itor cells and adult stem cells are capable of forming multiple cell types but are believed to have a more limited potential than ES cells.2 Three types of pluripotent stem cell lines have been established from mammalian embryos: embryonic carcinoma (EC), ES, and embryonic germ (EG) cells (Figure 1). The origin of ES cells from the preimplantation embryo (ICM or epiblast) is the defining feature that distinguishes these cells from other pluripotent embryonic cell lines. When returned to the embryonic environment by transfer into a host blastocyst or aggregation with blastomere-stage embryos, cultivated ES cells behave like normal embryonic cells. They can contribute to all tissues of the resulting chimeras, including germ cells. Thus, ES cells can be reincorporated into normal embryonic development and have the full potential to develop along all lineages of the embryo proper.3 EC cells are mainly feeder- cell independent, whereas mouse ES and EG cells require cultivation on feeder layers (mouse embryonic fibroblasts or STO cells) or the addition of a differentiation inhibitor factor, ie, leukemia inhibitory factor (LIF).4 Under these conditions, mouse ES and EG cells maintain a relatively normal and stable karyotype and have an unlimited ability to self-renew.5 Several groups have recently isolated and cultured human ES (hES)6 and EG7 cells. This has led to the suggestion that specific cell lineages derived from hES cells will be useful for therapeutic purposes. Mouse EC,8 ES,9,10 and EG11 cell cultures have been used to generate cardiomyocytes in vitro. The morphology, struc- ture, and function of these cardiomyocytes have been exten- sively studied, and the application of genetic and genomic approaches has led to recent insights into the program of cardiac development. hES cells can also differentiate to cardiomyocytes in culture, opening the possibility for con- trolled in vitro studies of developing human heart cells.12,13 The aims of the present review are to describe the in vitro process of ES cell differentiation into cardiomyocytes, dis- cuss recent results from genetic and gene manipulation studies, and describe the promise of postgenomic analyses in defining the molecular events responsible for cardiac differentiation. Mouse ES Cell���Derived Cardiomyocyte Differentiation Program In vitro differentiation of ES cells normally (except for neurogenesis) requires an initial aggregation step to form structures, termed embryoid bodies (EBs), which differentiate into a wide variety of specialized cell types, including cardiomyocytes (Figure 2). A number of parameters specifi- cally influence the differentiation potency of ES cells to form cardiomyocytes in culture: (1) the starting number of cells in the EB, (2) media, FBS, growth factors, and additives, (3) ES cell lines, and (4) the time of EB plating.14 Within the developing EB, cardiomyocytes are located between an epithelial layer and a basal layer of mesenchymal cells.15 Cardiomyocytes are readily identifiable, because within 1 to 4 days after plating, they spontaneously contract. With continued differentiation, the number of spontaneously beat- ing foci increases, and all the EBs may contain localized beating cells. The rate of contraction within each beating area rapidly increases with differentiation, followed by a decrease in average beating rate with maturation. Depending on the number of cells in the initial aggregation step, the change in beating rate and the presence of spontaneous contractions continue from several days to 1 month. Fully differentiated cardiomyocytes often stop contracting but can be maintained in culture for many weeks. Thus, developmental changes of cardiomyocytes can be correlated with the length of time in culture and can be readily divided into 3 stages of differen- tiation: early (pacemaker-like or primary myocardial���like cells), intermediate, and terminal (atrial-, ventricular-, nodal-, His-, and Purkinje-like cells).15 During early stages of differentiation, cardiomyocytes within EBs are typically small and round. The nascent myofibrils are sparse and irregularly organized or lacking, whereas others contain parallel bundles of myofibrils that show evidence of A and I bands.16 Adjacent cardiomyocytes often show different degrees of myofibrillar organization. With maturation, ES cell��� derived cardiomyocytes become elongated with well-developed myofibrils and sarcomeres. Beating cells are primarily mononucleated and rod-shaped, Figure 1. Pluripotent embryonic cell lines from mammals: EC, ES, and EG cells. ES cell lines are derived from the ICM and epiblast EC cell lines are derived from the undifferentiated embryonic components of germ cell tumors that arise spontane- ously or are experimentally induced by transfer of cells from the epiblast to extrauterine sites and EG cell lines are derived from primordial germ cells (PGCs) and are usually isolated from the genital ridge of 9.5- to 12.5-dpc embryos. The segregation of the germline in mouse embryos takes place in an extraembry- onic region where cell contacts and local signals induce differ- entiation of some epiblast cells to PGCs. After appropriate in vivo developmental pathways, ES and EG (but not EC) cells can contribute to all cells of a developing embryo, including the germ line. 190 Circulation Research August 9, 2002 by on June 4, 2010 circres.ahajournals.org Downloaded from

and they contain cell-cell junctions consistent with those observed in cardiomyocytes developing in the heart.16 During terminal differentiation stages, densely packed well- organized bundles of myofibrils can be observed, and the sarcomeres have clearly defined A bands, I bands, and Z disks.16,17 Nascent intercalated disks, fascia adherens, desmo- somes, and gap junctions have also been observed,9,16,18,19 and the spread of Lucifer yellow to adjacent cells after microinjection argues for functionally coupled gap junc- tions.16,20 Overall, the length, diameter, area, ultrastructure, and myofibrillar architecture of ES cell��� derived cardiomyo- cytes and sarcomere lengths with differentiation are similar to those reported for neonatal rodent myocytes. ES cell��� derived cardiomyocytes express cardiac gene products in a developmentally controlled manner. As in early myocardial development, mRNAs encoding GATA-4 and Nkx2.5 transcription factors appear in EBs before mRNAs encoding atrial natriuretic factor (ANF), myosin light chain (MLC)-2v, -myosin heavy chain ( -MHC), -myosin heavy chain ( -MHC), Na -Ca2 exchanger, and phospholamban (Figure 3A). Sarcomeric proteins of ES cell��� derived cardio- myocytes are also established developmentally in the follow- ing order: titin (Z disk), -actinin, myomesin, titin (M band), MHC, -actin, cardiac troponin T, and M protein (Figure 3B see review21). Cardiomyocytes with characteristics of fetal/ neonatal rodent cardiomyocytes express slow skeletal muscle troponin I isoforms and a greater proportion of -MHC versus -MHC, whereas cardiomyocytes that more rapidly contract preferentially express cardiac troponin I and -MHC.22 Thus, the appearance of cardiac-associated gene products is a function of differentiation time, similar to that seen in normal myocardial development. Spontaneously and rhythmically contracting cardiomyo- cytes can be isolated and studied in single cell assays (see Figure 2). Early ES cell��� derived cardiomyocytes have elec- trophysiological characteristics typical of primary myocardi- um (Figures 3C and 3D), whereas terminally differentiated ES cell��� derived cardiomyocytes have electrophysiological characteristics that are typical of those found in postnatal cardiomyocytes (see reviews15,23). Terminally differentiated Figure 2. ES cell���derived cardiogenic differentiation (adapted from Wobus et al14). When allowed to form cell aggre- gates, EC, ES, and EG cells spontane- ously differentiate into cells typical of all 3 primary germ layers (endoderm, ecto- derm, and mesoderm). Undifferentiated ES cells on primary cultures of embry- onic fibroblasts (feeder layers) are culti- vated as EBs in hanging drops for 2 days and in suspension for 3 to 5 days before being plated onto gelatin-coated tissue culture dishes. The morphology of 2-, 5-, and 7-day-old EBs by scanning electron microscopy (bar 50 m) is shown. After differentiation, individual properties of ES cell��� or EC cell���derived cardiomyocytes can be studied and assayed by using a variety of techniques, including RT-PCR (eg, -tubulin, -MHC, and MLC-2v), immunofluorescence (eg, anti���troponin T antibody), and electro- physiology (eg, action potential of 7 7- day cardiomyocyte). ES-derived cells in vitro can also be used to study embryo- genesis, abnormal development, func- tional genomics, pharmacotoxicology, and embryotoxicity of potential teratogens. Boheler et al ES Cell���Derived Cardiomyocytes 191 by on June 4, 2010 circres.ahajournals.org Downloaded from