Expression of cardiomyocytic markers on adipose tissue-derived cells in a murine model of acute myocardial injury

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1 Cytotherapy (2005) Vol. 7, No. 3, 282/291 Expression of cardiomyocytic markers on adipose tissue-derived cells in a murine model of acute myocardial injury BM Strem 1,2, M Zhu 1, Z Alfonso 1, EJ Daniels 1,3, R Schreiber 1, R Begyui 3,4, WR MacLellan 5, MH Hedrick 1,2,3 and JK Fraser 1 1 Macropore Biosurgery, San Diego, California, 2 Department of Bioengineering, 3 Department of Surgery, 4 Department of Cardiothoracic Surgery and 5 Departments of Medicine and Physiology, University of California, Los Angeles, USA Background Animal and early clinical studies have provided evidence suggesting that intracoronary administration of autologous bone marrow-derived cells results in improved outcome following myocardial infarction. Animal studies with cultured marrow stromal cells (MSC) have provided similar data. Cells with properties that are similar to MSC have been identified in adipose tissue. Other groups have demonstrated in vivo differentiation of adipose tissue-derived cells (ADC) into cells exhibiting biochemical and functional markers of cardiac myocytes, including spontaneous beating. Hypothesis Based on these observations, the objective of the present study was to determine whether ADC might undergo similar differentiation in vivo in the context of myocardial injury. Methods ADC were isolated from subcutaneous adipose tissue of Rosa26 mice (which express the beta-galactosidase transgene in almost every tissue) and injected into the intraventricular chamber of B6129S recipient mice immediately following induction of myocardial cryoinjury. Groups of recipients were euthanized at 24 hours, 7 and 14 days post surgery and examined for the presence of donor-derived cells within the heart. Results Beta-gal positive cells were identified in the infarcts of ADC-treated animals. No staining was observed in uninjured myocardium or in infarcts of control animals. Immunohistochemical analysis revealed coexpression of beta-gal with Myosin Heavy Chain, Nkx2.5 and with Troponin I. Co-expression of beta-galactosidase with Connexin 43, CD31, von Willebrand factor, MyoD or CD45 was not detected. Conclusion Thus, these data indicate that adipose tissue contains a population of cells that has the ability to engraft injured myocardium and that this engraftment is associated with expression of cardiomyocytic markers by donor-derived cells. Keywords Acute myocardial infarction, adipose tissue-derived cells, cell therapy. Introduction Despite the recent description of stem cells within the heart [1], it is clear that the limited endogenous regenerative capacity of the adult heart is a significant factor in the processes of ventricular remodeling and dilatation leading to progressive loss of cardiac function and, ultimately, to congestive heart failure [2]. A number of approaches designed to supplement limiting endogenous mechanisms have been developed and some have been brought to the clinic. For example, skeletal muscle-derived myoblast transfer has been applied for cellular therapy of the heart, with reports indicating improvement in cardiac function [3]. However, in these studies and the supporting small and large animal research there are limited data suggesting integration of donor-derived myocytes into the contractile function of the heart [4,5]. It is possible that improved function may derive from cell-mediated inhibition of collagen deposition and scar formation by provid- Correspondence to: Brian M Strem, Macropore Biosurgery Inc., 6740 Top Gun St, San Diego, CA 92121, USA ISCT DOI: /

2 Expression of cardiomyocytic markers on adipose tissue-derived cells 283 ing a body of viable cells within the infarct or by elaboration of angiogenic growth factors leading to improved perfusion. Furthermore, despite early clinical promise, there are concerns about timing of cell delivery and creation of arrhythmias [6]. BM represents another source of cells with potential to generate replacement cardiac myocytes. Indeed, a number of small clinical studies have generated data indicating significant promise for this approach, with sustained improvement in cardiac function in both acute and chronic settings [7,8]. However, the mechanism through which such benefits derive remains elusive. Data from a series of studies by Orlic et al. using purified (c-kit /Lin cells) BM-derived hematopoietic stem cells (HSC) indicated the ability of HSC to differentiate into cells with a cardiac myocyte phenotype [9/11]. However, two recent studies by other investigators have failed to reproduce these findings [12,13]. Yet another group has presented data consistent with transdifferentiation of human peripheral blood CD34 cells, a population that includes HSC, into a cardiomyocytic phenotype in the peri-infarct region of immunodeficient mice [14]. Others have examined a second multipotent cell population present in BM, marrow stromal cells (MSC), frequently referred to as mesenchymal stem cells. Thus, two groups have shown in vitro differentiation of MSC into cells expressing markers characteristic of cardiac myocytes [15,16], while in vivo studies in small and large animals have suggested clinical potential for these cells [15,17]. Cardiomyogenesis of donor cells offers much potential for improving cardiac function by replacing necrotic myocardium. However, this is not the only mechanism to achieve this goal. Angiogenic treatments offer much promise in the setting of acute myocardial infarctions, an injury that acts through ischemic pathways. We and others have demonstrated that BM is not the only source of cells with properties similar to MSC, and that adipose tissue represents a reservoir of cells with multipotent differentiation capacity [18]. Recently, three groups have reported in vitro differentiation of adipose tissue-derived cells (ADC) into cardiomyocytic-like cells [19 /21]. Although distinct differentiation induction methods were used, each group reported spontaneously beating cells and evidence of expression of cardiomyocytic cell surface markers. For this reason, we have examined the ability of ADC to engraft injured myocardium and to undergo differentiation into endothelial and cardiomyocytic phenotypes. Methods Cell preparation ADC were obtained as described previously [18]. Briefly, white subcutaneous adipose tissue was excised from the inguinal region of transgenic mice (ROSA) in which the beta-galactosidase gene product is expressed in almost every tissue (B6:129S-Gt(ROSA)26Sor) (Jackson Laboratories, Bar Harbor, ME, USA). Tissue was washed in 1 /PBS and digested in 0.075% Collagenase I (Sigma, St Louis, MO, USA) for 45 min at 378C shaking. The digest was passed through a gradient of cell straining filters (100 mm, 70 mm and 40 mm) and washed twice in 1/PBS by centrifugation at 400 g for 5 min. The cell pellet was resuspended in sterile saline and counted using a hemocytometer. Viability was determined using acridine orange live/dead stain (Molecular Probes, Eugene, OR, USA) under fluorescence microscopy. Cells were again centrifuged at 400 g for 5 min and resuspended at a concentration of 5/10 6 cells/ml. Cells were kept at 48C until experimental use or placed in vitro for cardiomyocytic differentiation. ADC characterization For the cytofluorometric assays, uncultured ADC were stained using Ab to Ly-6A/E (Sca-1) /FITC, CD34R /PE, CD117R /PE, CD81R /PE, CD44 APC, CD73R /PE and CD184R/PE for 20 minutes at 48C. After staining, the cells were washed twice in PBS and analyzed using a standard Becton-Dickinson FACSAria instrument (BD, San Jose, CA, USA), equipped with a 488 nm solid state laser and a 633 nm air cooled laser. The data were acquired and analyzed using the FACSDiVa software (BD, San Jose, CA, USA). All Ab and isotype controls were purchased from BD-Pharmingen (San Diego, CA, USA). In vitro cardiomyocytic differentiation Cells were grown for two passages and then replated in 100-mm tissue culture-treated dishes at a density of 5000/ cm 2 in complete media consisting of RPMI-1640, 15% FBS and 1% antibiotic/anti-mycotic. After 24 h, cells were exposed to 9 m m of 5-azacytidine (5-AZA; Sigma) for 24 h. After this transient exposure, the medium was replaced back to complete media and cells were collected at 0, 2 and 4 weeks for RT-PCR. Total RNA was extracted from adult

3 284 BM Strem et al. mouse heart (serving as a control) and ADC utilizing a commercially available kit (Qiagen, Valencia, CA, USA). Samples were treated with DNase I at 238C for 15 min and cdna was synthesized from 2 m g total RNA by using SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacture s instruction. The cardiac-specific genes a- and b-mhc, MEF-2c and -2d, MLC-2v, GATA-4 and TEF-1 were amplified using standard PCR techniques and products were size-fractionated by 2% agarose gel electrophoresis (PCR primers available upon request). ADC were also grown, as described above, on glass cover slips in six-well plates for 4 weeks. Cells were washed with 1/PBS and then fixed with methanol and acetone [50/50] at /208C for 10 min and permeabilized with 0.1% Triton X-100 (TX) for 10 min. After incubation with 5% goat serum at room temperature for 1 h to block non-specific labeling, a MAb against sarcomeric MHC (MF 20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied for 1 h at room temperature. After three 5-min washes with TX, cells were incubated with Texas Red 594- labeled goat anti-mouse IgG (Santa Cruz Biotechnology) for 1 h at room temperature. Cells were then washed with TX three times and mounted in medium contain DAPI. All solutions for Ab dilution were made in TX buffer containing 5% goat serum. Animal and study design All protocols involving animals were approved by the Animal Care and Use Committee of Macropore Biosurgery (San Diego, CA, USA). This investigation was performed in conformance with the principles described in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health [22]. Thirty-six mice were divided into three cohorts. One group received infarcts and cells, a second group received infarcts and no cells (negative control) and a third group received cells but no infarct (sham). Each of these cohorts was further subdivided equally into three groups harvested at 1, 7 and 14 days. B6129SF1/J mice (Jackson Laboratories) were anesthetized with an i.p. injection of 100/10 mg/kg ketamine/ xylazine (TW Medical Veterinary Supplies, Austin, TX, USA). A 20-gauge endotracheal tube was inserted and the mice were ventilated at 0.40 ml, 148 strokes/min, using a small animal ventilator (Harvard Apparatus, Holliston, MA, USA) to control respiration. Open-thoracic cardiac surgery was performed through a 1-cm left thoracotomy in the fourth intercostal space under sterile conditions. The left ventricle was visually confirmed by identification of the left antrioventricular descending artery. The left ventricular free wall was cryogenically injured by a 15- second direct application of a blunt probe pre-conditioned to /1968C; 200 ml of either 1/10 6 ADC (from the syngeneic ROSA donor described above) or saline was injected into the left ventricular chamber. The thoracotomy was closed with 5-O vicryl (TW Medical Veterinary Supplies) sutures and intrathoracic pressure was returned to normal. Remaining incisions were closed with running 5-O vicryl sutures. The animals were then allowed to recover in a warmed (308C) environment. Post-operative pain was minimized by intramuscular administration of 0.05 mg/kg Buprenex (TW Medical Veterinary Supplies) every 12 h post-surgery for 2 days and as needed thereafter. At 24 h, 7 days and 14 days post-surgery, mice were killed by isofluorane (TW Medical Veterinary Supplies) overdose. Retrograde perfusion was performed with 60 ml sterile saline through the descending aorta, and hearts were isolated, embedded in TBS tissue freezing medium (Fisher Scientific, Pittsburgh, PA, USA), frozen on dry ice and stored at /808C until cryosectioned. Histology Mice hearts were cut transversely into 5-mm sections and stained to identify donor-derived cells and lineage-specific differentiation markers. For b-galactosidase staining, slides were warmed to 258C in1/pbs, then immersed in 0.5% gluteraldahyde (Sigma) for 30 min at 48C. Post-fixation, slides were washed twice with 1/PBS for 5 min each, shaken at 258C then transferred to 1/ PBS, ph 8.5, until staining buffer was applied. Staining buffer consisted of 1 mg/ml 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (x-gal; Sigma), 5 mm potassium ferrocyanide, 5 mm potassium ferricyanide, 2 mm magnesium chloride, 0.01% sodium deoxycholate and 0.02% NP40 in 1 /PBS, ph 8.5, overnight at 378C. Slides were then counterstained with either immunohistochemical markers for angiogenesis or cardiomyocytic differentiation, Sirius Red or Masson s Trichrome for collagen, or hematoxylin and eosin for histomorphometry. For cardiomyocytic differentiation, four common markers were used: Troponin I (FL-210; Santa Cruz Biotechnology), myosin heavy chain (H-300; Santa Cruz Biotechnology), Nkx2.5 (N-19; Santa Cruz Biotechnology) and Connexin 43 (C-20; Santa Cruz

4 Expression of cardiomyocytic markers on adipose tissue-derived cells 285 Biotechnology). Skeletal myocytes were identified with MyoD (MAb 5.8A; BD PharMingen, San Diego, CA, USA). Vascular endothelial cells were identified with CD31 (MEC13.3; BD PharMingen) and von Willebrand Factor (AB6994; Abcam, Cambridge, MA, USA). Leukocytes were identified with CD45 expression (M-20; Santa Cruz Biotechnology). All staining used the Vectastain ABC system (Vector Laboratories, Burlingame, CA, USA). Slides were counterstained with Gill No. 1 Hematoxylin (Sigma) to identify nuclei. Results ADC characterization Average processing yields were 8.19/2.3/10 6 ADC/g of adipose tissue, which were characterized using flow cytometry for common stem cell surface markers. As noted in Table 1, a fraction of ADC was shown to be positive for the pluripotent markers CD34, c-kit and Sca-1. While the levels of CD34 and Sca-1 were comparable to BM, there were significantly more c-kit cells present in ADC. Upon placement of ADC into culture, their cell surface phenotype strongly correlated with that of MSC [23,24], expressing markers such as CD105 and CD166. In vitro cardiomyocytic differentiation As mentioned above, several groups have suggested that adult stem cells can differentiate into cardiac-like cells. To determine if murine ADC were capable of expressing cardiomyocytic markers in vitro, we treated them with the demethylating agent 5-azacytidine for 24 h. Cells were collected after 2 and 4 weeks and analyzed for the expression of a panel of cardiomyocytic markers. RT- PCR performed on total RNA prepared from parallel cultures of ADC revealed that expression of cardiacspecific markers MLC-2V, GATA-4 and a- and b-mhc was specifically up-regulated in ADC treated with 5-AZA (Figure 1a). To confirm that these mrna resulted in the cognate protein expression, ADC were immunostained with a MAb against sarcomeric MHC, 4 weeks after 5- AZA treatment. Only cells treated with 5-AZA demonstrated expression of sarcomeric MHC (Figure 1b). Therefore, we investigated further whether ADC could differentiate into cardiac-like cells in vivo. Myocardial injury Cryoinjury resulted in a reproducible transmural infarct that encompassed approximately 15% of the left ventricular free Table 1. Relative expression of common stem cell-associated surface molecules appears on subpopulations of non-buoyant cells within the heterogeneous ADC isolate Cell Surface Marker CD34 CD44 CD73 CD81 CD117 (c-kit) CD184 (CXCR4) Sca-1 % of ADCs Positive (range) 7.2 (1.3 /15.3) 42.9 (9.3 /83.6) 21.3 (11.1/25.9) 37.7 (NA) 14.6 (1.0 /18.0) 4.3 (1.0 /11.1) 58.7 (32.2/76.4) wall (Figure 2). In addition to the primary lesion, small periinfarct injured regions were frequently observed (Figure 2b). By day 14, the infarct region was largely populated by fibroblasts, with some residual leukocytes and extensive deposition of collagen, as evidenced by staining with Masson s Trichrome (Figure 2c). There was little mortality from this injury; all three groups (experimental, negative control, and sham) had /85% survival over the course of the study. Engraftment of donor-derived ADC Scattered clusters of beta-galactosidase cells were detected in close association with the infarcts of cryoinjured mice 24 h following injection (Figure 3a,b). No staining was observed in healthy, undamaged myocardium. Similarly, no staining was observed in sham-injured or in cryoinjured animals treated with saline alone. Seven days post-injection, beta-galactosidase-positivity was confined to small, injured peri-infarct regions (Figure 3c,d) that were largely confined to the area near the ventricular surface of the free wall. Co-staining with X-gal and Sirius Red (Figure 3e,f) demonstrated localization of donorderived cells in areas of scar formation. Similar regions were observed at day 14 (Figure 3g /j) after cell injection. Immunohistochemical characterization of donorderived cells Immunohistochemistry demonstrated expression of myosin heavy chain (Figure 4b), Troponin I (Figure 4c) and Nkx2.5 (Figure 5) by beta-galactosidase cells at day 14. Co-staining was most frequently detected on smaller cells within the region of engraftment (Figure 4). Larger cells tended to have lower X-gal staining, perhaps due to dilution of enzyme in the larger cytoplasmic volume, such that X-gal staining was sometimes obscured by the immunohistochemical stain. However, we did not observe

5 286 BM Strem et al. Figure 1. Murine ADC cultured in vitro after 5-AZA treatment demonstrate up-regulation of cardiac-specific genes by RT-PCR (a) and immunofluorescence demonstrating sarcomeric MHC protein expression (b). staining for the gap junction protein Connexin 43 on X-gal cells (data not shown). Similarly, we did not detect expression of the skeletal muscle marker MyoD (data not shown) or endothelial markers CD31 and vwf by betagalactosidase cells (Figure 6). However, while donorderived cells themselves did not express endothelial markers, the infarct regions immediately surrounding donor cells showed considerable staining for both markers (Figure 6). Finally, beta-galactosidase cells were uniformly negative for the leukocyte marker CD45 (data not shown). Discussion In the present study, we have verified previous findings of the ability of ADC to express cardiomyocytic-specific markers in vitro [19 /21]. Moreover, we are the first to report that freshly isolated syngeneic ADC, upon immediate delivery into the left ventricular chamber after Figure 2. Transmural injury at the site of probe contact (a, scale bar/200 mm) introduces pocket infarct regions on the endocardial side of the LVFW (b, scale bar/100 mm). (c) Trichrome staining (scale bar/200 mm).

6 Expression of cardiomyocytic markers on adipose tissue-derived cells 287 Figure 3. Donor-derived ADC engrafted in regions of pocket infarcts. Twenty-four hours post-injury, donor-derived ADC can be identified (a, b). One and 2 weeks following injury, donor-derived ADC are prevalent in these pocket infarct regions (c /j). cryoinjury, engraft within damaged myocardium. While this animal model of AMI has practical limitations, we believe it is a useful model to predict cell delivery, engraftment and initial repair in the hearts of patients receiving cell therapy, acutely after injury. Induction of myocardial damage using cryoinjury is a well-established experimental model [25 /28] that has been used to examine the therapeutic potential of cell transplantation using endothelial cells [29], BM cells [30], skeletal muscle myoblasts [28,31,32] and gene-modified cells [33]. Similarly, injection of cells into the ventricular chamber is considered an approximation of intracoronary artery administration of MSC, and thus has been used in small animal studies to enhance cell delivery over that achieved by i.v. administration [17]. In the present study, we applied this cell delivery route to investigate the ability of adipose tissue-derived cells to take up residence in and differentiate within injured myocardium. We evaluated syngeneic ADC because autologous cells will be immunologically tolerated and can be readily isolated from the adipose tissue of patients while they are receiving standard treatment in the clinic. However, for some patients who may be poor candidates for adipose tissue resection for various reasons, an allogeneic source of ADC might be preferred. The immune tolerance of allogeneic ADC needs to be confirmed if this is to be a clinically viable option. Multiple investigators have reported that cultured ADC do not express HLA-DR protein [24,34], suggesting their potential for allogeneic transplantation [35]. Our data demonstrate that, despite the influx of inflammatory cells that occurs in the first few days

7 288 BM Strem et al. Figure 4. Infarct regions showing donor ADC (a) co-express cardiac markers 14 days following injury. Staining of similar region for MHC (b) and Trop I (c) on consecutive slides, with dual staining pictured in insets (scale bars/20 mm, inset scale bars/10 mm). following the injury [28], adipose tissue-derived cells are capable of engrafting in pockets of injured myocardium. Coronary artery perfusion is approximately 5% of cardiac output at rest, thus only a fraction of injected cells are likely exposed to the injured area prior to exposure to the capillary beds of the liver and lungs, which have been shown to be major sites of entrapment of injected cells [17]. We also noted higher levels of engraftment at later time points, suggesting that either the engrafted cells may be proliferating or that donor cells might continue to circulate and progressively engraft the infarct over time. The observation that adipose tissue-derived cells delivered by an intravascular route engraft within injured myocardium and not the healthy myocardium suggests the presence of homing mechanisms that specifically recruit cells to the site of injury. One potentially operative Figure 5. LacZ donor-derived ADC express Nkx2.5. (a) Expression of lacz within infarct region.(b) Consecutive slide stained for Nkx2.5 in the nucleus of the cells (scale bars/100 mm). Overlay analysis depicts co-expression of both beta-galactosidase and Nkx2.5 cells, which can be identified by matching shape outlines.

8 Expression of cardiomyocytic markers on adipose tissue-derived cells 289 Figure 6. LacZ donor-derived ADC do not express endothelial markers; (a) CD31 (scale bar/100 mm), (b) von Willebrand factor (scale bar/100 mm). (c) Donor-derived cells (arrows) shown adjacent to von Willebrand factor endothelium (arrows, scale bar/25 mm). mechanism is the migration of cells in response to the chemokine SDF-1, which has been shown to play a role in homing and migration of HSC and other progenitor cells through interactions with CD184 [36 /38]. Other investigators have demonstrated that SDF-1 is expressed in ischemic myocardium [39,40] and that experimentally induced prolongation of SDF-1 expression within the infarct by implantation of fibroblasts engineered to express SDF-1 is associated with increased stem cell recruitment and homing [41]. It is possible that this, or a similar mechanism (such as stem cell factor and c-kit interactions), induces homing of the ADC to the site of injury. Cell entrapment in the myocardial wall due to the needle tract may have occurred; however, donor cells were not observed in uninjured hearts that received cells, disputing this possibility. Furthermore, donor cells were observed in multiple locations of injury and spanned approximately 1 mm in the vertical direction, further suggesting that a localized delivery that would be caused by a needle tract did not occur. It should also be noted that donor cells were not observed throughout the infarct but, rather, were largely confined to injured regions of tissue surrounded by healthy myocardium. This regional distribution may reflect the nature of the cryoinjury model, as it creates a region of severe and immediate damage, resulting in some areas of complete necrosis that are incapable of initiating or sustaining homing signals or of supporting traffic of cells. Our data further indicate that by 2 weeks after injection donor cells within the infarct region expressed markers consistent with differentiation towards cardiac myocytes. Thus, we observed expression of myosin heavy chain, troponin I and nkx2.5. The possibility of this staining being an artifact (donor cells overlapped by host myocytes) can be ruled out because positive co-staining could be observed on consecutive 5-mm sections. If overlapping was responsible for this expression, multiple dual positive staining would not have been observed. Expression of Connexin 43, a marker associated with electrical coupling of cardiac myocytes, was not observed in donor cells. It is possible that the process of differentiation of donor cells and their electrophysiologic incorporation into undamaged myocardium is incomplete 14 days after injection or donor cells may be fusing with host cardiac myocytes. In the present study, we were unable to examine the question of fusion between donor and host cells. Characterization of donor-derived cells in longer-term engraftment studies may shed light on these questions. We have demonstrated that donor-derived cells present within the infarct are not of hematopoietic origin, as demonstrated by lack of expression of CD45. This stands in contrast to recent data demonstrating that highly purified HSC transferred into myocardium following occlusion/reperfusion injury do not differentiate into cell-expressing cardiomyocytic markers but rather retain a hematopoietic phenotype evidenced by expression of CD45 [12]. Thus, expression of MHC, troponin I and nkx2.5, in the absence of expression of MyoD and CD45, is consistent with in vivo differentiation of multipotent adipose tissue-derived cells into cells of a cardiomyocytic phenotype. This finding is consistent with published reports demonstrating a similar cardiac myocyte differentiation phenomenon in vitro [19 /21]. ADC have recently been shown to differentiate into cells with an endothelial cell phenotype [42,43]. In contrast, we were unable to detect expression of endothelial markers on donor-derived cells. This suggests that donor cells were incapable of sustained endothelial

9 290 BM Strem et al. differentiation in this model. Hence, this cryoinjury model may not be suitable to support endothelial differentiation, since it is not an ischemia-induced injury. However, we did detect considerable numbers of endothelial cells in close proximity to donor cells consistent with the hypothesis that the presence of donor cells is inducing endogenous angiogenesis. While this hypothesis cannot be proven by the current data, similar observations have been made in animals injected with highly purified HSC [12] and skeletal muscle-derived myoblasts [44]. Interestingly, in this regard Rehman et al. have recently published data demonstrating expression of vascular endothelial growth factor (VEGF) by cultured adipose tissue-derived cells [45], which we have confirmed using an enzyme immunoassay (data not shown). Thus, our data are consistent with a model in which adipose tissue-derived cells induce angiogenesis at the site of engraftment through a VEGFmediated mechanism. In other preliminary assays, we have identified the potential of ADC to increase perfusion to surgically induced ischemic tissues (data not shown). In summary, in this study we have obtained data consistent with the hypothesis that adipose tissue contains a population of cells capable of homing to injured myocardium, of differentiating towards cardiac myocytes, and of inducing local angiogenesis. Thus, these cells may have the potential to contribute to improved function following AMI. We are currently engaged in comprehensive studies using a more clinically relevant small animal model and examining functional endpoints to test this hypothesis. These studies include investigating the acute functional effects of ADC delivery on reducing infarct size. As part of this aim, we are identifying the optimal time frame that ADC can be delivered acutely after injury and mediate functional improvements. Acknowledgements This research was supported in part by NHLBI Grant HL The authors would like to acknowledge the excellent technical assistance of Liu Wei, Zhengyu Zhou and Joel Mainit in animal care and Rebecca Symons and Mike DeEmedio in histology. References 1 Beltrami AP, Barlucchi L, Torella D et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763 /76. 2 Remme WJ. Overview of the relationship between ischemia and congestive heart failure. Clin Cardiol 2000;23:4 /8. 3 Menasche P, Hagege AA, Vilquin JT et al. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol 2003;41:1078 /83. 4 Reinecke H, Poppa V, Murry CE. Skeletal muscle stem cells do not transdifferentiate into cardiomyocytes after cardiac grafting. J Mol Cell Cardiol 2002;34:241 /9. 5 Murry CE, Wiseman RW, Schwartz SM et al. Skeletal myoblast transplantation for repair of myocardial necrosis. 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