Paracrine promotion of cardiomyogenesis in embryoid bodies by LIF modulated endoderm.

In the vertebrate embryo the heart is the first organ to form. Embryonic and extra-embryonic tissues are supposed to contribute to cardiac lineage commitment before and during gastrulation in a paracrine fashion. Evidence has accumulated that factors secreted by the anterior lateral endoderm and extra-embryonic endoderm contribute to cardiomyogenesis. Here we exploit in vitro differentiation of embryonic stem cells in embryoid bodies to study differentiation of the extraembryonic endodermal lineage, gastrulation-like processes, and the influence of endoderm on cardiomyogenesis. We demonstrate that in embryoid bodies primitive endoderm differentiates to visceral and parietal endoderm and that parietal endoderm influences onset of cardiomyogenesis in a concentration-dependent manner. Both increased concentrations of leukemia inhibitory factor and its absence in lif-/- embryoid bodies hampered parietal endoderm formation. Reduced differentiation of parietal endoderm correlated with an attenuation of cardiomyogenesis even in the presence of LIE These and previous results suggest that leukemia inhibitory factor is directly and indirectly, via endoderm formation, involved in the regulation of cardiomyogenesis. Increased proliferation of parietal endoderm in lifr -/- embryoid bodies and addition of conditioned lif -/- cell culture supernatant promoted cardiomyogenesis, demonstrating for the first time that parietal endoderm also contributes to cardiomyogenesis in embryoid bodies in a paracrine and leukemia inhibitory factor and its receptor independent pathway. New factors signaling independently of the leukemia inhibitory-factor receptor pathway may sustain cardiomyocyte cell proliferation and thus be a future target for gene therapy of cardiomyopathies and cell therapy of the myocardium.

Leukemia inhibitory factor modulates cardiogenesis in embryoid bodies in opposite fashions.

Cardiogenesis is a multistep process regulated by a hierarchy of factors defining each developmental stage of the heart. One of these factors, leukemia inhibitory factor (LIF), a member of the interleukin-6 family of cytokines, is expressed in embryonic and neonatal cardiomyocytes and induces cardiomyocyte hypertrophy. Many aspects of embryogenesis are faithfully recapitulated during in vitro differentiation of embryonic stem cells in embryoid bodies. We exploited this model to study effects of growth factors on commitment and differentiation of cardiomyocytes and on maintenance of their phenotype. We identified LIF as a factor affecting commitment and differentiation of cardiomyocytes in an opposite manner. Diffusible LIF inhibited mesoderm formation and hampered commitment of cardiomyocytes. Lack of both the diffusible and matrix-bound isoforms of LIF in lif-/- embryoid bodies did not interfere with commitment, but it severely suppressed early differentiation of cardiomyocytes. Onset of differentiation was rescued by very low concentrations of diffusible LIF; however, consecutive differentiation was attenuated in a concentration-dependent manner by diffusible LIF both in wild-type and lif-/- cardiomyocytes. Differentiation of cardiomyocytes was severely hampered but not completely blocked in lifr-/- embryoid bodies, suggesting additional, LIF-receptor ligand independent pathways for commitment and differentiation of cardiomyocytes. At the fully differentiated state, both paracrine and autocrine LIF promoted proliferation and increased longevity of cardiomyocytes. These findings suggest that both paracrine and autocrine and both diffusible and matrix-bound isoforms of LIF contribute to the modulation of cardiogenesis in a subtle, opposite, and developmental stage-dependent manner and control proliferation and maintenance of the differentiated state of cardiomyocytes.