Small molecule promoted feeder free and adherent differentiation of functional neurons from human embryonic and induced pluripotent stem cells.

Abstract

While human embryonic stem (hES) and induced pluripotent stem (hiPS) cells offer exciting prospects in the fields of regenerative medicine and developmental biology, efficient directed differentiation of these cells is still difficult. Neural induction protocols often include suspension culture or co-culture with other cell types, introducing heterogeneity and complicating analysis. In addition, expensive recombinant factors are often used over processes that take weeks to complete, making such experiments financially difficult. We have developed a fully adherent and feeder free neural differentiation protocol using small molecules such as dorsomorphin and common medium supplements. Using this protocol, we obtain >90% of cells developing into neural precursors, as measured by nestin staining. Neurons derived from these precursors are electrophysiologically active. After three weeks of terminal differentiation, we obtain functional neurons which fire high-amplitude action potentials upon depolarization. A subset of neurons also fires repetitive trains. This protocol offers a simpler and less expensive method for investigations involving the differentiation of neural precursors and neurons in culture.