Aggregation of embryonic stem cells gives rise to embryoid bodies (EBs) which undergo developmental processes reminiscent of early eutherian embryonic development. Development of the three germ layers suggests that gastrulation takes place. In vivo, gastrulation is a highly ordered process but in EBs only few data support the hypothesis that self-organization of differentiating cells leads to morphology, reminiscent of the early gastrula. Here we demonstrate that a timely implantation-like process is a prerequisite for the breaking of the radial symmetry of suspended EBs. Attached to a surface, EBs develop a bilateral symmetry and presumptive mesodermal cells emerge between the center of the EBs and a horseshoe-shaped ridge of cells. The development of an epithelial sheet of cells on one side of the EBs allows us to define an ‘anterior’ and a ‘posterior’ end of the EBs. In the mesodermal area, first cardiomyocytes (CMCs) develop mainly next to this epithelial sheet of cells. Development of twice as many CMCs at the ‘left’ side of the EBs breaks the bilateral symmetry and suggests that cardiomyogenesis reflects a local or temporal asymmetry in EBs. The asymmetric appearance of CMCs but not the development of mesoderm can be disturbed by ectopic expression of the muscle-specific protein Desmin. Later, the bilateral morphology becomes blurred by an apparently chaotic differentiation of many cell types. The absence of comparable structures in aggregates of cardiovascular progenitor cells isolated from the heart demonstrates that the self-organization of cells during a gastrulation-like process is a unique feature of embryonic stem cells.

No abstract

Compelling evidence for the existence of somatic stem cells in the heart of different mammalian species has been provided by numerous groups; however, so far it has not been possible to maintain these cells as self-renewing and phenotypically stable clonal cell lines in vitro. Thus, we sought to identify a surrogate stem cell niche for the isolation and persistent maintenance of stable clonal cardiovascular progenitor cell lines, enabling us to study the mechanism of self-renewal and differentiation in these cells. Using postnatal murine hearts with a selectable marker as the stem cell source and embryonic stem cells and leukemia inhibitory factor (LIF)-secreting fibroblasts as a surrogate niche, we succeeded in the isolation of stable clonal cardiovascular progenitor cell lines. These cell lines self-renew in an LIF-dependent manner. They express both stemness transcription factors Oct4, Sox2, and Nanog and early myocardial transcription factors Nkx2.5, GATA4, and Isl-1 at the same time. Upon LIF deprivation, they exclusively differentiate to functional cardiomyocytes and endothelial and smooth muscle cells, suggesting that these cells are mesodermal intermediates already committed to the cardiogenic lineage. Cardiovascular progenitor cell lines can be maintained for at least 149 passages over 7 years without phenotypic changes, in the presence of LIF-secreting fibroblasts. Isolation of wild-type cardiovascular progenitor cell lines from adolescent and old mice has finally demonstrated the general feasibility of this strategy for the isolation of phenotypically stable somatic stem cell lines.

There are considerable gaps in our knowledge on cell biological effects induced by the heavy metals mercury (Hg) and lead (Pb). In the present study we aimed to explore the effects of these toxicants on proliferation and cell size of primary human amniotic fluid stem (AFS) cells. Monoclonal human AFS cells were incubated with three dosages of Hg and Pb (single and combined treatment; ranging from physiological to cytotoxic concentrations) and the intracellular Hg and Pb concentrations were analyzed, respectively. At different days of incubation the effects of Hg and Pb on proliferation, cell size, apoptosis, and expression of cyclins and the cyclin-dependent kinase inhibitor p27 were investigated. Whereas we found Hg to trigger pronounced effects on proliferation of human AFS cells already at low concentrations, anti-proliferative effects of Pb could only be detected at high concentrations. Exposure to high dose of Hg induced pronounced downregulation of cyclin A confirming the anti-proliferative effects observed for Hg. Co-exposure to Hg and Pb did not cause additive effects on proliferation and size of AFS cells, and on cyclin A expression. Our here presented data provide evidence that the different toxicological effects of Pb and Hg on primary human stem cells are due to different intracellular accumulation levels of these two toxicants. These findings allow new insights into the functional consequences of Pb and Hg for mammalian stem cells and into the cell biological behavior of AFS cells in response to toxicants.