Differentiation of ReNcell VM197 cells is induced solely by growth factor removal and proceeds without any additional external stimulation.

This cell line differentiates into neurons and glial cells within 72 hours after growth factor removal, which might explain the faster time scale of our model compared to the Lee model.

It is the combination of these two WNT stimuli, that allows the cell to first generate an immediate response to the perturbation (removal of growth factor ) and in the following to keep the activation on a constant, but moderately incremented level (cf.

In response to growth factor removal, a transient increase of the intracellular ROS level activates DVL in a redox-dependent manner.

Thus in contrast to the previous model configuration we replaced the initial amount of WNT molecules with a onetime release of ROS molecules (nRos = 10000) in response to growth factor removal.

Within three days after growth factor removal, ReNcell VM197 cells differentiate into neurons, astrocytes, and oligodendrocytes without any additional exogenous stimulation.

Accordingly proliferating ReNcell VM197 cells were used as reference (Oh), whereas all following time points were measured after initiating the differentiation by growth factor removal.

Cells were kept in proliferative state by applying 10 ng/mL basic fibroblast growth factor (bFGF, Invitrogen) and 20 ng/mL epidermal growth factor (EGF, Sigma-Aldrich, Steinheim, Germany).

Upon growth factor removal ReNcell VM197 cells differentiate into neurons and glial cells within a few days and without any additional external stimulation.

However further evidence was provided in our previous study on ReNcell VM197 cells, as an early increase of mitochondrial ROS metabolism after growth factor removal was found to modulate DVL-mediated WNT/fi-catenin pathway and neurogenesis [21].