Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response

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1 The FASEB Journal Research Communication Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response Jeannette Nussbaum,*,,,1 Elina Minami,*,,,,1 Michael A. Laflamme,*,, Jitka A. I. Virag,*,, Carol B. Ware,, Amanda Masino,*,, Veronica Muskheli,*,, Lil Pabon,*,, Hans Reinecke,*,, and Charles E. Murry*,,,2 Departments of *Pathology, Medicine/Cardiology, and Comparative Medicine, Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA ABSTRACT Embryonic stem (ES) cells are promising for cardiac repair, but directing their differentiation toward cardiomyocytes remains challenging. We investigated whether the heart guides ES cells toward cardiomyocytes in vivo and whether allogeneic ES cells were immunologically tolerated. Undifferentiated mouse ES cells consistently formed cardiac teratomas in nude or immunocompetent syngeneic mice. Cardiac teratomas contained no more cardiomyocytes than hind-limb teratomas, suggesting lack of guided differentiation. ES cells also formed teratomas in infarcted hearts, indicating injury-related signals did not direct cardiac differentiation. Allogeneic ES cells also caused cardiac teratomas, but these were immunologically rejected after several weeks, in association with increased inflammation and up-regulation of class I and II histocompatibility antigens. Fusion between ES cells and cardiomyocytes occurred in vivo, but was rare. Infarct autofluorescence was identified as an artifact that might be mistaken for enhanced GFP expression and true regeneration. Hence, undifferentiated ES cells were not guided toward a cardiomyocyte fate in either normal or infarcted hearts, and there was no evidence for allogeneic immune tolerance of ES cell derivatives. Successful cardiac repair strategies involving ES cells will need to control cardiac differentiation, avoid introducing undifferentiated cells, and will likely require immune modulation to avoid rejection. Nussbaum, J., Minami, E., Laflamme, M. A., Virag, J. A. I., Ware C. B., Masino, A., Muskheli, V., Pabon, L., Reinecke, H., Murry, C. E. Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response. FASEB J. 21, (2007) Key Words: cardiomyocyte differentiation rejection tolerance fusion autofluorescence Coronary heart disease continues to be the greatest health problem in industrialized countries. In the United States nearly 1 million people a year suffer from myocardial infarction (1). It is well established that the heart has an insufficient intrinsic regenerative response; hence, infarcts heal by formation of noncontractile scar tissue (2, 3). So far, the only therapeutic option to replace tissue lost to infarction is transplantation of the entire organ, an approach severely limited due to the lack of donor organs and allograft rejection. Current research is focused on restoring cardiac function after myocardial infarction (MI) by transplanting a variety of cell types, including skeletal muscle (4 9), cardiomyocytes (10 12), or cells selected from bone marrow (13 15). Cardiomyocytes would seem to be the optimal cell type to repair a myocardial infarct based on their contractile properties and ability to form electromechanical junctions with host myocardium (10, 12, 15). Because primary cultures of cardiomyocytes do not proliferate and are difficult to obtain in humans, much recent research has focused on pluripotent embryonic stem (ES) cells. Various groups have investigated cardiac development within differentiating ES cells and found that they recapitulate normal embryological development (16 23). Furthermore, Field s group has shown that ES cell-derived cardiomyocytes will form stable grafts when implanted into normal hearts (22). These findings suggest that differentiating ES cells provide a potential source of donor cardiomyocytes suitable for transplantation. It was recently reported that transplantation of undifferentiated embryonic stem cells improves cardiac function after myocardial infarction and that ES cells could be transplanted into the heart across major histocompatibility and even species barriers (24 27). The authors of these studies suggested that functional 1 These authors contributed equally to this work. 2 Correspondence: Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, 815 Mercer St., Seattle, WA 98109, USA. murry@u.washington.edu doi: /fj com /07/ FASEB 1345

2 improvement was based on differentiation of the ES cells into cardiomyocytes. In contrast, when undifferentiated ES cells are implanted into many extracardiac sites, they form teratomas containing derivatives of all three germ layers (28 30). This seems to imply that the heart is an especially instructive environment compared with many other locations in the body, and that ES cells and their progeny enjoy an immune privilege not shared by other cells. Here we show that undifferentiated ES cells form teratomas when transplanted directly into the heart and that allogeneic ES cell derivatives elicit a vigorous immune response and are ultimately rejected. These findings indicate that it is still critical to develop efficient methods to guide cardiac differentiation. In addition, care must be taken to remove undifferentiated or noncardiogenic cells from any ES cell-derived population intended for therapy, and immunomodulatory therapies may be required to prevent allogeneic graft cell rejection. MATERIALS AND METHODS Cell culture Male murine ES cells of the R1 (from 129/Sv mouse) (31), CGR8 (from 129/Ola mouse) (32), and C57 (from C57Bl/6 mouse) lines (33) were maintained in an undifferentiated state in Dulbecco s modified Eagle medium (DMEM) high glucose (4.5 g/l), supplemented with 2 mm l-glutamine (Life Technologies, Inc., Carlsbad, CA, USA), 0.1 mm nonessential amino acids (Life Technologies.), 0.15 mm monothioglycerol (Sigma, St. Louis, MO, USA) 1 mm sodium pyruvate (Life Technologies), 50 U/ml penicillin, 50 g/ml streptomycin, 0.25 g/ml amphotericin (Life Technologies), 15% FBS (Schenk, Stanwood, WA, USA), and 1000 U/ml recombinant human leukemia inhibitory factor (LIF; Chemicon, Temecula, CA, USA). The C57 ES cells were stably transfected with the pcx- ( -actin) EGFP construct (34) to constitutively express green fluorescent protein under the control of the chicken -actin promoter. ES cells were grown on gelatin-coated tissue culture flasks (Corning, Corning, NY, USA) to 80% confluency, then passaged into fresh medium every 2 days. Surgical procedure All experiments were approved by the University of Washington Animal Care and Use Committee and were performed in accordance with federal guidelines. Studies were performed using male nude mice [CD-1 [ICR] nu/nu homozygotes, Charles River, Wilmington, MA, USA], C57Bl/6J mice, and male or female Balb/c mice as cell recipients (Jackson Laboratories, Bar Harbor, ME, USA). Coronary occlusion and cell implantation into the heart were performed as recently detailed (35, 36). In brief, mice were anesthetized with Avertin, supported on a ventilator and their chests opened aseptically to expose the heart. Undifferentiated ES cells (500,000 unless otherwise specified) were suspended in 7 l serum-free medium and injected into the anterior wall of the left ventricle with a Hamilton syringe. In most animals, the hearts were uninjured, but in some, myocardial infarction was induced by ligating the left anterior descending artery (LAD) using an 8 0 monofilament PE suture and a 6-mm tapered needle. In infarcted animals, undifferentiated ES cells were injected into one side of the border zone of the ischemic vascular bed. Animals were euthanized with an overdose of pentobarbital (Delmarva Laboratories, Inc., Midlothian, VA, USA) at 3 wk postinjection unless specified otherwise. The hearts were collected and fixed in methyl Carnoy s solution for 24 h, and routinely processed, paraffin-embedded and sectioned for histological evaluation (37, 38). Histology and immunohistochemistry Tissue sections were stained with hematoxylin-eosin to visualize the general morphology. For immunostaining, tissue sections were quenched for 30 min in 3% H 2 O 2 in methanol to block endogenous peroxidases. Slides were incubated 24 h at 4 C in a humidified chamber with the following antibodies: a rabbit polyclonal EGFP antibody (1:1500, Abcam, Cambridge, MA, USA) to track grafted cells within the host myocardium; a polyclonal -fetoprotein (AFP) antibody (1: 10,000; Dako) to detect endoderm-derived cells; a monoclonal sarcomeric actin (5c5) antibody (1:20,000 Sigma), using a blocking kit to reduce background (DAKO Ark-Kit), and monoclonal smooth muscle -actin (full-strength; Dako, Carpinteria, CA, USA) antibody to identify mesoderm structures. A polyclonal neurofilament antibody (NF-200) (1: 10,000; Sigma) was used to detect neuro-ectodermal differentiation. After application of appropriate secondary antibodies or reaction complexes, reaction products were visualized with diamino-benzidine (3,3 -diaminobenzidine; Sigma-Fast tablet sets). Cardiac differentiation in ES cell-derived teratomas was quantified from sarcomeric actin-stained sections using Scion Image Analysis Software (Scion Corporation, Frederick, MD, USA). The absence of skeletal muscle differentiation in ES cell-derived teratomas was confirmed by staining the tissue section with a monoclonal MY-32 antibody (Sigma, 1:400, using DAKO Ark-Kit) specific for fast skeletal myosin heavy chain. To detect a potential host immune response to the allogeneic ES cell grafts, we stained heart tissue sections with a CD45 antibody (PharMingen, San Diego, CA, USA; 1:2000, specific for leukocyte common antigen, Ly-5). The leukocyte density in the tumor, the intervening granulation tissue, and the surrounding myocardium were scored by a blinded observer (CEM) on a semiquantitative 0 to 4 scale. Morphometric analysis Cardiac differentiation in ES cell grafts was measured by immunostaining for myosin light chain 2v (polyclonal antiserum against MLC2V; a gift from Dr. Ken Chien) or sarcomeric actin (5C5 monoclonal antibody; Sigma). We studied C57Bl/6 mice receiving syngeneic, undifferentiated ES cells (heart, n 14; hind limb, n 4). Digital images were taken at 100 using a SPOT RT digital camera and SPOT imaging software (Diagnostic Instruments, Sterling Heights, MI, USA). The sarcomeric actin- or MLC-2V-positive areas of the tumor sections were traced using Scion Imaging software (Scion Corp.). Measurements were statistically analyzed using a 2-sample t test. Although the two antibodies gave generally comparable staining, the MLC-2V antibody showed some cross-reactivity with epithelial cells, so only morphometric data from sarcomeric actin stained sections are presented. Evaluation of ES cell-cardiomyocyte fusion To determine if fusion occurs in hearts grafted with ES cells, undifferentiated C57Bl/6 ES cells were transfected with Adfloxed-LacZ adenovirus ( 250 particles/cell; Microbix Bio Vol. 21 May 2007 The FASEB Journal NUSSBAUM ET AL.

3 systems, Toronto, ONT, Canada) as described (37, 38). 500,000 cells were prepared and injected into the uninjured heart of an -MHC/Cre FVB/N mouse in which Cre recombinase is expressed only in cardiomyocytes (37) (n 3). If fusion between ES cells and cardiomyocytes has taken place, the Cre recombinase will excise a floxed stop sequence, allowing expression of -galactosidase. After 3 days, hearts were collected and placed in 30% sucrose solution overnight and embedded in OCT embedding media (Sakura, Torrance, CA, USA). The entire heart was cryosectioned at 7 m thickness; every third section was fixed with 4% paraformadehyde for 5 min at room temperature and stained overnight with x-gal solution at 37 C. The sections were then counterstained with Contrast Red and visualized under bright-field microscopy with a 10 objective. Digital images of positive cells were taken with QCapture software. Evaluation for autofluorescenct artifact To distinguish between bona fide GFP signal and autofluorescent artifact, infarcted hearts from C57Bl/6 mice were injected with 500,000 undifferentiated C57Bl/6 EGFP-ES cells. After 3 wk, the hearts were dehydrated in 30% sucrose solution overnight and placed in OCT embedding media (Sakura) and cryosectioned. Each section was fixed in 4% paraformadehyde for 5 min and thoroughly rinsed with PBS. The sections were stained similarly as described above for EGFP, but the GFP signal was detected with Alexa 555- streptavidin (1:400; Molecular Probes, Carlsbad, CA, USA), counterstained with 4,6-diamidino-2-phenylindole, and visualized using fluorescent microscopy (Nikon Eclipse 80i). The EGFP antibody-derived signal was compared with intrinsic tissue fluorescence (native EGFP fluorescence plus tissue autofluorescence). RESULTS Undifferentiated ES cells cause tumor formation in the heart The initial hypothesis of this study was that the heart would provide an instructive environment to guide ES cell differentiation into cardiomyocytes. For our initial studies, we transplanted 500,000 undifferentiated ES cells into the uninjured left ventricles (LVs) of immunocompromised or syngeneic recipients. R1-ES cells from strain 129/sv and CGR8 ES cells from strain 129/Ola were grafted onto hearts of nude mice (n 4/ group) and C57Bl/6-derived ES cells were grafted into the LVs of syngeneic animals (n 14). To facilitate tracking of ES cells after transplantation, undifferentiated C57Bl/6-ES cells were stably transfected with a construct containing the chicken -actin promoter driving EGFP. Three weeks post-es cell injection, teratomas had formed in the hearts of all nude (Fig. 1A) and syngeneic animals (Fig. 1B). Teratomas were histologically analyzed to identify various cell types. All tumors, independent of host or ES cell type injected, consisted of structures derived from all three embry- Figure 1. Teratoma development in hearts of nude and C57Bl/6 mice. Hearts of nude and C57Bl/6 mice 3 wk postinjection of undifferentiated ES cells. A) Teratoma formation after injection of R1-ES cells into the heart of an immunocompromised nude mouse. The tumor replaces much of the LV wall and contains heterologous elements, including multiple epithelial-lined cysts. B) Teratoma formation after injection of syngeneic C57-ES cells into the heart of C57Bl/6 mouse. A necrotic core and multiple cystic and ductal structures are evident. C) Time course study of C57 syngeneic transplants. After 3 days, the ES cell graft consisted mostly of small blue cells without any organization. Necrotic cell debris is present within the graft. At 7 days postinjection, ES cell grafts expanded in size and were organized in clumps and cords. The graft cells were highly proliferative, displaying an abundance of mitotic figures (inset). At 14 days simple epithelial-lined duct-like structures were present. At 3 wk, the grafts had formed teratomas consisting of derivatives of ectoderm (a, squamous epithelium), mesoderm (b, cartilage), and endoderm (c, ciliated respiratory-like epithelium). CARDIAC ES CELL-DERIVED TERATOMAS 1347

4 onic germ lineages. Ectoderm-derived cells included keratinizing stratified squamous epithelium (Fig. 2A) and neurofilament-positive neuronal tissue (Fig. 2B, C). Mesoderm-derived cells included bone and cartilage (Fig. 2D) as well as smooth muscle (Fig. 2E, F). Endoderm-derived structures contained gut epithelium with goblet cells (Fig. 2G) and ciliated respiratory-like epithelium (Fig. 2H). Endoderm-derived cells also stained positive for -fetoprotein (Fig. 2I). We observed an increase in cartilage and bone structures in teratomas that resulted from injection of R1-ES cells into nude mice compared with CGR8 or C57Bl/6 ES cellderived tumors. Teratomas in the syngeneic group (C57Bl/6 animals with C57Bl/6-ES cell injections) showed an abundance of duct-like structures with stratified and pseudo-stratified epithelium. Time course study We next investigated the time course of tumor development with ES cell transplantation in syngeneic hosts (Fig. 1C). C57Bl/6 recipient mice were killed 3, 7, 14 and 21 days post-c57-es cell injection and analyzed histologically. At 3 days the ES cell grafts appeared as small clusters of cells with high nuclear-to-cytoplasmic ratios, and numerous mitotic figures were present. After 7 days of transplantation, the grafts appeared significantly larger and had abundant mitotic figures. After 2 wk, grafts in the syngeneic animals were larger still and displayed the first differentiated structures, such as epithelial ducts. After 3 wk, clear teratomas had formed with cell types derived from ectoderm (keratinized epithelium), endoderm (ciliated epithelium, goblet cells), and mesoderm (cartilage). Dose-response of undifferentiated ES cells transplanted into syngeneic recipients We next hypothesized that the differentiation pattern of ES cell grafts in the heart might be dose dependent, with tumors forming at high doses but cardiac differentiation occurring at lower doses. To test this, C57Bl/6 ES cells were injected into uninjured hearts of C57Bl/6 mice at doses ranging from 500 to 500,000 cells. Hearts were examined histologically 3 wk posttransplantation. As shown in Fig. 3A, teratoma formation showed a dose-response relationship, with no tumors forming at 50,000 cells, with 50% of animals developing tumors at 100,000 cells, and 80% of animals developing tumors at 250,000 cells. Tumor development was observed in 100% of animals that received 500,000 undifferentiated ES cells. Despite detailed histological evaluation, no surviving graft cells were detected in hearts receiving 50,000 undifferentiated ES cells (Fig. 3B). The absence of ES cell-derived grafts in these hearts was confirmed by the absence of immunostaining for EGFP. In contrast, teratomas stained intensely for EGFP (Fig. 3C). Cardiac differentiation in teratomas Although teratomas formed consistently in immunotolerant hearts, it remained a possibility that cardiomyocytes were among the mesodermal cell types that had differentiated and that the cardiac environment promoted this differentiation. To test this hypothesis, C57Bl/6-ES cells were introduced into the LV (n 14) or the hamstring muscle (n 4) of syngeneic C57Bl/6 mice. After 3 wk, the tumors were analyzed for cardiac differentiation by measuring the fraction of the tumors expressing sarcomeric actin (Fig. 4A). All tumors were consistently negative for the skeletal muscle marker, myosin heavy chain-fast. Therefore, sarcomeric actin staining indicates cardiac differentiation in this case (Fig. 4B). Teratomas in both the heart and the hind limb showed clusters of ES cell-derived cardiomyocytes. Quantitative morphometry revealed that % of the tumor mass in the hind limb was composed of cardiomyocytes, whereas % of tumor mass in the heart contained cardiomyocytes (P ns; Fig. 4C). We considered the possibility that cardiac differentiation might be enhanced at the graft-host border, but careful examination demonstrated no discernible increase in cardiac differentiation at the tumor s margin vs. its center. These studies suggest that the heart does not provide an environment conducive for cardiac differentiation, at least no more so than skeletal muscle. Infarction does not promote ES cell differentiation into cardiomyocytes We next asked whether ES cells might be guided toward cardiomyocyte differentiation in the infarcted heart, possibly replacing the lost myocardium as has been suggested recently (24 26, 39). Myocardial infarction was induced by LAD occlusion, and undifferentiated C57-ES cells were immediately injected into the ischemic border zone of C57Bl/6 (n 4) and Balb/c animals (n 5). After 3 wk of injection, teratoma development was observed in all animals independent of the recipient mouse strain (Fig. 5A). Morphometric analysis of sarcomeric actin-positive areas within the syngeneic teratomas showed a relatively small degree of cardiac differentiation in the infarcted heart ( %), not statistically different from that observed in noninfarcted recipients ( %) (Fig. 5B). This suggests that an injury environment does not prevent tumor formation or lead to increased cardiomyocyte differentiation. Immune response after allogeneic transplantation Another major question of this study was whether ES cells or their progeny would be immunologically tolerated in allogeneic hosts. For these studies, undifferentiated C57Bl/6-ES cells that stably expressed EGFP from the chicken -actin promoter were transplanted into the hearts of Balb/c recipients (n 5). A time 1348 Vol. 21 May 2007 The FASEB Journal NUSSBAUM ET AL.

5 Figure 2. Differentiation in ES cell-derived teratomas. Histological sections of ES cell-derived teratomas showing derivatives of ectoderm (A C), mesoderm (D F), and endoderm (G I). A) Stratified squamous keratinizing epithelium in R1-ES cells transplanted into the nude mouse heart (H&E). B) Nerve fibers in C57Bl/6-ES cells grafted into syngeneic host (H&E). C) Neurofilament-positive staining of serial section to panel B, identifying ES cell-derived neurons. D) R1-ES cell-derived teratomas in the nude mouse contained cartilage with calcification suggestive of endochondral bone formation (H&E) and E) smooth muscle cells (H&E) that stained positive for smooth muscle -actin (F). G) Endoderm-derived gut epithelium containing goblet cells (H&E) and H) ciliated respiratory-like epithelium (H&E) in R1 ES cell-derived tumor in the nude mouse heart. I) Endoderm-derived structures within the R1 ES-cell derived tumor in the nude mouse host, staining positive for -fetoprotein (AFP). CARDIAC ES CELL-DERIVED TERATOMAS 1349

6 Figure 3. Dose-response study. A) Incidence of tumor development 3 wk after injection of various doses of undifferentiated C57Bl/6-ES cells expressing EGFP into the LV of syngeneic mice. Tumors were observed in 50% of animals that received 100,000 ES cells and in 80% of animals that received 250,000 ES cells. All animals (100%) that received 500,000 ES cells developed teratomas. No tumor development could be observed in animals that received fewer than 100,000 ES cells. B) Histology of heart receiving 500 ESCs. No tumor is present. Scar tissue in injection site (high magnification in insets) contains no EGFP graft cells. C) Histology of heart receiving 100,000 ESCs. Endoluminal teratoma is present that stains intensely for EGFP (brown, inset). course study showed that teratomas formed in a pattern similar to the syngeneic model (data not shown). In brief, at 3 days the ES cell grafts were undifferentiated and highly proliferative. After 7 days, a significant increase in graft size could be observed, and, by 2 wk, the first signs of epithelial differentiation were apparent. At 3 wk, these allogeneic tumors could be characterized as teratomas, with cell types derived from all three embryonic germ lineages. Inflammation was apparent in the surrounding myocardium, intervening granulation tissue, and within the graft itself. By 5 wk, the inflammatory response was intense, and in some animals it obscured or replaced most of the graft cells. This inflammatory infiltrate suggested an immune response in the allogeneic setting. To compare inflammation between syngeneic and allogeneic grafts, we performed a blinded, semiquantitative assessment of leukocyte infiltration at 7, 14, and 21 days as well as 5 wk post-es cell transplantation by immunostaining for CD45 (common leukocyte antigen) and EGFP (for graft cells). Representative histological images are shown in Fig. 6 and the temporal analysis is shown in 1350 Vol. 21 May 2007 The FASEB Journal NUSSBAUM ET AL.

7 Figure 4. Cardiac differentiation in ES cell-derived teratomas. Cardiac differentiation in syngeneic C57-derived teratomas 3 wk postimplantation was compared in the heart vs. hind limb. A) Intraluminal cardiac teratoma surrounded by left ventricular myocardium, stained for sarcomeric actin (brown). Small islands of sarcomeric actin-positive myocardium are present within the tumor. Scale bar 100. B) Adjacent section stained for fast skeletal muscle myosin heavy chain. No skeletal muscle is present in the tumor, indicating sarcomeric actin staining is specific for cardiomyocytes. C) Quantitative morphometry demonstrated that cardiac teratomas derived from undifferentiated ESCs contain no more cardiomyocytes than do those in the hind limb. Fig. 7. In general, inflammatory cell content progressively increased over time in the allogeneic transplants and decreased in the syngeneic transplants. By 3 wk, the allogeneic grafts had significantly greater inflammation than did the syngeneic grafts (P 0.05) (Fig. 7C). There was a trend for greater inflammation in the surrounding granulation tissue and myocardium in the allogeneic grafts, which reached statistical significance after 5 wk (Fig. 7A, B). At 5 wk post-transplantation, 90% of the mice receiving allogeneic cells showed nearly complete elimination of the graft. In these hearts, the graft area was effaced by inflammatory cells and contained very few ES cell derivatives (Fig. 6). In contrast, syngeneic transplants at 5 wk remained large and had relatively little inflammation. These results indicate that transplantation of allogeneic ES cells leads to a significant host immune response after 3 wk, which leads to complete graft rejection at later time points. To test whether this rejection phenomenon was specific to the donor-host strains we had initially chosen, additional experiments were performed using undifferentiated CGR8 ES cells from strain 129/Ola introduced into the hearts of allogeneic C57Bl/6 recipient mice (n 10). Three weeks post-cgr8 ES cell injection, hearts from the allogeneic C57BL/6 donors did not exhibit any tumor formation; in fact, cell grafts could not even be detected (data not shown). This indicates that rejection of the allogeneic graft is independent of the host mouse strain or the ES cell type used. Major histocompatibility complex expression To understand the basis for rejection of allogeneic ES cell derivatives, we investigated class I and class II major histocompatibility antigen expression by flow cytometry. In these experiments, undifferentiated C57Bl/6 ES cells and EBs after 19 days of differentiation were analyzed in the presence or absence of IFN. Negative controls included duplicate samples stained with isotype-matched IgG and unstained samples (not shown). Primary spleen cell cultures were used as a broad positive control and treatment of primary kidney cultures was used as a positive control for IFN induction (not shown). As seen in the online supplemental Fig. 1, MHC levels could not be detected above background in undifferentiated ES cells even in the presence of IFN. Appreciable levels of MHC I and MHC II were observed in the differentiated EBs only in the presence of IFN. These data suggest that undifferentiated ES cells have a low immunogenic profile in the undifferentiated state; after differentiation, however, immunogenicity increases in the presence of inflammatory cytokines. Fusion of ES cells and host cardiomyocytes We employed a Cre-Lox system to determine if fusion occurred between ES cells and host cardiomyocytes after transplantation (37). ES cells were labeled with an adenovirus encoding a floxed LacZ reporter and trans- CARDIAC ES CELL-DERIVED TERATOMAS 1351

8 comparing EGFP immunostaining (using a red fluorescent label) to intrinsic tissue fluorescence (EGFP signal plus autofluorescent background). In the unstained sections, the teratomas had the expected green fluorescence (online supplemental Fig. 3). In the infarct, cardiomyocytes in the spared subendocardial region also exhibited green fluorescence, along with spindleshaped cells in the midwall. Based on intrinsic fluorescence, one might conclude that these green cells arose from the ES cells. In the immunostained sections, however, there was no red fluorescence in the cardiomyocytes or spindle-shaped cells in the infarct, indicating these cells did not contain the EGFP epitope. In contrast, the teratoma stained brightly with the EGFP antibody. Thus, these myocytes and spindle-shaped cells in the infarct region were clearly residual hostderived cells exhibiting autofluorescence. These results indicate the need for caution and careful examination of controls when using immunofluorescence to trace cell lineages in injured hearts. DISCUSSION Figure 5. Cardiac differentiation in infarcted and noninfarcted hearts. Undifferentiated ES cells were injected into the border zone of the ischemic region in syngeneic C57Bl/6 mice immediately after coronary occlusion. A) H&E staining of a 3-wk-old graft shows the development of a teratoma in LV wall adjacent to the infarct area (as outlined with arrows). The tumor extends from the LV wall into the LV lumen and shows differentiated structures such as epithelial lined cysts and ducts (inset). Note how the border of the endoventricular tumor matches that of the opposite ventricular wall. B) Morphometric analysis of cardiac differentiation based on sarcomeric actin staining in 3-wk-old grafts. Teratomas in the infarcted heart did not show a higher degree of cardiac differentiation than did the uninjured heart, indicating that cardiogenic signals were not increased after injury. planted into the hearts of transgenic mice expressing Cre recombinase in cardiomyocytes. Activation of LacZ expression indicates fusion has occurred, bringing the ES cell s DNA substrate into the same compartment as the cardiomyocyte s enzyme. At 3 days post-transplantation, exhaustive sectioning revealed a mean of 3 fusion events per heart (n 3 hearts; see online supplemental Fig. 2). This indicates that, while graft-host fusion can occur in this setting, it is rare. Evaluation for autofluorescence artifact Autofluorescence has emerged as a confounding factor in studies using EGFP fluorescence to track the fates of stem cells, particularly in injured tissues (40, 41). We explored possible autofluorescence in infarcted hearts receiving EGFP-expressing ES cells by systematically The main findings of this study are that 1) transplantation of undifferentiated mouse ES cells into the heart leads to teratoma formation; 2) teratomas formed in both normal and infarcted hearts; 3) cardiac teratomas contained no more cardiomyocytes than hind-limb teratomas; 4) teratomas were accepted in immunocompromised and syngeneic hosts but eventually were rejected in allogeneic hosts; 5) class I or II histocompatibility antigens could not be detected above background levels in undifferentiated ES cells, but these antigens were markedly up-regulated in differentiated cells after IFN treatment; 6) ES cell-cardiomyocyte fusion occurs rarely in this model; and 7) autofluorescence of cells in the infarct can be misjudged for EGFP expression unless appropriate controls are included. In the ensuing sections we compare these findings with the published literature and discuss possible relevance for ES cell-based cardiac repair. Differentiation of engrafted ES cells: tissue-specific or cell autonomous? Mouse ES cells are the best-studied pluripotent cell type. They can give rise to cells from all embryonic germ lineages and form chimeric mice after blastocyst injection, indicating that when appropriate environmental cues are present, their differentiation can be controlled precisely. On the other hand, ES cells have long been known to form teratomas when transplanted into the subcutaneous space (42), joint spaces (30), or spleen (27). In fact, the ability to form teratomas is one of the most rigorous criteria for defining pluripotentiality. These results imply that many adult tissues lack the signals needed to guide ES cells toward tissueappropriate fates. Our experiments suggest that the heart does not 1352 Vol. 21 May 2007 The FASEB Journal NUSSBAUM ET AL.

9 Figure 6. Histological demonstration of allogeneic immune rejection. C57 ES cells expressing EGFP were engrafted into hearts of C57Bl/6 or Balb/c mice. A) Leukocyte infiltration at 3 wk was identified by CD45 immunostaining (brown) in allogeneic (left panel) and syngeneic (right panel) hearts. Inflammation is much more intense surrounding and within the allogeneic graft, indicating immune rejection. B) Serial sections from an allogeneic graft showing nearly complete rejection by 5 wk. CD45 immunostaining (brown, left panel) shows an intense leukocyte infiltrate, nearly effacing the underlying tissue structure. EGFP immunostaining (brown, right panel) demonstrates a very small residual graft of allogeneic cells. offer signals to guide ES cells to form cardiomyocytes. Rather, we consistently observed teratomas when ES cells were implanted into the heart. This was true for three different lines of ES cells (CGR8 and R1 cells from strain 129, and another line from strain, C57Bl/6) and also for three separate strains of recipient mice (male CD-1 nudes, male C57Bl/6, and female Balb/c), including combinations of donor and host previously reported to result exclusively in cardiac differentiation (25). These experiments indicate that tumor formation is not dependent on the line of ES cells studied, host strain or gender, or a particular combination of ES cell line and host strain. We considered the possibility that a dose of cells exists that allows engraftment of ES-derived myocardium with no teratoma formation, reasoning that at high doses of cells, the majority of the graft was within an ES cell/teratoma environment rather than a cardiac environment. Dose-response studies did not support this hypothesis. Using a line of ES cells that constitutively expressed EGFP, we found that grafts were not detected when 50,000 cells were injected, whereas at higher doses all ES cell derivatives were contained within teratomas. Furthermore, cardiac differentiation was not enhanced at the graft-host interface, where potentially inductive host factors should have been concentrated the most. One explanation for the lack of any EGFP-positive cells in the heart after injection of 50,000 cells could be due to leakage of cells at the site of injection and washout. Cell retention has been a limitation in cell transplantation (43). After injection into beating myocardium, most cells either bleed back or wash out (43). In contrast to our findings, other studies support the hypothesis that ES cells are induced to form cardiomyocytes in the heart. Befahr et al. (25) reported that undifferentiated mouse ES cells became cardiomyocytes exclusively when transplanted into the hearts of allogeneic, immunocompetent C57Bl/6 hosts. They reported that undifferentiated tumors (not teratomas) formed when the ES cells expressed a dominant-negative TGF- receptor, suggesting that TGF- signaling was required for guided differentiation. Similarly, Wang et al. injected undifferentiated mouse ES cells expressing the EGFP transgene into the tail veins of allogeneic Balb/c recipients that exhibited viral myocarditis. They reported that ES cells homed to the host myocardium and formed cardiomyocytes exclusively, thereby increasing the survival of the diseased mice (44). Yang et al. reported that implantation of early- CARDIAC ES CELL-DERIVED TERATOMAS 1353

10 Figure 7. Time course of immune response to allogeneic ES cell grafts. C57 ES cells expressing EGFP were transplanted into the hearts of Balb/C or C57Bl/6 recipients. Tissue sections were stained for CD45 to detect leukocyte infiltration into the ES cell-derived tumors 7, 14, 21 days and 5 wk after transplantation. Inflammation was graded semiquantitatively by a blinded observer using a 0 4 scale (inflammatory score), with 4 representing maximal inflammation. Group sizes are given in parentheses above the bars. A) Leukocyte infiltration in the myocardium surrounding the tumor. Syngeneic grafts induced little inflammation in the surrounding myocardium, whereas inflammation steadily increased in this region with allogeneic grafts. B) Leukocyte infiltration in the granulation tissue between graft cells and host myocardium. In syngeneic grafts, inflammation steadily decreased over time as the injection site healed. In allogeneic grafts, inflammation was intense at all times. C) Leukocyte infiltration within the ES cell derived grafts. At 1 and 2 wk there was little inflammation in either group. In the allogeneic group, inflammation in the graft increased markedly at 3 wk and was maximal at 5 wk, while inflammation remained low in syngeneic grafts. Note that this underestimates severity of inflammation, because several animals where grafts were completely rejected were not included. *P differentiated mouse ES cells regenerated cardiac tissue and improved contractile function in the infarcted mouse heart (45). Most recently, Singla et al. reported that undifferentiated ES cells were directed toward a cardiomyocyte fate in the infarcted heart, resulting in improved mechanical function (24). The question then arises of why our data differ from the above-mentioned studies reporting guided differentiation of ES cells to cardiomyocytes. We have ruled out variations in ES cell lines, host strain and gender, ES cell dose, graft-host proximity, differentiation status at the time of transplantation, and the effect of myocardial injury. There are always minor differences between studies performed by different groups, and it is possible these could explain either the lack of guided differentiation or the lack of immune tolerance in our studies. For example, although all of these studies used similar conditions for culturing the ES cells, differences in serum quality or other seemingly minor culture conditions might explain the variation. Similarly, it remains a possibility that variations in mouse housing conditions could influence the fate of transplanted cells, although we have no data to support this idea. We tested the notion that predifferentiation of ES cells (without purification) might prevent tumor formation after transplantation, but found that teratomas arose after this procedure as well (data not shown). An alternative possibility that should be considered regarding the discrepancies cited above is that the ES cell derivatives in many studies may have been lost to rejection and host cardiomyocytes may have been mistaken for ES cell-derived cardiomyocytes. A critical element is how the lineage of the ES cells is followed. In all the above studies, the presence of the ES cell derivatives was based on the intrinsic fluorescence of EGFP or related proteins. Autofluorescence mimicking EGFP (or fluorescent immunostaining) has been described in normal and injured skeletal muscle (40), and we have reported similar autofluorescence in cardiomyocytes in the injured heart (46, 47). In the current study, we identified green fluorescent cardiomyocytes and spindle-shaped cells in the infarct that initially appeared to be derived from EGFP-expressing ES cells. When an anti-egfp antibody was used, however, neither the cardiomyocytes nor the spindle cells stained, indicating they exhibited artifactual autofluorescence (see online Supplemental Fig. 3). Thus, reliance solely on the intrinsic fluorescence of EGFP or other fluoroproteins is hazardous, especially when one is examining tissues with high levels of autofluorescence such as myocardial infarcts. Another possible confounding variable is fusion of ES cells with host cardiomyocytes, resulting in cardiomyocytes that express the ES cell lineage marker. We tested for fusion in this setting and found that ES cell-cardiomyocyte fusion occurred in three of three hearts tested. It should be noted, however, that fusion was an extremely rare event ( 3 cells/graft site). Based on its rarity, we think fusion is unlikely to explain the large-scale fluorescence reported in other studies (37). After the current manuscript had been accepted for publication, Kolossov et al. published a report showing teratoma formation after 4 wk in hearts that received injection of undifferentiated ES cells in the myocar Vol. 21 May 2007 The FASEB Journal NUSSBAUM ET AL.

11 dium, which confirms our findings (48). In addition, they created a transgenic mouse ES cell line containing the cardiac-specific -myosin heavy chain promoter driving both EGFP and the puromycin resistance gene. This transgene permits purification of ES-cell derived cardiomyocytes with puromycin and their subsequent tracking by EGFP expression. Cardiomyocytes purified from ES cells derivatives by puromycin selection did not form teratomas after intracardiac injection. This complements recent work from our group showing that when human embryonic stem cells were differentiated as embryoid bodies and subsequently enriched for cardiomyocytes, they did not form teratomas after transplantation into the heart, but rather formed stable grafts of human myocardium (47). This emphasizes the importance of predifferentiating ES cells and enriching the population for cardiomyocytes prior to cell injection to avoid teratoma formation. Immune privilege vs. immune rejection Immune rejection in response to antigenic differences is a well-described problem in solid organ transplantation and cell transplantation (e.g., islet cells) whereas its counterpart, graft-versus-host disease, occurs after bone marrow transplantation. A few tissues, such as corneas, have immune privilege that permits their transplantation across antigenic barriers. There is some evidence that certain stem cell populations, such as mesenchymal stem cells, may also have immune privilege based on their ability to suppress local immune responses (49). Several studies report immune privilege of embryonic stem cell derivatives in the heart based on graft cell survival across strain and even species barriers. Behfar et al. (25) reported 4 wk survival of ES cell derivatives from mouse strain 129 in hearts of allogeneic C57Bl/6 hosts. Wang et al. reported 2 wk survival of derivatives of 129-derived ES cells in hearts of allogeneic Balb/c recipients (44). Similarly, Yang et al. reported survival of EGFP graft cells in the infarcted hearts of allogeneic FVB/N mice 6 wk after implantation of early-differentiated 129-derived mouse ES cells (45). Data from several groups have even suggested that xenografting of EGFP-expressing mouse ES cells leads to significant improvement of cardiac function in the hearts of immunocompetent, postinfarcted rats due to the integration of functional ES-derived cardiomyocytes (25, 26, 39, 50). In contrast, our study found no evidence for immune tolerance of ES cells or their progeny in the heart. In 2 different allogeneic donor-host combinations (C57Bl/ 6-derived ES cells into BALB/c hosts and 129-derived ES cells into C57Bl/6 hosts), we observed substantial infiltration of the teratomas by host leukocytes at 3 wk and almost complete rejection by 5 wk. Syngeneic transplants, as expected, were well tolerated for up to 5 wk. Our in vitro studies suggest that up-regulation of class I and II histocompatibility antigens in response to local cytokine stimulation may contribute to the eventual rejection of the allogeneic graft cells. The difference between our findings and the above-cited literature cannot be explained by the degree of mismatch between donor and host, because we deliberately chose donor-host combinations previously reported to be tolerated (25). We conclude that ES cell derivatives are not immune privileged but rather face immune rejection similar to most other cell types. CONCLUSIONS Embryonic stem cells have the potential to generate virtually any cell type in the body, and this makes them extremely attractive for studies of tissue repair. The current study indicates that this same potential can also be problematic. The presence of undifferentiated cells in a transplant could result in the formation of cardiac teratomas rather than new myocardium. These findings underscore the importance of developing efficient strategies for selecting cardiomyocytes from other cell types in a mixed population. Indeed, when cardiomyocytes have been purified from predifferentiated ES cell cultures, their transplantation has resulted in the formation of new myocardium rather than teratomas (22, 47). The overall efficiency of this process would be greatly enhanced by developing methods to guide ES cells selectively to form cardiomyocytes or by isolating a progenitor population with a more restricted differentiation potential (e.g., a precardiac mesoderm cell). The vigorous rejection of ES cell derivatives in the heart also calls for further work to overcome the immune response to allogeneic transplantation. We thank Dr. Stephen Hauschka for his input to experimental design and for critically reading this manuscript. We thank Dr. Andrew Farr and Mr. James Dooley for advice on analysis of histocompatibility antigen expression. 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