OCT-3/4 and SOX-2 are Key Factors for SDIA Neurogenesis of Mouse Embryonic Stem Cells

Abstract

Utilizing a stromal-derived inducing activity (SDIA) model of neurogenesis, weinvestigated the effects of the targeted knockdown of Oct-3/4 and Sox-2 by shortinterfering RNAs (siRNAs) in mouse embryonic stem cells (mESC). Quantitativereal-time PCR showed a 40-90% knockdown of specific transcripts with cognate Oct-3/4 or Sox-2 siRNA transfection compared to FAM-labelled negative control (FAM)siRNAs or mock transfection, and was confirmed at the protein level by Western blotanalysis. Using PA6 SDIA co-cultures, neurogenesis was significantly diminished inOct-3/4 or Sox-2 targeted mESC upon differentiation. We observed that 45A?12%,65A?13% and 90A?8% (Mean +/- SD) of the colonies were stained with neuron-specificI?-tubulin III in Oct-3/4, Sox-2, and FAM siRNA transfected mESC respectively.Similar results were observed when differentiating mESC with neural-inducingfactors (Hep-NIF) collected from the surface of PA6 cells using heparin. In addition,differentiation of mESC using Hep-NIF but not Oct-3/4 and Sox-2 knockdown led tothe pronounced appearance of discrete, dark granular glial acidic fibrilary protein(GFAP)-positive cells which also expressed the glial cell marker Vimentin. Takentogether, these results extend the role of Oct-3/4 in SDIA, implicate a similar role forSox-2, and support emerging observations for the role of these factors and SDIA ingliogenesis.