Elucidation differentiation pathways using a novel combinatorial cell culture approach.

Embryonic stem cells because of their pluripotency and ability to self renew, offer the promise of being the starting material for novel cell therapies for a great many serious medical conditions. Harnessing this potential, however, requires the cells to be controllably differentiated in vitro. Though many protocols for differentiation have been published, they have very mixed outcomes and in general low efficiencies. Developing these protocols has traditionally been a slow process. Therefore the ability to test many protocols simultaneously would be of enormous benefit, but using conventional culture systems to achieve this is totally impractical. One way of circumventing this problem is to use combinatorial cell culture, which in its approach is not unlike combinatorial chemistry. In brief, combinatorial cell culture (CCC) is a multiplexing technology which allows millions of combinations of cell culture conditions to be tested in parallel, rapidly identifying an optimal protocol for any given outcome in cell biology. We have deployed this approach to find efficient methods for differentiating mouse embryonic stem cells into dopaminergic (DA) neurons (the cells lost in Parkinson’s disease) as a model system. By using CCC, we were able to test 2,400 differentiation pathways simultaneously in one simple experiment requiring only three 12-well plates. After a 28 day differentiation period, cells were stained for the presence of tyrosine hydroxylase. After analysis we were able to elucidate several successful pathways, which are highly efficient for the production of DA neurons. We will present data showing the advantages of this approach and the conclusions that can be drawn from the sequences of media compositions which are most efficient for neuronal differentiation in vitro