Neural differentiation from human embryonic stem cells in a defined adherent culture condition

Abstract

Understanding how to direct human embryonic stem cells (hESCs) toward a specific lineage pathway and generate appropriate cell types robustly is very important, not only for the study of developmental biology but also for potentially using these cells to treat human diseases. In this study, hESCs were differentiated to the neural lineage in defined adherent culture by retinoic acid and basic fibroblast growth factor. Our protocol seems to recapitulate the early steps of nervous system development in vivo in that undifferentiated hESCs organized into rosettes and then neural tube-like structures are formed. Differentiating cells expressed neuroectodermal and mature neuron markers during neural plate and tube formation and maturation, as shown by reverse transcriptase-PCR. More than 90% of differentiated cells expressed additional neuron-specific antigens (i.e., tubulin-III, MAP-2, synaptophysin and neurofilament protein). Ultrastructural analysis of differentiating neural tube-like structures in three dimensional collagen scaffolds showed an ependymal-like layer and neural structure with typical synapses. These results provide a simple and relatively defined system for differentiation of hESCs to neural lineages, particularly neurons with typical cellular, molecular and ultrastuctureal markers. The culture of neural precursor cells in a collagen scaffold may provide a new approach for the repair of spinal cord injury.