Murine embryonic stem cells (ESCs) were exposed to simulated microgravity using the principle of a fast 2D clinostat and hypergravity (1.8 g) to investigate the effects of (altered) gravity on cell differentiation. In this context, gravity-induced changes in the cell cycle distribution, the gene expression, the cytoskeleton, the embryoid body morphology and the differentiation potential have been investigated. Changes in the gene expression and embryoid body morphology as well as reduced beating patterns (correlated with a lower expression of Myh 7) of ESCs after exposure demonstrated the impact of simulated microgravity. The analysis showed changes in the actin framework of the cytoskeleton on day 6 of clinorotation as compared with the corresponding 1 g controls; the actin framework was less pronounced under simulated microgravity conditions. Cell cycle analysis of clinorotated ESCs revealed significant changes in the G1-phase: the number of cells in the 1 g control group was significant higher as compared with the clinostat group. ESCs exposed to hypergravity revealed changes in the actin framework after 24 hours; the microfilaments were denser and tightly arranged than under 1 g conditions. Furthermore, hypergravity induced significant differences in the morphology of embryoid bodies regarding area and diameter and resulted in an increased size compared with the 1 g control, whereas the cell cycle seemed to be unaffected by hypergravity. Location of the 1 g control near to the centrifuge versus location of the 1 g control on the bottom of a clinostat revealed different numbers of cells in the SubG1-phase. This result indicates a potential device-specific side effect of vibration and in turn a combined effect of hypergravity and vibration on the running centrifuge.
As a result of the insufficient RNA quality and the fluctuating expression values of the investigated genes (Oct 4, Nanog, CDH 1, Myh 6, cardiac Troponin and Klf 4), there were no consistent results obtained in this study. However, increased beating activities in EBs may indicate changes in the expression of differentiation markers and in genes, which are involved in the process of cardiomyogenesis. The handling procedures of embryonic stem cells used in this study, have to be revised for further studies since standard laboratory procedures include e.g. centrifugation steps in order to concentrate cells, which might interfere with the gravity-related experimental set-up. Overall, the results of this study suggest that embryonic stem cells are sensitive to altered gravity conditions and respond to gravity changes by e.g. forming a more/less powerful actin framework. Further investigations are necessary for understanding the effects of gravity and its effects on the actin cytoskeleton for the differentiation of ESCs and for understanding the development under altered gravity conditions
