Efficient neuronal differentiation of mouse ES and iPS cells using a rotary cell culture protocol

• Neuronal differentiation of moue ES and iPS cells under static or rotary conditions was compared.
• Rotation increased neurosphere uniformity and neural precursors.
• Rotation formed smaller neurospheres with significantly high cell survival and proliferation.
• Neural precursors under rotation matured faster and became functional neurons.
• Rotary culture procedures can be a routine technique for stem cell neuronal differentiation.

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells hold great promise in regenerative medicine for the treatment of neurodegenerative diseases. Current neuronal differentiation protocols however, are not optimized yet for the high scale production of neural precursors and terminally differentiated neurons. The present investigation reports a novel technique for the scalable production of highly uniformed neurospheres, neural precursors and terminal neurons from mouse ES and iPS cells using retinoic acid and a mechanical rotation procedure. We compared embryoid bodies (EB) and neurosphere morphology, yield of neural precursors and quality of neurons between rotary and static suspension cultures of mouse ES and iPS cells undergoing neural differentiation. Analysis of neurospheres formed under continuous rotation showed increased neurosphere uniformity and a high yield of neural precursors after neurosphere dissociation. Neurospheres formed under rotation conditions were relatively smaller, more uniform and had less dead cells and higher proliferation compared to those formed under static conditions. Neural precursors under rotation conditions matured faster, survived better, differentiated to functional neurons that stained positively for mature neuronal markers, and fired action potentials similar to the statically cultured neurons. This report thus provides a technique for the scalable production of neurons from ES and iPS cells and we suggest that rotation culture procedure can be a routine technique for stem cell neural and neuronal differentiation.