Development of effective isolation method of ES cells for analysis of differentiation

Abstract

Neuroectoderm development is a milestone of vertebrate neurogenesis. However, the molecular mechanism underlying the differentiation of neuroectoderm is still unclear, especially in mammals. ES cells co-cultured with PA6 cells can differentiate to neuroectoderm by the stromal cell-derived inducing activity method (SDIA method), but contamination of PA6 cells is an obstacle to the analysis of molecular mechanisms of differentiation. Here we describe a novel method by which differentiated ES cells are easily isolated from PA6 cells. We attempted to induce the differentiation of ES cells using paraformaldehyde-fixed PA6 cells. RT-PCR and DNA microarray analysis revealed that the background noise derived from contaminated PA6 cells disappeared when fixed PA6 cells were used. Furthermore, genes up-regulated during the differentiation of ES cells were expressed in a developing mouse embryo. Thus, our newly developed method will be very useful for identifying novel genes associated with mouse neuroectoderm development in vitro and in vivo.