Prospective isolation of human embryonic stemcell-derived cardiovascular progenitors thatintegrate into human fetal heart tissueReza Ardehalia,b,1, Shah R. Alic, Matthew A. Inlayc, Oscar J. Abilezd, Michael Q. Chend, Timothy A. Blauwkampc,Masayuki Yazawac, Yongquan Gongc, Roeland Nussec, Micha Drukkerc, and Irving L. Weissmanc,1

aDivision of Cardiology, Department of Internal Medicine, University of California Los Angeles School of Medicine, Los Angeles, CA 90095; bBroad Stem CellResearch Center, University of California Los Angeles School of Medicine, Los Angeles, CA 90095; and cInstitute for Stem Cell Biology and RegenerativeMedicine, and dDepartment of Bioengineering, Stanford University School of Medicine, Stanford, CA 94305

Contributed by Irving L. Weissman, January 7, 2013 (sent for review May 2, 2012)

A goal of regenerative medicine is to identify cardiovascular progen-itors from human ES cells (hESCs) that can functionally integrate intothe human heart. Previous studies to evaluate the developmentalpotential of candidate hESC-derived progenitors have deliveredthese cells into murine and porcine cardiac tissue, with inconclusiveevidence regarding the capacity of these human cells to physiolog-ically engraft in xenotransplantation assays. Further, the potentialof hESC-derived cardiovascular lineage cells to functionally coupleto human myocardium remains untested and unknown. Here, wehave prospectively identified a population of hESC-derived ROR2+/CD13+/KDR+/PDGFRα+ cells that give rise to cardiomyocytes, endo-thelial cells, and vascular smooth muscle cells in vitro at a clonallevel. We observed rare clusters of ROR2+ cells and diffuse expres-sion of KDR and PDGFRα in first-trimester human fetal hearts. Wethen developed an in vivo transplantation model by transplantingsecond-trimester human fetal heart tissues s.c. into the ear pinna ofa SCID mouse. ROR2+/CD13+/KDR+/PDGFRα+ cells were deliveredinto these functioning fetal heart tissues: in contrast to traditionalmurine heart models for cell transplantation, we show structuraland functional integration of hESC-derived cardiovascular progeni-tors into human heart.

engraftment | surface markers | Stem cells | mature cardiomyocytes |clonal analysis

Human cardiomyocytes are derived from proliferating pre-cursors during development, but there is little evidence to

support a robust postnatal regenerative capacity (1, 2). As a con-sequence, myocardial injury or disease in adult humans results inirreversible cardiomyocyte loss that can lead to progressive heartfailure. Cell transplantation may be an effective therapy to com-pensate for myocardial loss in an attempt to improve the pumpingability of the damaged heart (3). The expected mechanism bywhich the grafted cells may restore function is to couple with thenative host myocardium, thereby functionally replacing the deadtissue, with the assumption that the grafted cells or tissue adopta cardiovascular fate in situ (4, 5). However, no study to date hasdemonstrated the delivery of a pure population of tissue-specificstem cells capable of generating functioning cardiomyocytes in theinjured or healthy myocardium. Although adult stem cells havefailed to convincingly regeneratemyocardial tissue, consistent withmany reports that demonstrate the nonplasticity of adult organ-specific stem cells (6, 7), human ES cells (hESCs) have proven tobe a potential and unlimited source for generating cardiomyocytesin vitro as a result of their pluripotent nature (8).Although several studies have reported efficient differentiation

of hESCs toward cardiovascular lineages, the two most significantbarriers to therapy remain the heterogeneity of the putative pro-genitor cells and the fate of these cells upon transplantation, whichremains insufficiently characterized for prospective clinical appli-cation (9–11). The current method to evaluate the in vivo de-velopmental potential and functional properties of hESC-derivedcardiovascular progenitors is to transplant them into animal hearts

[commonly murine (muridae includes mouse and rat) or porcinemodels] (12, 13). Although the transplanted cells engraft into theseheart models, it is unclear whether they functionally integrate orwhether demonstration of such a proof-of-principle concept wouldhave relevance to the human. A recent report has shown electro-mechanical integration of hESC-derived cardiomyocyte grafts inguinea pig hearts (14). A system to prospectively isolate cardio-vascular stem cells/progenitors and to evaluate their in vivo de-velopmental potential in functioning human hearts will be animportant step in clinical translation for myocardial regeneration.Identification of surface markers that are uniquely expressed on

hESC-derived cardiovascular progenitors allows for their pro-spective isolation. Here, we prospectively isolate an enriched pop-ulation of cardiovascular progenitors by using a combination of foursurface markers. We then demonstrate the structural and func-tional integration of the hESC-derived cardiovascular progenitorsin beating human fetal heart tissues.

ResultsImproved Culture Conditions to Promote Differentiation of hESCs toPrimitive Streak, Mesoderm, and Cardiac Mesoderm Progenitors. Topromote efficient cardiovascular differentiation from hESCs, it isnecessary to elucidate the signaling pathways that regulate cardio-genesis during embryonic development and to manipulate them invitro. To this end, we established a protocol based on stage-specificactivation and then inhibition of the canonical Wnt/β-cateninpathway: we generated embryoid bodies (EBs) from the hBCL2-hESC line [enforced expression of antiapoptotic B-cell lymphoma 2in this transgenic line greatly improves the survival of hESCs inculture and experiments (15)] in serum-free media and temporallyexposed them to activin A, BMP4, Vascular Endothelial GrowthFactor (VEGF), and Fibroblast Growth Factor 8 (FGF8) (Fig. S1)(16). Gene expression analysis of the populations generated atdifferent stages of the differentiation protocol revealed induction ofmesodermal genes initially (T or Brachyury and Mix-Like Homeo-box), followed by cardiac mesodermal genes (MESP1), and car-diovascular progenitor genes (NKX2-5, TBX5, andMEF2C) at laterstages (Fig. S1). Despite this robust differentiation assay, many cellswith noncardiovascular developmental fates remained in the finalproduct.

hESC-Derived Cardiovascular Progenitors Express a Unique SurfaceMarker Expression Signature. We recently screened a large panelof mAbs to identify early lineage-committed precursors that emerge

Author contributions: R.A., M.A.I., T.A.B., M.D., and I.L.W. designed research; R.A., S.R.A.,O.J.A., M.Q.C., M.Y., and Y.G. performed research; R.A. contributed new reagents/analytictools; R.A., T.A.B., R.N., M.D., and I.L.W. analyzed data; and R.A., S.R.A., and I.L.W. wrotethe paper.

The authors declare no conflict of interest.1To whom correspondence may be addressed. E-mail: rardehali@mednet.ucla.edu or irv@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1220832110/-/DCSupplemental.

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from differentiating hESCs (17). Several markers were shown to beexpressed individually or in combination on mesodermal cells. Amember of the receptor tyrosine kinase-like orphan receptor family,ROR2, and aminopeptidase-N (i.e., CD13) were individuallyshown to allow FACS to enrich for mesodermal progenitors. Be-cause cardiac cells develop from a Flk-1 (KDR) (11)-expressingpopulation, and the embryonic heart expresses platelet-derivedgrowth factor receptor-α (PDGFR-α) (18), we added these mAbsto the screening protocol.To determine whether cardiovascular progenitors develop from

a subpopulation of differentiating hESCs that expresses one ormore of these surface markers, EBs were analyzed for expressionof ROR2, CD13, KDR, and PDGFR-α after 5 d of differentiation.As shown in Fig. 1A and Fig. S2A, a distinct population marked bycoexpression of ROR2 and CD13 emerges temporally as hESCsdifferentiate. This population exhibited a transcriptional profilesimilar to primitive streak/mesodermal cells (Fig. 1B and Fig. S2).Induction of genes such as T (Brachyury), MIXL, FOXA2, andSOX17 revealed patterns consistent with generation of cells ofmesodermal as well as endodermal lineages.The ROR2+/CD13+ population was sorted, and expression of

KDR and PDGFRα was examined and confirmed. We then eval-uated the lineage commitment of the ROR2+/CD13+/KDR+/PDGFRα+ population [hereafter referred to as the quadruple-positive (QP) population]. The QP population expressed highlevels of cardiac mesoderm and cardiac developmental genes,including mesoderm posterior 1 (MESP1), the earliest knownmarker for cardiogenesis, and key cardiac transcription factorsof the primary and secondary heart fields, including TBX5,GATA4,MEF2C,NKX2.5, and ISL1 (Fig. 1B and Fig. S2) (19, 20).In contrast, the fraction of cells in the EBs that were negative for allfour markers had the highest expression of pluripotency genes,indicative of residual undifferentiated hESCs. Although the QPpopulation highly expressed cardiac lineage genes, it also expressed

genes corresponding to the primitive streak and endoderm, al-though to a much lesser degree (Fig. S2). Enrichment for cardiaclineage cells in the sortedQP population was confirmed by protein-level detection of NKX2-5, MEF2C, and GATA4 (Fig. 1C).

Expression of ROR2, KDR, and PDGFRα During Early Human FetalHeart Development. To elucidate the expression of ROR2, CD13,KDR, and PDGFRα during in utero heart development, humanfetal hearts were sectioned and immunostained for these proteins.KDR and PDGFRα were broadly expressed in 9- to 10-wk-oldhuman fetal cardiac tissue, including in the vasculature. Rare,distinct areas of ROR2 expression were detected in the myocar-dium and interventricular septum, but not in the epicardium (Fig. 1D and E). In contrast, we could not detect CD13 expression. Ex-pression of ROR2 was limited to the human fetal hearts from thefirst trimester, as second-trimester hearts did not stain positively forROR2. Furthermore, examination of adult human myocardialtissues revealed no expression of ROR2 proteins.

QP Progenitor Population Gives Rise to Cardiomyocytes and Endothelialand Vascular SmoothMuscle Cells.To further characterize the in vitrodevelopmental potential of the QP progenitor population, freshlysorted QP cells were cultured as aggregates in suspension for 7 to10 d in the presence of Wnt11 and FGF8 in serum-free media.Consistent with the gene expression profile described earlier, theQP population gave rise to cells of the cardiovascular lineage basedon immunostaining and gene expression (Fig. 2 A and B and Fig.S3A). We consistently detected a high frequency of cardiomyocytesbeating spontaneously as a synchronous mass (Movie S1). Whenplated on Matrigel-coated dishes and treated with a high concen-tration of VEGF, the QP cells acquired the morphology of endo-thelial cells and formed a lattice (Fig. S3B). These cells expressedCD31 and von Willebrand factor and efficiently incorporated Dil-

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Fig. 1. Identification of a cardiac mesoderm population marked by four surface markers—ROR2, CD13, KDR, and PDGFRα—and their expression in humanfetal hearts. (A) Flow cytometric analysis of EBs at different time points of differentiation. On day 5, a distinct population defined by coexpression of ROR2and CD13 (II) appeared, which was further analyzed for expression of KDR and PDGFRα. (B) Quantitative RT-PCR gene expression analysis of the QP (III), ROR2+

CD13+ (II), and QN (I) cells isolated from day-5 EBs. The average expression is normalized to GAPDH. Data represent mean ± SD for three biologically in-dependent experiments (P < 0.05, one-way ANOVA, populations III vs. I and II vs. I). (C) Presence of NKX2-5 (Left), MEF2C (Middle), and GATA-4 (Right)immunostaining in the QP population 24 h after sorting (Fig. S2). (Scale bar, 50 μm.) (D) Immunofluorescence staining of first trimester human hearts revealedpockets of ROR2-positive cells and diffuse KDR and PDGFRα staining in the left ventricle (arrows). (Scale bar, 120 μm.) (E) An area of the left ventricle witha cluster of ROR2+ cells that also costain with NKX2-5. (Scale bar, 120 μm.)

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labeled acetylated LDL (Dil-Ac-LDL), confirming their endothe-lial phenotype functionally (Fig. 2 C and D).To quantify the extent of cardiomyocyte generation, a cardiac

troponin-GFP reporter hESC line was used and the QP pop-ulation was maintained in culture for more than 30 d. More than55% of the derived cells developed into cardiomyocytes, based ontroponin expression, reflecting efficient enrichment of progenitorsin the QP population (Figs. S3C and S4 A and B; Tables S1 andS2). To determine the nature of the other cell types that arise fromQP cells, we performed immunostaining for endodermal, ecto-dermal, and hematopoietic (i.e., mesodermal) lineages. The ma-jority of these cells stained positive for vimentin, a markerexpressed on many cell types, including fibroblasts (Fig. S3D).These cells appear to expand rapidly over time (Fig. S3C).The functional properties of theQP-derived cardiomyocytes were

evaluated 10 d after sorting, using field potential measurements, aswell as whole-cell patch-clamp recording (Fig. 3A). Synchronousmultifocal field potential recordings performed on microelectrodearrays showed homogeneous spread of electrical activity through-out the adherent cultures (Fig. S5 A–C). Additionally, action po-tential (AP) recordings from single cells revealed the presence ofpacemaker-, atrial-, and ventricular-like patterns characterizedpredominantly by a fast phase 1 depolarization. More than 90% of

the single cells studied exhibited a ventricular-like AP morphology.These results confirm that the QP population can differentiate tocontractile cardiomyocytes with a fetal-like AP phenotype (21).To further understand the mechanism by which QP cells commit

to the cardiovascular fate, we tested whether the addition of con-ditioned media from cultured QP cells to control (i.e., unsorted)EBs could recapitulate the cardiac specification of QP cells. Thedaily addition of the QP conditioned media did not promotecardiac differentiation in control cells, indicating that QP cells donot secrete soluble proteins that enhance cardiovascular specifi-cation, arguing against a cell-nonautonomous mechanism. Wenext confirmed the specification of the QP population to a car-diomyocyte fate by transferring freshly sorted QP cells derivedfrom a GFP-expressing hESC line into a synchronous EB derivedfrom unlabeled hESCs. The majority of the GFP+ cells in thechimeric EB developed into contracting foci (Fig. S5 D–F andMovie S2). These results suggest that QP cells are a distinctpopulation with commitment to cardiovascular fate, most likelythrough a cell-autonomous mechanism.

Single QP Cell Is Multipotent for Cardiovascular Lineages. To de-termine further whether the QP cells are multipotential in a clonalmanner, a single QP cell from a GFP-expressing hESC line wassorted directly into each well of a 96-well plate containing 1,000WT QP cells. This approach was taken as a result of the poorsurvival of single QP cells when grown individually compared withtheir growth in clusters, in which they have better survival. Aftera few days in culture, colonies emerged that included foci of GFP+

cells, which were analyzed to determine the differentiation po-tential of a single QP cell (Fig. S6 A–D). Immunostaining of thesecolonies confirmed the presence of GFP+ cardiomyocyte, endo-thelial, and vascular smooth muscle cells in these culture clones(Fig. 3B and Fig. S6D). Taken together, the clonal analysis showsthat a singleQP cell can generate progeny ofmultiple lineages, i.e.,cardiomyocytes, endothelial, and vascular smooth muscle cells invitro, as would be expected of an embryonic cardiac progenitor.

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Fig. 2. In vitro characterization of QP cells. (A) Immunofluorescence analysisof QP cells 6 d after sorting for markers of cardiomyocytes and smooth muscleand endothelial cells. (Scale bar, 25 μm.) (B) Quantitative RT-PCR analysis of QPcells grown in culture after 13 d after sorting for cardiac genes. Data representmean± SD for three biologically independent experiments. (C) Upon exposureto VEGF after sorting, QP cells (derived from hBCL2-hESC line and thereforeexpressing GFP) formed a lattice of tubular structures. (Scale bar, 100 μm.) (D)Endothelial phenotype was further confirmed by Dil-labeled acetylated LDLuptake. (Scale bar, 50 μm.)

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500 ± 39 37 ± 1.3 -43 ± 1.4 21 ± 2.4

ventricular atrial nodal (n=16) (n=1) (n=1)

Fig. 3. Developmental potential of QP cells. (A) Whole-cell current-clamprecordings of spontaneous APs demonstrate ventricular-, atrial-, and nodal-like APs in the cultured QP population (Fig. S5). (B) Immunostaining of cellsgrown from a single GFP-QP cell indicates the presence of cardiomyocytes andendothelial and smooth muscle cells. (Top Left) Corresponding GFP cells (Fig.S6). (Scale bar, 50 μm.).

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QP Cells Transplanted into Mouse Hearts Mature to Cardiomyocytesbut Fail to Integrate. To test their in vivo developmental potential,day-5 EB-derived QP cells from a GFP-hESC line were trans-planted into healthy or injured hearts of nonobese diabetic/SCIDmice with common γ-chain KO. Approximately 5 to 10 × 105 QPcells were sorted and immediately transplanted by direct injectioninto the left ventricle of healthy mice or into the periinfarct area ofmice following occlusion of the left anterior descending artery. Ascontrols, quadruple-negative (QN) cells from the same EB cultureas described earlier were also sorted and transplanted in similarareas in healthy and injured nonobese diabetic/SCID mice withcommon γ-chain KO. The animals were euthanized after 8 wk, andhistological analyses of the explanted QP-transplanted heartsshowed clusters of GFP-positive cells throughout the injected area(Fig. 4A). Although no teratomas were observed in any of theanimals transplanted with the QP cardiovascular progenitors, oneof the seven mice transplanted with the QN cells developed ter-atomas in the heart (Fig. 4D and Fig. S7D), demonstrating thateven EB cells exposed to as much as 5 d of potent differentiationfactors harbor some teratogenic cells, which can be excluded on thebasis of the QP markers. The GFP-positive QP cells were detectedonly as clusters in the injection sites without significant migration,but exhibited cardiac differentiation as evidenced by expression oftroponin and myosin heavy chain (Fig. 4 B and C). Despite theirengraftment and differentiation into mature cells, detailed histo-logical examination of the explanted hearts showed no gap junctionformation between hESC-derived cardiovascular progenitors andthe host myocardium: the human QP cells failed to integrate withthemouse myocardium.We hypothesized that the failure of hESC-

derived cardiomyocytes to structurally and functionally integrateinto the adult mouse host may be a result of several factors, in-cluding: (i) interspecies differences that prevent the coupling ofhuman andmouse cells, (ii) inability of an adult heart to provide anoptimal environment for maturation and integration of the cardiacprogenitors, and/or (iii) an inherent inability of QP-derived car-diovascular progenitors to functionally integrate.

Structural and Functional Integration of QP Cells in Viable Fetal HumanHeart. To address these issues, we developed a transplantationmodel that allows us to assess the functional development ofhESC-derived cardiovascular progenitors in fetal human hearts (22,23). The ventricular tissues from a second-trimester human fetalheart (15 wk gestation) were implanted s.c. into a pouch formed inthe ear pinna of an SCID mouse (Fig. S7A and Movie S3). Graftviability was confirmed by the presence of autonomous beatingdetermined by visual inspection and electrocardiography∼7 to 10 dafter implantation (Fig. S7B and Movie S3). Two weeks later, ∼5 ×105 freshly sorted QP cardiovascular progenitors (and QN cells ascontrol) from a GFP-hESC line were transplanted into the heartgraft. The animals were euthanized after 8 wk, and confocal mi-croscopy of the explanted heart receiving QP cells revealed clustersof GFP-positive cells spread throughout the myocardium, includingareas distant from the injection site (Fig. 5A). Such a distribution ofthe grafted cells is most likely a result of migration or simplespreading, in contrast to the possibility of the cells passivelyspreading along the injection site (5). The GFP-positive cellscoexpressed troponin, α-actinin, or CD31, which suggests in vivodifferentiation of the progenitors into cardiomyocyte and endo-thelial lineages (Fig. 5 B and C and Fig. S7C). No teratoma for-mation was observed in animals receiving QP cells (n = 4). Incontrast, transplantation of QN cells resulted in teratoma forma-tion in one ear–heart model (n = 4), and transplanted QN cells didnot differentiate into cardiomyocyte or endothelial lineages.A careful histological examination of the explanted QP-recipient

heart tissues revealed typical punctate staining for connexin-43along the regions of intimate cell-to-cell contact between hESC-derived cardiomyocytes (GFP+) and host cardiomyocytes (Fig. 5 Band C). These results indicate that, when transplanted into a hu-man fetal heart, hESC-derived cardiovascular progenitors not onlymature to cardiomyocytes, they can also migrate and couplestructurally to their neighboring allogeneic cells. We seldom ob-served phosphohistone H3-positive QP cells in explanted sections(phosphohistone H3 is a marker for mitotic activity), suggestingthat, at some point within 8 wk after transplantation, the QP cellslose the robust proliferative activity they exhibit in vitro (Fig. S7E).Although junctional proteins connecting QP cells to the sur-

rounding cells were readily detected, it is important to determinewhether the transplanted cells were also electrically connected tothe host myocardium. Human fetal heart tissues with QPtransplantation were removed from the mouse ear and immedi-ately sectioned, and real-time Ca2+ transients were measured inareas with QP-derived GFP-positive cells. GFP-positive cells dem-onstrated periodic Ca2+ oscillations similar to—and synchronizedwith—the host cells (24). The Ca2+ oscillations responded to in-creasing frequencies of external electrical stimulation. Theserecordings showed conduction of Ca2+ signals from the host myo-cardium into areas of GFP-positive transplanted cells resemblinga continuous electrical propagation (Fig. 6A and Movie S4). Al-though these results indicate functional integration of the trans-planted QP cells into the human myocardium, the present modelhas limitations. We cannot rule out the possibility that the graft canbe transilluminated by calcium transients in the surrounding tissue.As some studies have reported fusion between transplanted cells

and host myocardium (25, 26), we sought to determine whether theextent of cell fusion was significant enough to explain our findings.Because the H9 ESC line is of female origin and the transplantedfetal heart wasmale, we were able to perform sexmismatch analysisby evaluating for the presence of the Y chromosome in the GFPtransplanted cells (derived from XX H9 cells). We traced donorGFP-positive cells for FISH with X- and Y- chromosome paints to

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Fig. 4. Engraftment of QP cells in mouse myocardium. (A) (Left) Wholemouse heart explanted 8 wk after injection of GFP+ QP cells. The dotted circleindicates the approximate location of the infarct zone. (Scale bar, 0.5 mm.)GFP-hESC–derivedQP cells engraft into the periinfarct regions ofmouse hearts(Middle and Right). (Scale bar, 120 μm.) (B and C ) Immunofluorescencestaining of mouse hearts 8 wk after QP transplantation reveals presenceof GFP-hESC–derived QP cells that costain with troponin (B) and withhuman cardiomyocyte-specific β-myosin heavy chain (C). (Scale bar, 100 μm.)(D) Immunohistochemical evidence for teratoma formation after 8 wk upontransplantation of QN cells. The QN-derived cells gave rise to all three germlayers including columnar epithelium (Left), cartilage (Center), and neuralrosette (Right). (Scale bar, 100 μm.)

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assess karyotype (Fig. 6B). Fewer than 3.8% of the transplantedcells expressed the Y in addition to the X chromosome, which maybe an overestimate for degree of cell fusion as a result of theoverlap of nuclei of unique cells on 6-μm sections contributing tothis value. This experiment indicates that, although rare fusionevents may occur, they cannot account for the overall integration ofthe transplanted cardiovascular progenitors into the host tissue.

DiscussionWehave identified four surfacemarkers, ROR2, CD13, KDR, andPDGFRα, that mark early cardiovascular progenitors among dif-ferentiating hESCs. These cells, designated QP cells, can give riseto three distinct cell populations, namely cardiomyocytes, endo-thelial cells, and vascular smooth muscle cells.The possibility that progenitors marked by QP markers repre-

sent only a distinct, transitory state of differentiating hESCs can-not be formally excluded. We have provided evidence that ROR2/CD13 cells may resemble a population of primitive-streak/meso-dermal cells. This claim is based on several lines of evidence fromour work and that of others: (i) the similarity of the gene expres-sion profiles of ROR2/CD13 cells and primitive streak cells (i.e.,expression of MIXL, T, GSC, Mesp1, KDR, GATA4); (ii) in vitrophenotype of a subpopulation of ROR/CD13 cells that differen-tiate to cardiomyocytes, vascular smooth muscle cells, and endo-thelial cells, which are mesoderm-derived progeny; (iii) expressionof ROR2 in the first-trimester human fetal heart; (iv) transplanta-tion into the human fetal heart of a subpopulation of ROR2/CD13cells resulting in robust commitment toward cardiovascular line-ages; and (v) expression of ROR2 in the entire region of theprimitive streak in embryonic day 7.5 mouse embryo and sub-sequent ROR2 mutation in mice leading to defects in somito-genesis and cardiogenesis (27, 28). To formally show that QP cellsalso exist in utero is ethically impermissible, as it would requireaccess to early human embryos to comprehensively screen forROR2 and CD13 expression in early human development (i.e.,during primitive streak and lateral plate mesoderm formation).Nonetheless, the identification of cells that can generate threemajor cell types in the heart provides a unique opportunity toinvestigate the mechanisms that regulate the onset of humancardiac development as well as those that control their specifica-tion to the cardiac and vascular cells.The role of the ROR2 protein has been studied in developmental

processes, cell migration, and polarity (29, 30). It has been shownthat ROR2 is expressed in the entire primitive streak region duringmouse embryonic development and later in the developing limbs,brain, heart, and lungs (27). In humans, mutations in the ROR2

gene have been associated with autosomal-recessive Robinowsyndrome, characterized by short stature, mesomelic limb short-ening, abnormal craniofacial features, and distinct cardiac anom-alies affecting the myocardium (31). The observed phenotypesarising from deficient or mutated ROR2 emphasizes its essentialroles in morphogenetic and developmental processes.Although the potential of hESCs to generate cardiovascular

cells is indisputable, a set of challenges remains that limit thetherapeutic application of hESC-derived cells. A major concern

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Fig. 5. Structural Integration of QP cellsin a human fetal heart. (A) Myocardialsections from human fetal heart tissues 8wk after transplantation s.c. into mouseear and delivery of QP cells shows clustersof GFP+ cells spread throughout theventricle. The site of injection of QP cellsin the first micrograph is in the left uppercorner. (Right, Inset) High-magnificationimage of sarcomeric structure. (Scale bar,100 μm.) (B and C) Coexpression of GFPwith cardiac-specific markers troponin (B)or α-actinin (C) and Connexin43 stainingbetween host and transplanted GFP+

cells. (Right) Overlay with Connexin43staining depicted in red and troponin orα-actinin in white. (Scale bar, 100 μm.)

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Fig. 6. QPcells integrate into fetal humanmyocardium. (A)Myocardial sectionsshow evoked calcium signals when paced electrically ex vivo. Fluo-4 calcium dyewas added to tissue, whichwas then electrically paced at 2 Hz. (Right) Same areaafter treatment with anti-GFP antibody reveals a GFP+ area. This region wasanalyzed for dye intensity changes (f) and results are plotted normalized to theintensity of the initial movie frame (f0). Real-time Ca2+ flux through the tissueindicate functional integration of GFP+ cells into the host tissue. (B) GFP+ cells,derived from XX H9 ESCs, were traced for FISH analyses to reveal XX karyotype(white arrowhead, Inset), whereas the host myocardium, from a male donor,expresses XY karyotype (white arrowhead, Inset). (Scale bar, 50 μm.)

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is their capacity to from teratomas after transplantation (32).The progenitor population we isolated based on the expressionof distinct surface markers did not form teratomas when trans-planted into mouse or human hearts. On the contrary, thepopulation characterized by absence of the QP surface proteinsresulted in teratoma formation upon transplantation into mouseor human hearts, which indicates that pluripotent stem cells canremain in culture even after 5 d of differentiation.Another significant challenge to clinical application relates to

the fate of the hESC-derived cardiac cells upon transplantation(12). Several investigators have reported engraftment of hESC-derived cells in infarcted mouse, rat, guinea pig, and pig hearts(14, 33, 34). Aside from the recent report of electromechanicalintegration of hESC-derived cardiomyocytes into guinea pighearts, careful examination of animal models has revealed thatthe transplanted cells form islands of nascent myocardium withinthe scar zone (12). Furthermore, the rapid heart rate of mice(∼600 beats/min) may prevent the human cardiac cells, whichhave an intrinsic rate of 60 to 100 beats/min, from keeping pacewith the mouse cardiomyocytes. In our experiments involving QPcell transplantation, we failed to see coupling of these cellswithin the mouse heart. In contrast, transplantation of QP cellsinto the human fetal heart revealed maturation, migration,coupling with resident cardiomyocytes, and electrophysiologicalactivity in concert with the host myocardium.Our findings clarify the ambiguity regarding the fate of trans-

planted hESC-derived cardiovascular cells. We show here thatcardiovascular progenitors generated from hESCs engraft intomouse hearts, but fail to integrate. However, when transplantedinto a human fetal heart tissue, they integrate into the host myo-cardium. A major limitation of the described model is that it is notamenable to a variety of physiological activities (i.e., hemody-namics or sinoatrial and atrioventricular conduction), the lack ofwhich may influence the development of hESC-derived cardio-vascular progenitors. Fetal hearts also have much less developedconnective tissue, which may promote integration of the QP cellsthat would be absent in adult hearts. Additionally, the present

model represents transplantation in an uninjured heart, whichmaybe a different environment for engraftment than an injured heart,and, moreover, does not allow us to investigate whether theengrafted cells could provide any functional improvement afterinjury. Nevertheless, the data herein establish the transplantabilityof hESC-derived cardiovascular progenitors into human fetalhearts as a proof of concept of functional allogeneic integration.Taken together, the data presented in this article suggest that

hESC-derived cardiovascular progenitors, defined by four surfacemarkers, can structurally and functionally integrate into the elec-trical syncytia of human fetal heart tissue upon transplantation.Additionally, our finding of ROR2 as an early marker for cardiaclineage specification highlights a previously unknown role of ROR2expression in cardiac development. Further research to delineatethe mechanism by which ROR2 is involved in early cardiovascularprogenitor formation is warranted. These valuable results providethe basis for future hESC-based cardiac therapy by identification ofa progenitor population capable of engraftment and regenerationwithout risk of teratoma formation.

Materials and MethodsHuman ES cells were maintained in standard ES culture as described. EBswere differentiated in the presence of Wnt 3a, BMP4, VEGF, activin A, sFz 8,FGF8, and Wnt11 Tables S3 and S4. Fetal heart tissues were obtained fromAdvanced Bioscience Resources. The Stanford institutional review boardapproved the use of one fetal heart for transplantation into a mouse ear,followed by injection of hESC-derived cardiovascular progenitors. A moredetailed discussion is provided in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Dr. Yoav Soen for valuable advice, Dr.Mirko Corselli for his help in immunostaining, Libuse Jerabek for excellentlaboratory management, and members of the I.L.W laboratory for adviceand assistance. This work was supported by California Institute for Re-generative Medicine Grant RCI 00354 (to I.L.W); National Institutes ofHealth/National Heart, Lung, and Blood Institute Fellowship 5T32 HL00708;an American College of Cardiology/Pfizer Career Development Award (toR.A.); the Howard Hughes Medical Institute (to S.R.A.); and the CaliforniaInstitute for Regenerative Medicine (to R.N.).

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