Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types

Mechanisms of Development, 44 (1993) 41-50

41

© 1993Elsevier ScientificPublishers Ireland, Ltd. 0925-4773/93/$06.00 MOD 191

Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types Victor A. Maltsev a, Jiirgen Rohwedel a, Jiirgen Hescheler b and A n n a M. Wobus a,, a Institut fiir Pflanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, D-06466 Gatersleben, Germany and b Institut far Pharmakologie, Freie Universitiit Berlin, D-14195 Berlin, Germany

(Received 1 June 1993;revisionreceived 3 August1993;accepted6 August1993)

Pluripotent embryonic stem cells (ESC, ES ceils) of line D3 were differentiated in vitro via embryo-like aggregates (embryoid bodies) of defined cell number into spontaneously beating cardiomyocytes. By using RT-PCR technique, a- and /3-cardiac myosin heavy chain (MHC) genes were found to be expressed in embryoid bodies of early to terminal differentiation stages. The exclusive expression of the/3-cardiac MHC gene detected in very early differentiated embryoid bodies proved to be dependent on the number of ES cells developing in the embryoid body. Cardiomyocytes enzymatically isolated from embryoid body outgrowths at different stages of development were further characterized by immunocytological and electrophysiological techniques. All cardiomyocytes appeared to be positive in immunofluorescence assays with monoclonal antibodies against cardiac-specific a-cardiac MHC, as well as muscle-specific sarcomeric myosin heavy chain and desmin. The patch-clamp technique allowed a more detailed characterization of the in vitro differentiated cardiomyocytes which were found to represent phenotypes corresponding to sinusnode, atrium or ventricle of the heart. The cardiac cells of early differentiated stage expressed pacemaker-like action potentials similar to those described for embryonic cardiomyocytes. The action potentials of terminally differentiated cells revealed shapes, pharmacological characteristics and hormonal regulation inherent to adult sinusnodal, atrial or ventricular cells. In cardiomyocytes of intermediate differentiation state, action potentials of very long duration (0.3-1 s) were found, which may represent developmentally controlled transitions between different types of action potentials. Therefore, the presented ES cell differentiation system permits the investigation of commitment and differentiation of embryonic cells into the cardiomyogenic lineage in vitro.

Cardiomyocyte; Embryonic stem cell differentiation; Cardiac-specific gene expression; Action potential

Introduction

A considerable amount of data has been accumulated on the morphology and physiology of adult heart ceils, but only limited information is available about the origin, commitment and differentiation of the cells that will make up the myocardium (Litvin et al., 1992). In contrast to the well-described process of skeletal muscle cell development (Buckingham, 1992) the temporally and spatially controlled events of commitment and differentiation of cardiomyocytes remain to be

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investigated. In this respect it is relevant to emphasize that these two muscle cell types arise from different progenitor cells. Skeletal muscle cells develop from somites, but cardiac cells from anterior lateral plate mesoderm at an earlier stage of embryonic development. Recent data suggest that a very short interval exists between mesodermal commitment and phenotypic manifestation of differentiated cardiomyocytes (Han et al., 1992). One of the most important obstacles to study commitment and differentiation of cardiac myocytes is the lack of permanent cell lines to model and analyze the earliest stages of cardiomyogenesis. Physiological studies of developing heart cells have been mainly carried out on organ-cultured chick hearts (Sperelakis, 1982; Sperelakis and Shigenobu, 1974),

42 precardiac areas of blastoderm (McLean et al., 1987; Rosenquist and de Haan, 1966), and cultured mammalian heart cells of embryonic and neonatal origin (Wollenberger 1984; 1985). These studies on primary cardiac cell cultures are limited since cell properties and normal cardiogenesis apparently are disturbed during cultivation. For example, when 3-day-old chick embryonic heart cells are placed into culture, the cells do not continue to differentiate with respect to their electrical and morphological properties (Sperelakis, 1978). Differentiated ventricular cells from 16-day-old chick embryos cultured as monolayers revert back to the young embryonic state (Sperelakis and Pappano, 1983). Cultivated adult ventricular myocytes lose myofibrillar proteins and highly organized morphology (Nag et al., 1983; Claycomb and Palazzo, 1980). They retain only a few properties of the differentiated heart (e.g. lack of automaticity) during a limited culture period (Bugaisky and Zak, 1989). Up to now, only a few permanent cell lines (e.g. H9c2 from embryonic rat heart) expressing some cardiac-specific properties including L-type Ca 2+ current were characterized (Hescheler et al., 1991; Sipido and Marban, 1991). A new approach may be offered by using cardiomyocytes differentiated in vitro from pluripotent mouse embryonic stem cells (ES ceils, ESC) (Doetschmann et al., 1985; Wobus et al., 1991). These permanent cell lines derived by in vitro culturing undifferentiated cells of early embryos are capable to take part in the embryonic development in vivo after retransfer into blastocysts (e.g. Bradley et al., 1984). In vitro, ES cells have been shown to spontaneously differentiate into derivatives of all three primary germ layers, endoderm, ectoderm and mesoderm (Evans and Kaufman, 1981; Martin, 1981; Wobus et al., 1984; 1988). Our previous results with ES cell-derived cardiomyocytes (lines D3 and Bl17) revealed the functional expression of adrenoceptors, cholinoceptors and L-type Ca 2 ~ channels (Wobus et al., 1991). In the present study we used the ES cell differentiation system to investigate the cardiomyocyte differentiation in vitro with respect to expression of tissue-specific genes, proteins and the developmentally controlled functional expression of electrophysiological properties. The expression of cardiac-specific genes was demonstrated by RT-PCR analysis, the formation of cardiac-specific intracellular proteins was detected by indirect immunofluorescence, and the development of action potentials was characterized by patch-clamp technique. Our data proved that ES cell-derived cardiomyocytes expressed cardiac-specific genes and proteins. Furthermore, a more detailed characterization of cardiomyocytes differentiating into sinusnodal, atrial and ventricular cells was only possible by electrophysiological measurements of action potentials.

Results

Expression of cardiac-.V)eciJic genes i.s del'elopmenta/b" regula ted Because the differentiation capacity is clearly dependent on the number of cells differentiating in the embryoid bodies (see Smith et al., 1987: Wobus ct al., 1991), all experiments were done with a standardized differentiation protocol, i.e. cultivation of cmbryoid bodies with a defined cell number in hanging drops (Fig. 1). During different stages of cultivation single embryoid bodies were analyzed by RT-PCR wilh oligonucleotide primers specific for (v- and /3-cardiac and embryonic skeletal MHC genes (Fig. 2). The first transcripts of /3-cardiac MHC could be detected in "4 d' embryoid bodies originated from 1000 cells but not from 400 cells. At this stage beating areas in embryoid bodies originated from 400 ES cells wcrc never observed.

Embryonic stem cells cultivated on feeder layer

1 Cultivation of 400 or 1000 cells/2Olal medium in hanging drops

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