Application Note: An Automated Method for the Expansion and Neural Differentiation of Embryonic Stem Cells
6 October 2008

The study of embryonic stem cells (ESCs) is a growing area. The ability of ESCs to differentiate into the three somatic germ layers and eventually into different tissues will be increasingly pivotal in tissue engineering, drug discovery and regenerative medicine. The capability to automate differentiation procedures in higher throughout formats will be needed in these areas.

Typically, ESCs are grown manually by direct co-culture with mouse embryonic fibroblasts (MEFs) which allow the ESCs to remain un-differentiated. Differentiation is initiated by the removal of the fibroblasts with subsequent protocol-specific ESC manipulations. A common differentiation method is to form three dimensional cell clusters [embryoid bodies (EBs)] either in suspension or “hanging drop” culture methods. The EBs allow for the initial formation of the three germ layers which later develop into different tissue types. The methods for forming EBs are labor intensive and not amendable to automation.

We have investigated alternatives to EB formation in the expansion and neural differentiation of ESCs colonies and/or mono-layers. Mouse embryonic stem cells (mESCs) were maintained in an undifferentiated state with a Tecan Freedom EVO® workstation and an integrated Liconic® incubator. The MEFs were seeded in a layer separated from the mESCs by a 1μm PET membrane. This setup (indirect co-culture) allows easy automated removal of the MEF layer that, in turns, allows the cells to differentiate along neural lineages as demonstrated by the formation of neurites. The formation of neuritis was confirmed by morphology and expression of neurite markers (neurofilament, β-Tubulin and nestin).