Defined Human Pluripotent Stem Cell Culture Enables Highly Efficient Neuroepithelium Derivation Without Small Molecule Inhibitors

Abstract

Abstract The embryonic neuroepithelium gives rise to the entire central nervous system in vivo, making it an important tissue for developmental studies and a prospective cell source for regenerative applications. Current protocols for deriving homogenous neuroepithelial cultures from human pluripotent stem cells (hPSCs) consist of either embryoid body-mediated neuralization followed by a manual isolation step or adherent differentiation using small molecule inhibitors. Here, we report that hPSCs maintained under chemically defined, feeder-independent, and xeno-free conditions can be directly differentiated into pure neuroepithelial cultures ([mt]90% Pax6+/N-cadherin+ with widespread rosette formation) within 6 days under adherent conditions, without small molecule inhibitors, and using only minimalistic medium consisting of Dulbecco’s modified Eagle’s medium/F-12, sodium bicarbonate, selenium, ascorbic acid, transferrin, and insulin (i.e., E6 medium). Furthermore, we provide evidence that the defined culture conditions enable this high level of neural conversion in contrast to hPSCs maintained on mouse embryonic fibroblasts (MEFs). In addition, hPSCs previously maintained on MEFs could be rapidly converted to a neural compliant state upon transfer to these defined conditions while still maintaining their ability to generate all three germ layers. Overall, this fully defined and scalable protocol should be broadly useful for generating therapeutic neural cells for regenerative applications. Stem Cells  2014;32:1032–1042