EMBRYONIC STEM-CELLS DIFFERENTIATE IN-VITRO INTO CARDIOMYOCYTES REPRESENTING SINUSNODAL, ATRIAL AND VENTRICULAR CELL-TYPES

Pluripotent embryonic stem cells (ESC, ES cells) of line D3 were diffe rentiated in vitro via embryo-like aggregates (embryoid bodies) of def ined cell number into spontaneously beating cardiomyocytes. By using R T-PCR technique, alpha- and beta-cardiac myosin heavy chain (MHC) gene s were found to be expressed in embryoid bodies of early to terminal d ifferentiation stages. The exclusive expression of the beta-cardiac MH C gene detected in very early differentiated embryoid bodies proved to be dependent on the number of ES cells developing in the embryoid bod y. Cardiomyocytes enzymatically isolated from embryoid body outgrowths at different stages of development were further characterized by immu nocytological and electrophysiological techniques. All cardiomyocytes appeared to be positive in immunofluorescence assays with monoclonal a ntibodies against cardiac-specific alpha-cardiac MHC, as well as muscl e-specific sarcomeric myosin heavy chain and desmin. The patch-clamp t echnique allowed a more detailed characterization of the in vitro diff erentiated cardiomyocytes which were found to represent phenotypes cor responding to sinusnode, atrium or ventricle of the heart. The cardiac cells of early differentiated stage expressed pacemaker-like action p otentials similar to those described for embryonic cardiomyocytes. The action potentials of terminally differentiated cells revealed shapes, pharmacological characteristics and hormonal regulation inherent to a dult sinusnodal, atrial or ventricular cells. In cardiomyocytes of int ermediate differentiation state, action potentials of very long durati on (0.3-1 s) were found, which may represent developmentally controlle d transitions between different types of action potentials. Therefore, the presented ES cell differentiation system permits the investigatio n of commitment and differentiation of embryonic cells into the cardio myogenic lineage in vitro.