transplantation of undifferentiated murine embryonic stem cells in

The FASEB Journal • Research Communication

Transplantation of undifferentiated murine embryonicstem cells in the heart: teratoma formation andimmune 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 forCardiovascular Biology, �Institute for Stem Cell and Regenerative Medicine, University ofWashington, Seattle, Washington, USA

ABSTRACT Embryonic stem (ES) cells are promisingfor cardiac repair, but directing their differentiationtoward cardiomyocytes remains challenging. We inves-tigated whether the heart guides ES cells toward cardi-omyocytes in vivo and whether allogeneic ES cells wereimmunologically tolerated. Undifferentiated mouse EScells consistently formed cardiac teratomas in nude orimmunocompetent syngeneic mice. Cardiac teratomascontained no more cardiomyocytes than hind-limb ter-atomas, suggesting lack of guided differentiation. EScells also formed teratomas in infarcted hearts, indicat-ing injury-related signals did not direct cardiac differ-entiation. Allogeneic ES cells also caused cardiac tera-tomas, but these were immunologically rejected afterseveral weeks, in association with increased inflamma-tion and up-regulation of class I and II histocompati-bility antigens. Fusion between ES cells and cardiomy-ocytes occurred in vivo, but was rare. Infarctautofluorescence was identified as an artifact that mightbe mistaken for enhanced GFP expression and trueregeneration. Hence, undifferentiated ES cells werenot guided toward a cardiomyocyte fate in either nor-mal or infarcted hearts, and there was no evidence forallogeneic immune tolerance of ES cell derivatives.Successful cardiac repair strategies involving ES cellswill need to control cardiac differentiation, avoid intro-ducing undifferentiated cells, and will likely requireimmune 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 mu-rine embryonic stem cells in the heart: teratoma forma-tion and immune response. FASEB J. 21, 1345–1357(2007)

Key Words: cardiomyocyte differentiation � rejection � tolerance� fusion � autofluorescence

Coronary heart disease continues to be the great-est health problem in industrialized countries. In theUnited States nearly 1 million people a year suffer from

myocardial infarction (1). It is well established that theheart has an insufficient intrinsic regenerative re-sponse; hence, infarcts heal by formation of noncon-tractile scar tissue (2, 3). So far, the only therapeuticoption to replace tissue lost to infarction is transplan-tation of the entire organ, an approach severely limiteddue to the lack of donor organs and allograft rejection.Current research is focused on restoring cardiac func-tion after myocardial infarction (MI) by transplanting avariety of cell types, including skeletal muscle (4–9),cardiomyocytes (10–12), or cells selected from bonemarrow (13–15).

Cardiomyocytes would seem to be the optimal celltype to repair a myocardial infarct based on theircontractile properties and ability to form electrome-chanical junctions with host myocardium (10, 12, 15).Because primary cultures of cardiomyocytes do notproliferate and are difficult to obtain in humans, muchrecent research has focused on pluripotent embryonicstem (ES) cells. Various groups have investigated car-diac development within differentiating ES cells andfound that they recapitulate normal embryologicaldevelopment (16–23). Furthermore, Field’s group hasshown that ES cell-derived cardiomyocytes will formstable grafts when implanted into normal hearts (22).These findings suggest that differentiating ES cellsprovide a potential source of donor cardiomyocytessuitable for transplantation.

It was recently reported that transplantation of un-differentiated embryonic stem cells improves cardiacfunction after myocardial infarction and that ES cellscould be transplanted into the heart across majorhistocompatibility 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, In-

stitute for Stem Cell and Regenerative Medicine, University ofWashington, 815 Mercer St., Seattle, WA 98109, USA. E-mail:[email protected]

doi: 10.1096/fj.06-6769com

13450892-6638/07/0021-1345 © FASEB

improvement was based on differentiation of the EScells into cardiomyocytes. In contrast, when undifferen-tiated ES cells are implanted into many extracardiacsites, they form teratomas containing derivatives of allthree germ layers (28–30). This seems to imply that theheart is an especially instructive environment com-pared with many other locations in the body, and thatES cells and their progeny enjoy an immune privilegenot shared by other cells.

Here we show that undifferentiated ES cells formteratomas when transplanted directly into the heartand that allogeneic ES cell derivatives elicit a vigorousimmune response and are ultimately rejected. Thesefindings indicate that it is still critical to developefficient methods to guide cardiac differentiation. Inaddition, care must be taken to remove undifferenti-ated or noncardiogenic cells from any ES cell-derivedpopulation intended for therapy, and immunomodula-tory therapies may be required to prevent allogeneicgraft 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/6mouse) lines (33) were maintained in an undifferentiatedstate in Dulbecco’s modified Eagle medium (DMEM) highglucose (4.5 g/L), supplemented with 2 mM l-glutamine(Life Technologies, Inc., Carlsbad, CA, USA), 0.1 mM non-essential amino acids (Life Technologies.), 0.15 mM mono-thioglycerol (Sigma, St. Louis, MO, USA) 1 mM sodiumpyruvate (Life Technologies), 50 U/ml penicillin, 50 �g/mlstreptomycin, 0.25 �g/ml amphotericin (Life Technologies),15% FBS (Schenk, Stanwood, WA, USA), and 1000 U/mlrecombinant human leukemia inhibitory factor (LIF; Chemi-con, Temecula, CA, USA). The C57 ES cells were stablytransfected with the pCX- (�-actin) EGFP construct (34) toconstitutively express green fluorescent protein under thecontrol of the chicken �-actin promoter. ES cells were grownon gelatin-coated tissue culture flasks (Corning, Corning, NY,USA) to 80% confluency, then passaged into fresh mediumevery 2 days.

Surgical procedure

All experiments were approved by the University of Washing-ton Animal Care and Use Committee and were performed inaccordance with federal guidelines. Studies were performedusing male nude mice [CD-1 [ICR] nu/nu homozygotes,Charles River, Wilmington, MA, USA], C57Bl/6J mice, andmale or female Balb/c mice as cell recipients (JacksonLaboratories, Bar Harbor, ME, USA). Coronary occlusion andcell implantation into the heart were performed as recentlydetailed (35, 36). In brief, mice were anesthetized withAvertin, supported on a ventilator and their chests openedaseptically to expose the heart. Undifferentiated ES cells(500,000 unless otherwise specified) were suspended in 7 �lserum-free medium and injected into the anterior wall of theleft ventricle with a Hamilton syringe. In most animals, thehearts were uninjured, but in some, myocardial infarction wasinduced 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 wereinjected into one side of the border zone of the ischemicvascular bed. Animals were euthanized with an overdose ofpentobarbital (Delmarva Laboratories, Inc., Midlothian, VA,USA) at 3 wk postinjection unless specified otherwise. Thehearts were collected and fixed in methyl Carnoy’s solutionfor 24 h, and routinely processed, paraffin-embedded andsectioned for histological evaluation (37, 38).

Histology and immunohistochemistry

Tissue sections were stained with hematoxylin-eosin to visual-ize the general morphology. For immunostaining, tissuesections were quenched for 30 min in 3% H2O2 in methanolto block endogenous peroxidases. Slides were incubated 24 hat 4°C in a humidified chamber with the following antibodies:a rabbit polyclonal EGFP antibody (1:1500, Abcam, Cam-bridge, MA, USA) to track grafted cells within the hostmyocardium; a polyclonal �-fetoprotein (AFP) antibody (1:10,000; Dako) to detect endoderm-derived cells; a monoclo-nal sarcomeric actin (5c5) antibody (1:20,000 Sigma), using ablocking kit to reduce background (DAKO Ark-Kit), andmonoclonal smooth muscle �-actin (full-strength; Dako,Carpinteria, CA, USA) antibody to identify mesoderm struc-tures. A polyclonal neurofilament antibody (NF-200) (1:10,000; Sigma) was used to detect neuro-ectodermal differen-tiation. After application of appropriate secondary antibodiesor reaction complexes, reaction products were visualized withdiamino-benzidine (3,3�-diaminobenzidine; Sigma-Fast tabletsets).

Cardiac differentiation in ES cell-derived teratomas wasquantified from sarcomeric actin-stained sections using ScionImage Analysis Software (Scion Corporation, Frederick, MD,USA). The absence of skeletal muscle differentiation in EScell-derived teratomas was confirmed by staining the tissuesection with a monoclonal MY-32 antibody (Sigma, 1:400,using DAKO Ark-Kit) specific for fast skeletal myosin heavychain. To detect a potential host immune response to theallogeneic ES cell grafts, we stained heart tissue sections witha CD45 antibody (PharMingen, San Diego, CA, USA; 1:2000,specific for leukocyte common antigen, Ly-5). The leukocytedensity in the tumor, the intervening granulation tissue, andthe surrounding myocardium were scored by a blindedobserver (CEM) on a semiquantitative 0 to 4� scale.

Morphometric analysis

Cardiac differentiation in ES cell grafts was measured byimmunostaining for myosin light chain 2v (polyclonal anti-serum against MLC2V; a gift from Dr. Ken Chien) or sarco-meric actin (5C5 monoclonal antibody; Sigma). We studiedC57Bl/6 mice receiving syngeneic, undifferentiated ES cells(heart, n�14; hind limb, n�4). Digital images were taken at100� using a SPOT RT digital camera and SPOT imagingsoftware (Diagnostic Instruments, Sterling Heights, MI,USA). The sarcomeric actin- or MLC-2V-positive areas of thetumor sections were traced using Scion Imaging software(Scion Corp.). Measurements were statistically analyzed usinga 2-sample t test. Although the two antibodies gave generallycomparable staining, the MLC-2V antibody showed somecross-reactivity with epithelial cells, so only morphometricdata 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 Ad-floxed-LacZ adenovirus (�250 particles/cell; Microbix Bio-

1346 Vol. 21 May 2007 NUSSBAUM ET AL.The FASEB Journal

systems, Toronto, ONT, Canada) as described (37, 38).500,000 cells were prepared and injected into the uninjuredheart of an �-MHC/Cre FVB/N mouse in which Cre recom-binase is expressed only in cardiomyocytes (37) (n�3). Iffusion between ES cells and cardiomyocytes has taken place,the Cre recombinase will excise a floxed stop sequence,allowing expression of �-galactosidase. After 3 days, heartswere collected and placed in 30% sucrose solution overnightand embedded in OCT embedding media (Sakura, Torrance,CA, USA). The entire heart was cryosectioned at 7 �mthickness; every third section was fixed with 4% paraforma-dehyde for 5 min at room temperature and stained overnightwith x-gal solution at 37°C. The sections were then counter-stained with Contrast Red and visualized under bright-fieldmicroscopy with a 10� objective. Digital images of positivecells were taken with QCapture software.

Evaluation for autofluorescenct artifact

To distinguish between bona fide GFP signal and autofluores-cent artifact, infarcted hearts from C57Bl/6 mice were in-jected with 500,000 undifferentiated C57Bl/6 EGFP-ES cells.After 3 wk, the hearts were dehydrated in 30% sucrosesolution 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 forEGFP, 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 visu-alized using fluorescent microscopy (Nikon Eclipse 80i). TheEGFP antibody-derived signal was compared with intrinsic

tissue fluorescence (native EGFP fluorescence plus tissueautofluorescence).

RESULTS

Undifferentiated ES cells cause tumor formation inthe heart

The initial hypothesis of this study was that the heartwould provide an instructive environment to guide EScell differentiation into cardiomyocytes. For our initialstudies, we transplanted 500,000 undifferentiated EScells into the uninjured left ventricles (LVs) of immu-nocompromised or syngeneic recipients. R1-ES cellsfrom strain 129/sv and CGR8 ES cells from strain129/Ola were grafted onto hearts of nude mice (n�4/group) and C57Bl/6-derived ES cells were grafted intothe LVs of syngeneic animals (n�14). To facilitatetracking of ES cells after transplantation, undifferenti-ated C57Bl/6-ES cells were stably transfected with aconstruct containing the chicken �-actin promoterdriving EGFP. Three weeks post-ES cell injection, tera-tomas had formed in the hearts of all nude (Fig. 1A)and syngeneic animals (Fig. 1B). Teratomas were his-tologically analyzed to identify various cell types. Alltumors, independent of host or ES cell type injected,consisted of structures derived from all three embry-

Figure 1. Teratoma development in hearts ofnude and C57Bl/6 mice. Hearts of nude andC57Bl/6 mice 3 wk postinjection of undifferen-tiated ES cells. A) Teratoma formation afterinjection of R1-ES cells into the heart of animmunocompromised nude mouse. The tumorreplaces much of the LV wall and containsheterologous elements, including multiple epi-thelial-lined cysts. B) Teratoma formation afterinjection of syngeneic C57-ES cells into the heartof C57Bl/6 mouse. A necrotic core and multiplecystic and ductal structures are evident. C) Timecourse study of C57 syngeneic transplants. After3 days, the ES cell graft consisted mostly of smallblue cells without any organization. Necrotic celldebris is present within the graft. At 7 dayspostinjection, ES cell grafts expanded in size andwere organized in clumps and cords. The graftcells were highly proliferative, displaying anabundance of mitotic figures (inset). At 14 dayssimple epithelial-lined duct-like structures werepresent. At 3 wk, the grafts had formed terato-mas consisting of derivatives of ectoderm (a,squamous epithelium), mesoderm (b, cartilage),and endoderm (c, ciliated respiratory-likeepithelium).

1347CARDIAC ES CELL-DERIVED TERATOMAS

onic germ lineages. Ectoderm-derived cells includedkeratinizing 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 epitheliumwith goblet cells (Fig. 2G) and ciliated respiratory-likeepithelium (Fig. 2H). Endoderm-derived cells alsostained positive for �-fetoprotein (Fig. 2I). We observedan increase in cartilage and bone structures in terato-mas that resulted from injection of R1-ES cells intonude mice compared with CGR8 or C57Bl/6 ES cell-derived tumors. Teratomas in the syngeneic group(C57Bl/6 animals with C57Bl/6-ES cell injections)showed an abundance of duct-like structures with strat-ified and pseudo-stratified epithelium.

Time course study

We next investigated the time course of tumor devel-opment with ES cell transplantation in syngeneic hosts(Fig. 1C). C57Bl/6 recipient mice were killed 3, 7, 14and 21 days post-C57-ES cell injection and analyzedhistologically. At 3 days the ES cell grafts appeared assmall clusters of cells with high nuclear-to-cytoplasmicratios, and numerous mitotic figures were present.After 7 days of transplantation, the grafts appearedsignificantly larger and had abundant mitotic figures.After 2 wk, grafts in the syngeneic animals were largerstill and displayed the first differentiated structures,such as epithelial ducts. After 3 wk, clear teratomas hadformed with cell types derived from ectoderm (keratin-ized epithelium), endoderm (ciliated epithelium, gob-let cells), and mesoderm (cartilage).

Dose-response of undifferentiated ES cellstransplanted into syngeneic recipients

We next hypothesized that the differentiation patternof ES cell grafts in the heart might be dose dependent,with tumors forming at high doses but cardiac differ-entiation occurring at lower doses. To test this,C57Bl/6 ES cells were injected into uninjured hearts ofC57Bl/6 mice at doses ranging from 500 to 500,000cells. Hearts were examined histologically 3 wk post-transplantation. As shown in Fig. 3A, teratoma forma-tion showed a dose-response relationship, with notumors forming at �50,000 cells, with 50% of animalsdeveloping tumors at 100,000 cells, and 80% of animalsdeveloping tumors at 250,000 cells. Tumor develop-ment was observed in 100% of animals that received500,000 undifferentiated ES cells. Despite detailed his-tological evaluation, no surviving graft cells were de-tected in hearts receiving �50,000 undifferentiated EScells (Fig. 3B). The absence of ES cell-derived grafts inthese hearts was confirmed by the absence of immuno-staining for EGFP. In contrast, teratomas stained in-tensely for EGFP (Fig. 3C).

Cardiac differentiation in teratomas

Although teratomas formed consistently in immuno-tolerant hearts, it remained a possibility that cardiomy-ocytes were among the mesodermal cell types that haddifferentiated and that the cardiac environment pro-moted 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/6mice. After 3 wk, the tumors were analyzed for cardiacdifferentiation by measuring the fraction of the tumorsexpressing sarcomeric actin (Fig. 4A). All tumors wereconsistently negative for the skeletal muscle marker,myosin heavy chain-fast. Therefore, sarcomeric actinstaining indicates cardiac differentiation in this case(Fig. 4B). Teratomas in both the heart and the hindlimb showed clusters of ES cell-derived cardiomyocytes.Quantitative morphometry revealed that 2.1 0.5% ofthe tumor mass in the hind limb was composed ofcardiomyocytes, whereas 1.1 0.3% of tumor mass inthe heart contained cardiomyocytes (P�ns; Fig. 4C).We considered the possibility that cardiac differentia-tion might be enhanced at the graft-host border, butcareful examination demonstrated no discernible in-crease in cardiac differentiation at the tumor’s marginvs. its center. These studies suggest that the heart doesnot provide an environment conducive for cardiacdifferentiation, at least no more so than skeletal mus-cle.

Infarction does not promote ES cell differentiationinto cardiomyocytes

We next asked whether ES cells might be guided towardcardiomyocyte differentiation in the infarcted heart,possibly replacing the lost myocardium as has beensuggested recently (24–26, 39). Myocardial infarctionwas induced by LAD occlusion, and undifferentiatedC57-ES cells were immediately injected into the isch-emic border zone of C57Bl/6 (n�4) and Balb/c ani-mals (n�5). After 3 wk of injection, teratoma develop-ment was observed in all animals independent of therecipient mouse strain (Fig. 5A). Morphometric analy-sis of sarcomeric actin-positive areas within the synge-neic teratomas showed a relatively small degree ofcardiac differentiation in the infarcted heart(2.41.4%), not statistically different from that ob-served in noninfarcted recipients (1.30.5%) (Fig.5B). This suggests that an injury environment does notprevent tumor formation or lead to increased cardio-myocyte differentiation.

Immune response after allogeneic transplantation

Another major question of this study was whether EScells or their progeny would be immunologically toler-ated in allogeneic hosts. For these studies, undifferen-tiated C57Bl/6-ES cells that stably expressed EGFPfrom the chicken �-actin promoter were transplantedinto the hearts of Balb/c recipients (n�5). A time


Figure 2. Differentiation in ES cell-derived teratomas. Histological sections of ES cell-derived teratomas showing derivatives ofectoderm (A–C), mesoderm (D–F), and endoderm (G–I). A) Stratified squamous keratinizing epithelium in R1-ES cellstransplanted 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 teratomasin the nude mouse contained cartilage with calcification suggestive of endochondral bone formation (H&E) and E) smoothmuscle cells (H&E) that stained positive for smooth muscle �-actin (F). G) Endoderm-derived gut epithelium containing gobletcells (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).


course study showed that teratomas formed in a patternsimilar to the syngeneic model (data not shown). Inbrief, at 3 days the ES cell grafts were undifferentiatedand highly proliferative. After 7 days, a significantincrease in graft size could be observed, and, by 2 wk,the first signs of epithelial differentiation were appar-ent. At 3 wk, these allogeneic tumors could be charac-terized as teratomas, with cell types derived from allthree embryonic germ lineages. Inflammation was ap-parent in the surrounding myocardium, interveninggranulation tissue, and within the graft itself. By 5 wk,

the inflammatory response was intense, and in someanimals it obscured or replaced most of the graft cells.

This inflammatory infiltrate suggested an immuneresponse in the allogeneic setting. To compare inflam-mation between syngeneic and allogeneic grafts, weperformed a blinded, semiquantitative assessment ofleukocyte infiltration at 7, 14, and 21 days as well as 5 wkpost-ES cell transplantation by immunostaining forCD45 (common leukocyte antigen) and EGFP (forgraft cells). Representative histological images areshown in Fig. 6 and the temporal analysis is shown in

Figure 3. Dose-response study. A) Incidence of tumor development 3 wk after injection of various doses of undifferentiatedC57Bl/6-ES cells expressing EGFP into the LV of syngeneic mice. Tumors were observed in 50% of animals that received 100,000ES cells and in 80% of animals that received 250,000 ES cells. All animals (100%) that received 500,000 ES cells developedteratomas. No tumor development could be observed in animals that received fewer than 100,000 ES cells. B) Histology of heartreceiving 500 ESCs. No tumor is present. Scar tissue in injection site (high magnification in insets) contains no EGFP� graftcells. C) Histology of heart receiving 100,000 ESCs. Endoluminal teratoma is present that stains intensely for EGFP (brown,inset).


Fig. 7. In general, inflammatory cell content progres-sively increased over time in the allogeneic transplantsand decreased in the syngeneic transplants. By 3 wk, theallogeneic grafts had significantly greater inflammationthan did the syngeneic grafts (P0.05) (Fig. 7C). Therewas a trend for greater inflammation in the surround-ing granulation tissue and myocardium in the alloge-neic grafts, which reached statistical significance after 5wk (Fig. 7A, B). At 5 wk post-transplantation, 90% ofthe mice receiving allogeneic cells showed nearly com-plete elimination of the graft. In these hearts, the graftarea was effaced by inflammatory cells and containedvery few ES cell derivatives (Fig. 6). In contrast, synge-neic transplants at 5 wk remained large and hadrelatively little inflammation. These results indicatethat transplantation of allogeneic ES cells leads to asignificant host immune response after 3 wk, whichleads to complete graft rejection at later time points.

To test whether this rejection phenomenon wasspecific to the donor-host strains we had initially cho-sen, additional experiments were performed using un-differentiated CGR8 ES cells from strain 129/Ola intro-duced into the hearts of allogeneic C57Bl/6 recipientmice (n�10). Three weeks post-CGR8 ES cell injection,hearts from the allogeneic C57BL/6 donors did notexhibit any tumor formation; in fact, cell grafts couldnot even be detected (data not shown). This indicatesthat rejection of the allogeneic graft is independent ofthe host mouse strain or the ES cell type used.

Major histocompatibility complex expression

To understand the basis for rejection of allogeneic EScell derivatives, we investigated class I and class II majorhistocompatibility antigen expression by flow cytom-etry. In these experiments, undifferentiated C57Bl/6ES cells and EBs after 19 days of differentiation wereanalyzed in the presence or absence of IFN�. Negativecontrols included duplicate samples stained with iso-type-matched IgG and unstained samples (not shown).Primary spleen cell cultures were used as a broadpositive control and treatment of primary kidney cul-tures 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 inundifferentiated ES cells even in the presence of IFN�.Appreciable levels of MHC I and MHC II were observedin the differentiated EBs only in the presence of IFN�.These data suggest that undifferentiated ES cells have alow immunogenic profile in the undifferentiated state;after differentiation, however, immunogenicity in-creases in the presence of inflammatory cytokines.

Fusion of ES cells and host cardiomyocytes

We employed a Cre-Lox system to determine if fusionoccurred between ES cells and host cardiomyocytesafter transplantation (37). ES cells were labeled with anadenovirus encoding a floxed LacZ reporter and trans-

Figure 4. Cardiac differentiation in ES cell-derived tera-tomas. Cardiac differentiation in syngeneic C57-derivedteratomas 3 wk postimplantation was compared in theheart vs. hind limb. A) Intraluminal cardiac teratomasurrounded by left ventricular myocardium, stained forsarcomeric actin (brown). Small islands of sarcomericactin-positive myocardium are present within the tumor.Scale bar � 100 �. B) Adjacent section stained for fastskeletal muscle myosin heavy chain. No skeletal muscleis present in the tumor, indicating sarcomeric actinstaining is specific for cardiomyocytes. C) Quantitativemorphometry demonstrated that cardiac teratomas de-rived from undifferentiated ESCs contain no more car-diomyocytes than do those in the hind limb.


planted into the hearts of transgenic mice expressingCre recombinase in cardiomyocytes. Activation of LacZexpression indicates fusion has occurred, bringing theES cell’s DNA substrate into the same compartment asthe cardiomyocyte’s enzyme. At 3 days post-transplan-tation, exhaustive sectioning revealed a mean of �3fusion events per heart (n�3 hearts; see online supple-mental Fig. 2). This indicates that, while graft-hostfusion can occur in this setting, it is rare.

Evaluation for autofluorescence artifact

Autofluorescence has emerged as a confounding factorin studies using EGFP fluorescence to track the fates ofstem cells, particularly in injured tissues (40, 41). Weexplored possible autofluorescence in infarcted heartsreceiving EGFP-expressing ES cells by systematically

comparing EGFP immunostaining (using a red fluores-cent label) to intrinsic tissue fluorescence (EGFP signalplus autofluorescent background). In the unstainedsections, the teratomas had the expected green fluores-cence (online supplemental Fig. 3). In the infarct,cardiomyocytes in the spared subendocardial regionalso exhibited green fluorescence, along with spindle-shaped cells in the midwall. Based on intrinsic fluores-cence, one might conclude that these green cells arosefrom the ES cells. In the immunostained sections,however, there was no red fluorescence in the cardio-myocytes or spindle-shaped cells in the infarct, indicat-ing these cells did not contain the EGFP epitope. Incontrast, the teratoma stained brightly with the EGFPantibody. Thus, these myocytes and spindle-shapedcells in the infarct region were clearly residual host-derived cells exhibiting autofluorescence. These resultsindicate the need for caution and careful examinationof controls when using immunofluorescence to tracecell lineages in injured hearts.

DISCUSSION

The main findings of this study are that 1) transplanta-tion of undifferentiated mouse ES cells into the heartleads to teratoma formation; 2) teratomas formed inboth normal and infarcted hearts; 3) cardiac teratomascontained no more cardiomyocytes than hind-limbteratomas; 4) teratomas were accepted in immunocom-promised and syngeneic hosts but eventually wererejected in allogeneic hosts; 5) class I or II histocom-patibility antigens could not be detected above back-ground levels in undifferentiated ES cells, but theseantigens were markedly up-regulated in differentiatedcells after IFN� treatment; 6) ES cell-cardiomyocytefusion occurs rarely in this model; and 7) autofluores-cence of cells in the infarct can be misjudged for EGFPexpression unless appropriate controls are included. Inthe ensuing sections we compare these findings withthe published literature and discuss possible relevancefor ES cell-based cardiac repair.

Differentiation of engrafted ES cells: tissue-specificor cell autonomous?

Mouse ES cells are the best-studied pluripotent celltype. They can give rise to cells from all embryonicgerm lineages and form chimeric mice after blastocystinjection, indicating that when appropriate environ-mental cues are present, their differentiation can becontrolled precisely. On the other hand, ES cells havelong been known to form teratomas when transplantedinto the subcutaneous space (42), joint spaces (30), orspleen (27). In fact, the ability to form teratomas is oneof the most rigorous criteria for defining pluripotenti-ality. These results imply that many adult tissues lackthe signals needed to guide ES cells toward tissue-appropriate fates.

Our experiments suggest that the heart does not

Figure 5. Cardiac differentiation in infarcted and nonin-farcted hearts. Undifferentiated ES cells were injected intothe border zone of the ischemic region in syngeneic C57Bl/6mice immediately after coronary occlusion. A) H&E stainingof a 3-wk-old graft shows the development of a teratoma in LVwall adjacent to the infarct area (as outlined with arrows).The tumor extends from the LV wall into the LV lumen andshows differentiated structures such as epithelial lined cystsand ducts (inset). Note how the border of the endoventricu-lar tumor matches that of the opposite ventricular wall. B)Morphometric analysis of cardiac differentiation based onsarcomeric actin staining in 3-wk-old grafts. Teratomas in theinfarcted heart did not show a higher degree of cardiacdifferentiation than did the uninjured heart, indicating thatcardiogenic signals were not increased after injury.


offer signals to guide ES cells to form cardiomyocytes.Rather, we consistently observed teratomas when EScells were implanted into the heart. This was true forthree different lines of ES cells (CGR8 and R1 cellsfrom 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 previouslyreported to result exclusively in cardiac differentiation(25). These experiments indicate that tumor formationis not dependent on the line of ES cells studied, hoststrain or gender, or a particular combination of ES cellline and host strain.

We considered the possibility that a dose of cellsexists that allows engraftment of ES-derived myocar-dium with no teratoma formation, reasoning that athigh doses of cells, the majority of the graft was withinan ES cell/teratoma environment rather than a cardiacenvironment. Dose-response studies did not supportthis hypothesis. Using a line of ES cells that constitu-tively expressed EGFP, we found that grafts were notdetected when �50,000 cells were injected, whereas athigher doses all ES cell derivatives were containedwithin teratomas. Furthermore, cardiac differentiationwas not enhanced at the graft-host interface, where

potentially inductive host factors should have beenconcentrated the most. One explanation for the lack ofany EGFP-positive cells in the heart after injection of50,000 cells could be due to leakage of cells at the siteof injection and washout. Cell retention has been alimitation in cell transplantation (43). After injectioninto beating myocardium, most cells either bleed backor wash out (43).

In contrast to our findings, other studies support thehypothesis that ES cells are induced to form cardiomy-ocytes in the heart. Befahr et al. (25) reported thatundifferentiated mouse ES cells became cardiomyo-cytes exclusively when transplanted into the hearts ofallogeneic, immunocompetent C57Bl/6 hosts. Theyreported that undifferentiated tumors (not teratomas)formed when the ES cells expressed a dominant-nega-tive TGF-� receptor, suggesting that TGF-� signalingwas required for guided differentiation. Similarly,Wang et al. injected undifferentiated mouse ES cellsexpressing the EGFP transgene into the tail veins ofallogeneic Balb/c recipients that exhibited viral myo-carditis. They reported that ES cells homed to the hostmyocardium and formed cardiomyocytes exclusively,thereby increasing the survival of the diseased mice(44). Yang et al. reported that implantation of “early-

Figure 6. Histological demonstration of alloge-neic immune rejection. C57 ES cells expressingEGFP were engrafted into hearts of C57Bl/6 orBalb/c mice. A) Leukocyte infiltration at 3 wkwas identified by CD45 immunostaining(brown) in allogeneic (left panel) and synge-neic (right panel) hearts. Inflammation is muchmore intense surrounding and within the allo-geneic graft, indicating immune rejection. B)Serial sections from an allogeneic graft showingnearly complete rejection by 5 wk. CD45 immu-nostaining (brown, left panel) shows an intenseleukocyte infiltrate, nearly effacing the underly-ing tissue structure. EGFP immunostaining(brown, right panel) demonstrates a very smallresidual graft of allogeneic cells.


differentiated” mouse ES cells regenerated cardiac tis-sue and improved contractile function in the infarctedmouse heart (45). Most recently, Singla et al. reportedthat undifferentiated ES cells were directed toward a

cardiomyocyte fate in the infarcted heart, resulting inimproved mechanical function (24).

The question then arises of why our data differ fromthe above-mentioned studies reporting guided differ-entiation of ES cells to cardiomyocytes. We have ruledout variations in ES cell lines, host strain and gender,ES cell dose, graft-host proximity, differentiation statusat the time of transplantation, and the effect of myo-cardial injury. There are always minor differences be-tween studies performed by different groups, and it ispossible these could explain either the lack of guideddifferentiation or the lack of immune tolerance in ourstudies. For example, although all of these studies usedsimilar conditions for culturing the ES cells, differencesin serum quality or other seemingly minor cultureconditions might explain the variation. Similarly, itremains a possibility that variations in mouse housingconditions could influence the fate of transplantedcells, although we have no data to support this idea. Wetested the notion that predifferentiation of ES cells(without purification) might prevent tumor formationafter transplantation, but found that teratomas aroseafter this procedure as well (data not shown).

An alternative possibility that should be consideredregarding the discrepancies cited above is that the EScell derivatives in many studies may have been lost torejection and host cardiomyocytes may have been mis-taken for ES cell-derived cardiomyocytes. A criticalelement is how the lineage of the ES cells is followed. Inall the above studies, the presence of the ES cellderivatives was based on the intrinsic fluorescence ofEGFP or related proteins. Autofluorescence mimickingEGFP (or fluorescent immunostaining) has been de-scribed in normal and injured skeletal muscle (40), andwe have reported similar autofluorescence in cardiomy-ocytes in the injured heart (46, 47). In the currentstudy, we identified green fluorescent cardiomyocytesand spindle-shaped cells in the infarct that initiallyappeared to be derived from EGFP-expressing ES cells.When an anti-EGFP antibody was used, however, nei-ther the cardiomyocytes nor the spindle cells stained,indicating they exhibited artifactual autofluorescence(see online Supplemental Fig. 3). Thus, reliance solelyon the intrinsic fluorescence of EGFP or other fluoro-proteins is hazardous, especially when one is examiningtissues with high levels of autofluorescence such asmyocardial infarcts.

Another possible confounding variable is fusion ofES cells with host cardiomyocytes, resulting in cardio-myocytes that express the ES cell lineage marker. Wetested for fusion in this setting and found that EScell-cardiomyocyte fusion occurred in three of threehearts tested. It should be noted, however, that fusionwas an extremely rare event (�3 cells/graft site). Basedon its rarity, we think fusion is unlikely to explain thelarge-scale fluorescence reported in other studies (37).

After the current manuscript had been accepted forpublication, Kolossov et al. published a report showingteratoma formation after 4 wk in hearts that receivedinjection of undifferentiated ES cells in the myocar-

Figure 7. Time course of immune response to allogeneic EScell grafts. C57 ES cells expressing EGFP were transplantedinto the hearts of Balb/C or C57Bl/6 recipients. Tissuesections were stained for CD45 to detect leukocyte infiltrationinto the ES cell-derived tumors 7, 14, 21 days and 5 wk aftertransplantation. Inflammation was graded semiquantitativelyby a blinded observer using a 0–4� scale (inflammatoryscore), with 4� representing maximal inflammation. Groupsizes are given in parentheses above the bars. A) Leukocyteinfiltration in the myocardium surrounding the tumor. Syn-geneic grafts induced little inflammation in the surroundingmyocardium, whereas inflammation steadily increased in thisregion with allogeneic grafts. B) Leukocyte infiltration in thegranulation tissue between graft cells and host myocardium.In syngeneic grafts, inflammation steadily decreased overtime as the injection site healed. In allogeneic grafts, inflam-mation was intense at all times. C) Leukocyte infiltrationwithin the ES cell derived grafts. At 1 and 2 wk there was littleinflammation in either group. In the allogeneic group, in-flammation in the graft increased markedly at 3 wk and wasmaximal at 5 wk, while inflammation remained low in synge-neic grafts. Note that this underestimates severity of inflam-mation, because several animals where grafts were completelyrejected were not included. *P 0.05.


dium, which confirms our findings (48). In addition,they created a transgenic mouse ES cell line containingthe cardiac-specific �-myosin heavy chain promoterdriving both EGFP and the puromycin resistance gene.This transgene permits purification of ES-cell derivedcardiomyocytes with puromycin and their subsequenttracking by EGFP expression. Cardiomyocytes purifiedfrom ES cells derivatives by puromycin selection did notform teratomas after intracardiac injection. This com-plements recent work from our group showing thatwhen human embryonic stem cells were differentiatedas embryoid bodies and subsequently enriched forcardiomyocytes, they did not form teratomas aftertransplantation into the heart, but rather formed stablegrafts of human myocardium (47). This emphasizes theimportance of predifferentiating ES cells and enrichingthe population for cardiomyocytes prior to cell injec-tion to avoid teratoma formation.

Immune privilege vs. immune rejection

Immune rejection in response to antigenic differencesis a well-described problem in solid organ transplanta-tion and cell transplantation (e.g., islet cells) whereas itscounterpart, graft-versus-host disease, occurs after bonemarrow transplantation. A few tissues, such as corneas,have immune privilege that permits their transplanta-tion across antigenic barriers. There is some evidencethat certain stem cell populations, such as mesenchy-mal stem cells, may also have immune privilege basedon their ability to suppress local immune responses(49). Several studies report immune privilege of embry-onic stem cell derivatives in the heart based on graft cellsurvival across strain and even species barriers. Behfar etal. (25) reported 4 wk survival of ES cell derivativesfrom mouse strain 129 in hearts of allogeneic C57Bl/6hosts. Wang et al. reported 2 wk survival of derivatives of129-derived ES cells in hearts of allogeneic Balb/crecipients (44). Similarly, Yang et al. reported survival ofEGFP� graft cells in the infarcted hearts of allogeneicFVB/N mice 6 wk after implantation of early-differen-tiated 129-derived mouse ES cells (45). Data fromseveral groups have even suggested that xenografting ofEGFP-expressing mouse ES cells leads to significantimprovement of cardiac function in the hearts ofimmunocompetent, postinfarcted rats due to the inte-gration of functional ES-derived cardiomyocytes (25,26, 39, 50).

In contrast, our study found no evidence for immunetolerance of ES cells or their progeny in the heart. In 2different allogeneic donor-host combinations (C57Bl/6-derived ES cells into BALB/c hosts and 129-derivedES cells into C57Bl/6 hosts), we observed substantialinfiltration of the teratomas by host leukocytes at 3 wkand almost complete rejection by 5 wk. Syngeneictransplants, as expected, were well tolerated for up to 5wk. Our in vitro studies suggest that up-regulation ofclass I and II histocompatibility antigens in response tolocal cytokine stimulation may contribute to the even-

tual rejection of the allogeneic graft cells. The differ-ence between our findings and the above-cited litera-ture cannot be explained by the degree of mismatchbetween donor and host, because we deliberately chosedonor-host combinations previously reported to betolerated (25). We conclude that ES cell derivatives arenot immune privileged but rather face immune rejec-tion similar to most other cell types.

CONCLUSIONS

Embryonic stem cells have the potential to generatevirtually any cell type in the body, and this makes themextremely attractive for studies of tissue repair. Thecurrent study indicates that this same potential can alsobe problematic. The presence of undifferentiated cellsin a transplant could result in the formation of cardiacteratomas rather than new myocardium. These findingsunderscore the importance of developing efficientstrategies for selecting cardiomyocytes from other celltypes in a mixed population. Indeed, when cardiomyo-cytes have been purified from predifferentiated ES cellcultures, their transplantation has resulted in the for-mation of new myocardium rather than teratomas (22,47). The overall efficiency of this process would begreatly enhanced by developing methods to guide EScells selectively to form cardiomyocytes or by isolating aprogenitor population with a more restricted differen-tiation potential (e.g., a precardiac mesoderm cell).The vigorous rejection of ES cell derivatives in the heartalso calls for further work to overcome the immuneresponse to allogeneic transplantation.

We thank Dr. Stephen Hauschka for his input to experi-mental design and for critically reading this manuscript. Wethank Dr. Andrew Farr and Mr. James Dooley for advice onanalysis of histocompatibility antigen expression. These ex-periments were supported by National Institutes of Healthgrants R01 HL61553, P01 HL03174, and R24 HL64387.

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Received for publication July 18, 2006.Accepted for publication December 6, 2006.


transplantation of undifferentiated murine embryonic stem cells in

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