Over-expression of bHLH genes facilitate neural formation of mouse embryonic stem (ES) cells in vitro

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Abstract
Mouse embryonic stem (ES) cells are useful tools for investigating differentiation into neurons and glial cells in vitro. In order to induce ES cells to differentiate into neural cells, many researchers have investigated the efficiency of induction. Embryoid body (EB) formation and retinoic acid are potent differentiation inducers known to be a trigger at the early stage of development. Basic helix-loop-helix (bHLH) is one of the important transcription factors, which is essential for premature neural formation. In NeuroD2 and Mash1-transfected cells, neural formation was observed at day 6 after the plating of embryoid bodies in culture. Nestin was detected in NeuroD2- and Mash1-transfected cells at day 10, and strong signal was detected in Mash1 transfectants by RT-PCR analysis. Map2 and Nurr1 were also detected strongly at the early stage in transfected cells compared with the wild type control, especially in the Mash1 transfectant. In immunocytochemical analysis, Tuj1-positive neurons were detected at high frequency in Mash1 transfectants and some cells were stained by tyrosine hydrogenase (TH), a marker of dopaminergic neurons. These results demonstrate that bHLH has a potential activity at an early stage for ES cells and can induce effective and rapid neural differentiation in vitro.
Funding Information
  • Scientific Frontier program of the Ministry of Education, Culture, Sports, Sciences and Technology