Bone marrow stem cells have myogenic potential and are

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1 Basic Science Reports Systemic Delivery of Bone Marrow Derived Mesenchymal Stem Cells to the Infarcted Myocardium Feasibility, Cell Migration, and Body Distribution Israel M. Barbash, MD; Pierre Chouraqui, MD; Jack Baron, MS; Micha S. Feinberg, MD; Sharon Etzion, PhD; Ariel Tessone, BMedSc; Liron Miller, BSc; Esther Guetta, PhD; Dov Zipori, PhD; Laurence H. Kedes, MD; Robert A. Kloner, MD, PhD; Jonathan Leor, MD Background Systemic delivery of bone marrow derived mesenchymal stem cells (BM-MSCs) is an attractive approach for myocardial repair. We aimed to test this strategy in a rat model after myocardial infarction (MI). Methods and Results BM-MSCs were obtained from rat bone marrow, expanded in vitro to a purity of 50%, and labeled with 99m Tc exametazime, fluorescent dye, LacZ marker gene, or bromodeoxyuridine. Rats were subjected to MI by transient coronary artery occlusion or to sham MI. 99m Tc-labeled cells ( ) were transfused into the left ventricular cavity of MI rats either at 2 or 10 to 14 days after MI and were compared with sham-mi rats or MI rats treated with intravenous infusion. Gamma camera imaging and isolated organ counting 4 hours after intravenous infusion revealed uptake of the 99m Tc-labeled cells mainly in the lungs, with significantly smaller amounts in the liver, heart, and spleen. Delivery by left ventricular cavity infusion resulted in drastically lower lung uptake, better uptake in the heart, and specifically higher uptake in infarcted compared with sham-mi hearts. Histological examination at 1 week after infusion identified labeled cells either in the infarcted or border zone but not in remote viable myocardium or sham-mi hearts. Labeled cells were also identified in the lung, liver, spleen, and bone marrow. Conclusions Systemic intravenous delivery of BM-MSCs to rats after MI, although feasible, is limited by entrapment of the donor cells in the lungs. Direct left ventricular cavity infusion enhances migration and colonization of the cells preferentially to the ischemic myocardium. (Circulation. 2003;108: ) Key Words: cells myocardial infarction nuclear medicine transplantation Bone marrow stem cells have myogenic potential and are therefore promising candidates for multiple cell-based therapies for myocardial disease. 1 5 A critical step for the clinical success of stem cell based therapy for myocardial repair is an efficient method for cell delivery. Intravenous delivery of bone marrow derived stem cells, as performed during bone marrow transplantation, to patients recovering from myocardial infarction (MI) is an attractive noninvasive strategy that allows repeated administration of large numbers of cells. Furthermore, it may be applicable to patients with diffuse myocardial disease, such as idiopathic dilated cardiomyopathy. However, there are insufficient data to support systemic delivery of bone marrow mesenchymal stem cells (BM-MSCs) as a strategy for myocardial repair. The purpose of the present study was therefore to test the feasibility and efficacy of several strategies of systemic BM-MSC administration and to evaluate cell kinetics, body distribution, and colonization in the infarcted myocardium. Methods The study was performed in accordance with the guidelines of the Animal Care and Use Committee of Sheba Medical Center, Tel-Aviv University, which conforms to the policies of the American Heart Association and the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, NIH publication No ). Mesenchymal Stem Cell Isolation and Culture BM-MSC cultures were prepared according to the protocol developed in the Caplan laboratory. 6 Briefly, under sterile conditions, the femur and tibia of 2-month-old female Sprague-Dawley rats (Harlan, Jerusalem, Israel) were excised, with special attention given to remove all connective tissue attached to bones. Bone marrow plugs were extracted from the bones by flushing the bone marrow cavity Received February 11, 2002; de novo received February 10, 2003; revision received April 17, 2003; accepted April 25, From the Neufeld Cardiac Research Institute (I.M.B., P.C., J.B., M.S.F., S.E., A.T., L.M., J.L.) and Danek Gertner Institute of Human Genetics (E.G.), Sheba Medical Center, Tel-Hashomer, Israel; Department of Molecular Cell Biology (D.Z.), Weizmann Institute of Science, Rehovot, Israel; Institute for Genetic Medicine (L.H.K., R.A.K.), Department of Biochemistry & Molecular Biology, Los Angeles, Calif; Department of Medicine (L.H.K.), Keck School of Medicine of the University of Southern California, Los Angeles, Calif; and Heart Institute (R.A.K.), Good Samaritan Hospital, Los Angeles, Calif. Correspondence to Jonathan Leor, MD, FACC, FESC, Neufeld Cardiac Research Institute, Sheba Medical Center, Tel-Hashomer 52621, Israel. leorj@post.tau.ac.il 2003 American Heart Association, Inc. Circulation is available at DOI: /01.CIR A 863

2 864 Circulation August 19, 2003 Experimental Groups Delayed (10 14 days) cell transfer MI IV infusion MI LV cavity infusion MI RV cavity infusion Sham MI LV cavity infusion Early (2 days) cell transfer MI LV cavity infusion Sham MI LV cavity infusion IV indicates intravenous; RV, right ventricular. with complete culture medium. Marrow plug suspension was dispersed by passing it through subsequent pipettes of decreasing sizes. After a homogenous cell suspension was achieved, the cells were centrifuged (1400 rpm, 8 minutes), resuspended in complete culture medium, plated ( cells per 75-cm 2 culture flask), and incubated at 37 C humidified atmosphere with 5% CO 2 for 3 days before the first medium change. The mesenchymal population was isolated on the basis of its ability to adhere to the culture plate. 6 8 At 90% confluence, the cells were trypsinized (0.25% trypsin-edta, Sigma) and were passaged to 225-cm 2 flasks at 1:3 ratios. First-passage BM-MSCs were used in all experiments. To assess the percentage of BM-MSCs among the total cells that were transfused, we used the polyclonal antibody to the BM-MSC surface antigen SB-10 (activated leukocyte-cell adhesion molecule [ALCAM]; Santa Cruz Biotechnology). 9 Cell Labeling To monitor cell kinetics and detect distribution in the body, representative samples of cultured cells were labeled by several methods. (1) For gamma camera in vivo imaging and organ counting, the cells were labeled with technetium 99m ( 99m Tc) by incubation with 99m Tc exametazime (Amersham Healthcare) according to a modified procedure developed and validated in our laboratory. Viability of the cells was assessed by both trypan blue exclusion test and by replating and continuous culture for an additional week. (2) Twenty-four hours before cell infusion, cultured cells were incubated with mol/l of a green fluorescent dye PKH2 (Sigma) followed by fetal calf serum wash and overnight incubation with complete culture medium. (3) In another group, cultured cells were labeled with the thymidine analogue 5-bromo-2 deoxyuridine (BrdU; Zymed) as described previously. 10 The cells were incubated with 1% BrdU in complete culture medium overnight. (4) By a previously described method, 10 2 cell samples were genetically labeled by transfection with recombinant E1a-deleted adenovirus-5 encoding the nuclear LacZ reporter gene under the control of the cytomegalovirus promoter. Rat Model of MI and Cell Transfer Rats (Sprague-Dawley) were anesthetized with a combination of ketamine (50 mg/kg) and xylazine (10 mg/kg). We induced MI by transient (1 hour) left coronary artery occlusion as described previously. 11 Cells were prepared for infusion by detaching the cells from the culture plates by 1-minute incubation with 0.25% trypsin-edta (Sigma), centrifugation (1400 rpm for 8 minutes), and resuspension in 0.9% saline. The Table describes the experimental groups. We tested 3 strategies of cell delivery. In the first group, BM-MSCs ( ) were infused via the femoral vein for 1 minute. In the second group, BM-MSCs ( ) were aspirated into scalp vein set (Vasuflo). The rats were placed in supine position, and left ventricular (LV) cavity infusion was performed with the guidance of an echocardiography system (Sonos 5500, Hewlett Packard) equipped with a 12.5-MHz phased-array transducer. The transducer was placed above the left side of the chest, and a 23-gauge needle of scalp vein set (Vasuflo) was introduced gently into the LV via the right Figure 1. Echocardiographic short-axis view of rat heart during infusion into LV cavity. A, Needle (arrow) inserted in LV cavity. B, Bubbles (arrow) indicate that cell infusion was performed in LV cavity. fourth intercostal space, and the cells were infused within 30 seconds. In the third group of MI rats, we tested the feasibility of infusion into the right ventricular cavity by a similar method used for LV infusion. However, shortly after infusion into the right ventricular cavity, all animals died of massive pulmonary emboli and were excluded from final analysis. We took several precautions to avoid direct intramyocardial injections. First, the leading tip of the needle was filled with saline. Second, the precise location of the needle tip in the LV cavity was confirmed by echocardiography before initiation of infusion (Figure 1), blood pulsation was identified in the scalp vein set, and intracavity bubbles were viewed during the infusion. Third, the needle was flushed with saline before its withdrawal from the LV cavity. To evaluate the best time interval for cell transfer, we compared the efficacy of systemic cell delivery early (2 days) versus delayed (10 to 14 days) after MI (Table). Nuclear Imaging and Counting At 4 hours after infusion, whole-body distribution of the 99m Tclabeled cells was imaged with a gamma camera (Varicam camera, General Electric). Then, the rats were killed, and the heart, lungs, liver, spleen, and bones were excised. The organs were weighed and counted on the camera for organ uptake with regions of interest corrected for background activity to calculate the specific activity of each sample. Considering the possibility that slight variations in the amounts of the infused cells to each animal do occur, we used liver uptake as a reference value for calculating the relative specific activity of each organ. To determine the percentage of infused cells in the myocardium, uptake in the heart was calculated as a percentage of the total whole-body count. Histological and Immunohistochemical Examination To assess the percentage of BM-MSCs among the total cells that were transfused, cells were deposited onto positively charged glass slides by centrifugation (Cytospin 3, Shandon). Slides were air-dried overnight and immunostained with polyclonal antibody to the BM-MSC surface antigen SB-10 (ALCAM). 9 Histological examination was performed in MI and sham-mi rats that were treated with BrdU, LacZ, or fluorescent dye labeled cells. One week after cell infusion, representative samples of the heart, lungs, liver, spleen, kidneys, bladder, and femur bone were frozen sectioned or embedded in paraffin. Representative sections of lung were also obtained 1 day after injection. To identify LacZ expression in treated animals, slides were stained with X-gal solution. 10 For immunohistochemical examination, adjacent blocks were sectioned into 5- m slices and stained with hematoxylin and eosin. Biotinylated mouse anti-brdu (Zymed) was used to localize the labeled cells in the heart and other organs. Representative lung sections were subjected to immunohistostaining with an antibody against ED-1 (Chemicon International), an antigen that is expressed by tissue macrophages. Statistical Analysis All variables are expressed as mean SEM. Differences in organ activity between groups were compared by unpaired t test (InStat,

3 Barbash et al Systemic Delivery of Stem Cells 865 Figure 2. Microscopic view of cultured BM-MSCs. BM-MSCs were labeled by (A) green fluorescent dye (arrows; magnification 400) or (B) transfected with recombinant adenovirus encoding reporter gene LacZ (blue nuclei; magnification 200). Histology sections of MI rat hearts at 1 week after BM-MSC infusion showed (C) green fluorescent dye labeled cells (arrow; magnification 200) or (D) LacZ-labeled cells (blue cells; arrow) colonized in ischemic myocardium (magnification 400). To assess percentage of BM-MSCs among total cells that were transfused, we used polyclonal antibody to BM-MSC surface antigen SB-10 (ALCAM). E, Immunostaining of cell samples used for transplantation showed that 51 9% of cells were stained positive for BM-MSC surface antigen SB-10 (ALCAM; magnification 400). F, At 24 hours after cell transfer, immunostaining of lung section with anti-ed1 identified a few macrophages (arrows) among cells entrapped in lung (original magnification 400). version 3.01; GraphPad Software Inc). Data of cell distribution among various organs were not normally distributed and were compared with nonparametric ANOVA, Kruskal-Wallis test. All tests were 2-tailed, and significance was accepted at P Results A total of 39 rats were included in the present study. Nuclear studies were performed in 19 rats both early and late after MI. 99m Tc-labeled cells were transfused into the LV cavity of MI rats at either 2 days (n 5) or 10 to 14 days (n 8) after MI and were compared with sham-mi rats (n 3) after LV cavity infusion and with MI rats after intravenous infusion (n 3). Histological studies were performed in representative organs of an additional 20 rats. Peri-MI mortality was 20%, and these animals were excluded from the final analysis. In Vitro Studies Four days after bone marrow derived stem cell seeding in culture plates, a contrast phased microscope revealed adherent cells in small colonies with fibroblast-shaped morphology. The hematopoietic cells, which constituted the majority of the cells, did not stick to the culture plate and were removed with subsequent medium changes. The fibroblastlike morphology was also maintained after cell passages and throughout the culture period. After 2 weeks, the adherent Figure 3. A, Whole-body scans of rats 4 hours after intravenous (IV) infusion (left) vs LV cavity (IC) infusion (right) of Tc 99m -labeled BM-MSCs. Intravenous infusion resulted predominantly in lung uptake and lower uptake in liver, kidneys, and bladder. Lower lung activity and significant uptake in heart were obtained after infusion into LV cavity (IC). Immunohistochemical staining for BrdU of recipient lung 1 (B) and 7 (C) days after injection of BrdU prelabeled cells. Nuclear brown staining indicates trapping and engraftment of donor cells in lung (original magnification 400). cells reached 90% confluence and were passaged for the first time. The 99m Tc-labeled cells demonstrated a viability of 99% as assessed by the trypan blue exclusion test and their ability to readhere to the culture plate and proliferate. The efficiency of BM-MSC labeling with fluorescent green dye and the adenoviral transfection efficiency were both 95% (Figure 2, A and B). The efficiency of BM-MSC labeling with BrdU was 30%. Immunostaining of cell samples before transplantation showed that 51 9% of the cells were stained positive for the BM-MSC surface antigen SB-10 (Figure 2E). Intravenous Versus LV Cavity Infusion To determine the best route for systemic cell delivery to the infarcted myocardium, we compared intravenous versus LV cavity infusion into MI rats. Relative specific activity of the labeled cells in the organs revealed the highest activity in the lungs compared with other organs, particularly after intravenous infusion (P 0.001; Figures 3 and 4A). LV cavity infusion was significantly more effective than intravenous infusion (Figures 3 and 4A), with significantly lower activity in the lungs ( versus , respectively; P 0.01) and increased uptake in the heart ( versus , respectively; P 0.001). We also observed increased uptake in the spleen after LV cavity versus intravenous infusion ( versus , respectively; P 0.001; Figure 4A). Migration and Colonization of Infused BM-MSCs To investigate the influence of infarction on migration and colonization of BM-MSCs in the heart, we compared LV

4 866 Circulation August 19, 2003 Figure 4. Relative specific activity (mean SEM) of various organs in animals treated with 99m Tc-labeled BM-MSCs at 10 to 14 days after MI. A, Specific activity of various organs 4 hours after infusion, comparing intravenous (IV; n 3) vs LV cavity infusion (n 8) of cells. B, Specific activity of various organs, comparing MI (n 8) vs sham-mi (n 3) rats 4 hours after cell infusion into LV cavity. cavity infusion of BM-MSCs in MI versus sham-mi rats. Gamma camera imaging revealed that BM-MSC infusion into MI rats resulted in significantly higher uptake in the heart than in sham-mi rats ( versus ; P 0.03; Figure 4B). Specific activity of hearts indicated that fewer than 1% of the infused cells resided in the infarcted heart 4 hours after infusion. There was also higher activity in the spleen of MI rats than in that of sham-mi rats ( versus respectively; P 0.02; Figure 4B). Timing of Cell Transfer We compared the distribution of BM-MSCs after LV infusion at 2 days versus 10 to 14 days after MI. As shown in Figure 5A, LV cavity infusion at 48 hours after MI resulted in significantly higher activity in the heart and spleen than in sham-mi rats (P 0.05). Organ distribution in animals transplanted 2 days after MI showed minor activity in the bone marrow 4 hours after infusion (Figure 5A). No significant difference in cell distribution patterns was found between early versus delayed infusion. Still, a trend toward higher uptake in the heart ( versus ; P 0.29) and spleen ( versus ; P 0.28) was observed after 2-day infusion (Figure 5B). Histological Analysis To evaluate the specific colonization site of the labeled BM-MSCs, histological studies were performed on tissue sections at 1 week after infusion (Figures 2 and 6). Fluorescent localization confirmed the presence of donor cells within the infarcted hearts (Figure 2C). In addition, we were able to identify, at the border zone, clusters of donor cells that were stained positively for LacZ marker gene (Figure 2D). In specimens in which the implanted cells were prelabeled with BrdU, immunostaining confirmed that the infarcted border zone contained the donor cells after either early (2 days) or delayed (10 to 14 days) post-mi infusion (Figure 6, A and B), but there were no donor cells in the remote intact myocardium or sham-mi hearts (Figure 6C). Sections of lung, obtained at 24 hours or 1 week after cell injection, were subjected to hematoxylin and eosin staining and immunohistostaining with an antibody against BrdU or ED-1, an antigen that is expressed by rat macrophages. Microscopic examination of hematoxylin and eosin stained slides showed congestion and lymphocyte aggregates. At 1 and 7 days after systemic injection, BrdU immunostaining confirmed that the lung parenchyma contained many of the donor cells (Figure 3, B and C). However, only % of cells in the lung parenchyma were stained positive for the macrophage marker ED-1 (Figure 2F), which suggests that most of the entrapped cells were BM-MSCs. A few labeled Figure 5. Relative specific activity (mean SEM) of various organs in animals treated with 99m Tc-labeled BM-MSC transfusion at 48 hours after MI. A, Specific activity of various organs, comparing MI (n 5) vs sham-mi (n 3) rats at 4 hours after cell infusion into LV cavity. B, Comparison of infusion at 48 hours (n 5) vs 10 to 14 days (n 8) after MI shows generally similar specific activity of various organs at 4 hours after infusion.

5 Barbash et al Systemic Delivery of Stem Cells 867 Figure 6. Microscopic view of myocardial specimens obtained at 1 week after cell transfer, in which donor cells were prelabeled with BrdU. Positive brown nuclei (arrows) were identified in infarcted and border zone both after infusion at 2 (A) or 10 to 14 (B) days after MI ( 400). C, We did not identify labeled cells in remote intact (I) myocardium or sham-mi hearts ( 400). Labeled brown nuclei were identified in (D) bone marrow (arrows; 400), (E) liver (arrows; 200), and (F) spleen (arrows; 400). BM-MSCs were also identified in histological slides obtained from the bone marrow (Figure 6D), liver (Figure 6E), and spleen (Figure 6F). At 1 week after infusion, we did not identify BrdU-positive cells in kidney or bladder. Discussion The major new findings of the present study are as follows: (1) Systemic intravenous delivery of BM-MSCs to rats after MI, although feasible, is limited by entrapment of donor cells mainly in the lungs. (2) BM-MSCs delivered by LV cavity infusion migrate to and colonize the infarcted heart in significantly higher amounts than after intravenous infusion. (3) BM-MSCs are preferentially attracted to and retained in the ischemic tissue but not in the remote or intact myocardium. (4) Within 4 hours after infusion, fewer than 1% of cells migrate to the infarcted myocardium. (5) BM-MSCs can be successfully labeled with 99m Tc without affecting cell viability, and this technique is useful for tracking whole-body distribution. Thus, the present study illustrates both the potential application and the difficulties of systemic BM- MSC delivery for the repair of myocardial disease. Comparison With Previous Studies Recent reports have suggested that there is a noncirculating bone marrow derived cell population with remarkable plasticity: the mesenchymal stem cells. 4,12,13 BM-MSCs reside within the bone marrow cavity and can be isolated on the basis of their adhesive properties. 6,12,14,15 In culture, BM- MSCs can maintain an undifferentiated, stable phenotype over many generations. However, controversy still exists regarding their precise phenotype and markers to select purified cell populations. 13,14,16 Kocher et al 17 reported that infusion of human bone marrow progenitor cells enhances neovascularization, attenuates remodeling, and improves cardiac function in athymic rat with MI. Chen et al 18,19 reported that systemic administration of bone marrow stromal cells improves function and angiogenesis in rat model of stroke. On the basis of these reports, an attractive clinical strategy has been proposed: systemic administration of BM-MSCs to repair infarcted myocardium. 5 Once delivered, the cells would migrate through the systemic circulation, resettle in the infarcted myocardium, and receive local signals that would direct myogenic differentiation. 5,13 At 1 week after injection, examination of the heart identified donor cells in the ischemic zone only. These findings suggest that BM-MSCs are preferentially attracted to and retained in the ischemic tissue, colonize there, and might contribute to healing of the heart. 5,13 It is possible that the injured tissue expresses specific receptors or ligands to facilitate trafficking, adhesion, and infiltration of stem cells to the site of injury. 13 In the present study, however, fewer than 1% of the infused cells trafficked to the heart in the first hours after transplantation. The low percentage of cell migration to the heart is in agreement with several recent reports on human sex-mismatched transplanted hearts Still, it is possible that after longer follow-up, the percentage of cell migration to the heart will be increased by circulating donor cells. The present findings about donor cell entrapment in the lungs are in accordance with previous reports. Gao and colleagues 15 studied the potential of BM-MSC infusion for bone repair. Similar to the present study, they found that most of the infused cells were trapped in the lungs. Pretreatment with the vasodilator sodium nitroprusside decreased the number of cells entrapped. 15 Noort et al 23 examined the distribution of intravenous transplanted human MSCs in NOD/SCID mice. Reverse transcription polymerase chain reaction for human 2 -microglobulin detected transplanted MSCs expression in the lung up to 48 hours after injection. 23 Cell entrapment in the lung might be explained because expanded MSCs are relatively large, activated, and express adhesion molecules. Because of this, they may not be able to pass the capillaries in the lung, and many of them become trapped. By LV cavity injection, this trapping is bypassed and prevented. In the present study, we identified donor cells in the lung parenchyma after 1 week. This is consistent with previous works in models of lung injury 24,25 or intact lung. 18,26 Kotton et al 25 found that compared with normal lung, engraftment is enhanced after bleomycin-induced lung injury. It is possible that our surgical procedure or even MI created lung injury that contributed to entrapment and engraftment of the infused cells in the lung. It is of interest that some of the donor cells resided in the spleen of infarcted rats. The present findings suggest that the spleen may be another barrier of the infused cells on their voyage to repair the infarcted myocardium.

6 868 Circulation August 19, 2003 Study Limitations We tested an adherent population of bone marrow cells that were previously characterized. 6,15 Only 50% of the infused cells were stained positive for the BM-MSC antigen SB-10 (ALCAM). It is possible that by sorting the cells into subpopulations, we may have achieved better results of migration and colonization. A previous report, 5 however, suggested that sorting is not essential for homing and myogenic differentiation. The present study was not designed to confirm or refute the ability of the transfused cells to differentiate into cardiomyocytes or vascular cells after colonization in the ischemic myocardium. On the basis of the most recent report, 5 1 week after infusion may be too early for differentiation and integration of donor cells. Implications and Future Research The present study suggests that BM-MSCs are able to colonize in the infarcted myocardium when transfused either early or late after MI. This approach is relevant for myocardial repair and site-specific therapeutic gene targeting. However, only a very small percentage of the infused cells colonized in the heart. Recent reports from animal model and human patients suggest that intracoronary delivery of marrow or progenitor cells may overcome some of these barriers Better understanding of the signaling mechanism that attracts marrow cells to the ischemic heart and promotes differentiation may enhance the prospects of systemic delivery of BM-MSCs becoming a therapeutic strategy for myocardial repair. Acknowledgments This work was supported by grant No from the United States-Israel Binational Science Foundation. Dr Kloner is supported by a grant from the National Heart, Lung, and Blood Institute (HL 61488). Dr Kedes is supported by a grant from the National Heart, Lung, and Blood Institute. We thank Parvin Zarin for extensive histopathology work, Radka Holbova for technical assistance, and Prof Willem E. 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