title: Uni-directional differentiation of mouse embryonic stem cells into neurons by the neural stem sphere method.
abstract: We previously showed that our neural stem sphere (NSS) method promotes the neuronal differentiation of mouse, monkey and human embryonic stem (ES) cells. Here we analyzed changes in expression of marker genes and proteins during neuronal differentiation. When cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, colonies of ES cells formed floating cell spheres, which, within 4 days, gave rise to NSSs. In the spheres, the expression of ES cell marker genes was consistently down-regulated, while expression of an epiblast marker was transiently up-regulated, beginning on day 2, and the expression of neuroectoderm, neural stem cell and neuron markers was up-regulated, beginning on days 3, 4 and 6, respectively. The expression of the marker genes was consistent with that of marker proteins. The time course of expression of these markers in the spheres resembled that of neuronal differentiation from the inner cell mass (ICM) cells of blastula. In contrast, the expression of endoderm, mesoderm, epidermis, astrocyte and oligodendrocyte markers was low and not up-regulated during differentiation. Only a small number of apoptotic cells were present in the spheres. These results suggest that mouse ES cells uni-directionally differentiate into neurons via epiblast cells, neuroectodermal cells and neural stem cells.
authors.affiliation: Laboratory of Regenerative Neurosciences, Department of Frontier Health Sciences, Graduate School of Human Health Sciences, Tokyo Metropolitan University, 7-2-10 Higashiogu, Arakawa-ku, Tokyo 116-8551, Japan. otsu masa@ks.kyorin-u.ac.jp
authors.forename: Masahiro
authors.initials: M
authors.lastname: Otsu
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Sai
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakayama
authors.forename: Koji
authors.initials: K
authors.lastname: Murakami
authors.forename: Nobuo
authors.initials: N
authors.lastname: Inoue
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D020869:Gene Expression Profiling; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D058953:Neural Stem Cells / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction