Differentiation of neural precursors and dopaminergic neurons from human embryonic stem cells

Abstract

Directed differentiation of human embryonic stem cells (hESCs) to a functional cell type, including neurons, is the foundation for application of hESCs. We describe here a reproducible, chemically defined protocol that allows directed differentiation of hESCs to nearly pure neuroectodermal cells and neurons. First, hESC colonies are detached from mouse fibroblast feeder layers and form aggregates to initiate the differentiation procedure. Second, after 4 days of suspension culture, the ESC growth medium is replaced with neural induction medium to guide neuroectodermal specification. Third, the differentiating hESC aggregates are attached onto the culture surface at day 6-7, where columnar neural epithelial cells appear and organize into rosettes. Fourth, the neural rosettes are enriched by detaching rosettes and leaving the peripheral flat cells attached and expanded as neuroepithelial aggregates in the same medium. Finally, the neuroepithelial aggregates are dissociated and differentiated to nearly pure neurons. This stepwise differentiation protocol results in the generation of primitive neuroepithelia at day 8-10, neural progenitors at the second and third week, and postmitotic neurons at the fourth week, which mirrors the early phase of neural development in a human embryo. Identification of the primitive neuroepithelial cells permits efficient patterning of region-specific progenitors and neuronal subtypes such as midbrain dopaminergic neurons.

Fig. 1

Five morphologically distinct steps of neuronal differentiation from human ESCs. (A-F) Phase contrast image showing ESC colony, ESC aggregate, primitive neuroepithelia, definitive neuroepithelia, neuroepithelial aggregate and postmitotic neuron stages. Arrows show rosette formation in primitive or definitive neuroepithelial stages. (G-K) Phase contrast image showing a partially differentiated ESC colony (arrow), differentiated ESC aggregates, asynchronized neural differentiation with neurons appearing at the neuroepithelial stage, neuroepithelial aggregates and postmitotic neurons.