Efficient Generation of Neural Stem Cells from Embryonic Stem Cells Using a Three-Dimensional Differentiation System

Abstract

Mouse embryonic stem cells (ESCs) are useful tools for studying early embryonic development and tissue formation in mammals. Since neural lineage differentiation is a major subject of organogenesis, the development of efficient techniques to induce neural stem cells (NSCs) from pluripotent stem cells must be preceded. However, the currently available NSC differentiation methods are complicated and time consuming. This study aimed to propose an efficient method for the derivation of NSCs from mouse ESCs; early neural lineage commitment was achieved using a three-dimensional (3D) culture system, followed by a two-dimensional (2D) NSC derivation. To select early neural lineage cell types during differentiation, Sox1-GFP transgenic ESCs were used. They were differentiated into early neural lineage, forming spherical aggregates, which were subsequently picked for the establishment of 2D NSCs. The latter showed a morphology similar to that of brain-derived NSCs and expressed NSC markers, Musashi, Nestin, N-cadherin, and Sox2. Moreover, the NSCs could differentiate into three subtypes of neural lineages, neurons, astrocytes, and oligodendrocytes. The results together suggested that ESCs could efficiently differentiate into tripotent NSCs through specification in 3D culture (for approximately 10 days) followed by 2D culture (for seven days).