The Characterization Of Stable Expression Of Transcription Factor FoxA2 In Pluripotent Stem Cells

Transcription factor FoxA2 plays a critical role in neural differentiation during embryonic development. Our study has found that FoxA2 is expressed during neural differentiation of P19 pluripotent embryonal carcinoma cells. To further understand the biological roles of FoxA2 during this process, we have established the stable expression of a GFP-rFoxA2 fusion protein in P19 pluripotent embryonal carcinoma cells, named P19-GFP-rFoxA2 cells. The stable expression of GFP-rFoxA2 fusion protein results in decreasing expression of pluripotency-related transcription factors Nanog and Sox2 and reducing activity of alkaline phosphatase, though the cells still express pluripotency-related transcription factors Oct4. Increased Nestin expression levels and Prominin-1+ populations have been observed in P19-GFP-rFoxA2 cells, implicating that the stable expression of FoxA2 leads P19 cells to losing pluripotency and gaining partial characteristics of neural stem cells. The expression of neurogenic transcription factor NeuroD1 also expresses in P19-GFP-rFoxA2 cells. We confirm that the promoter of NeuroD1 is bound and activated by FoxA2 directly, suggesting that NeuroD1 is one of FoxA2 downstream target genes. Furthermore, a more rapid elevation of NeuroD1 expression is found during RA-induced P19-GFP-rFoxA2 cell neural differentiation and a same phenomemon is observed in suspension culture of P19-GFP-rFoxA2 cells without RA, implicating that FoxA2 can promote pluripotent stem cells to neural differentiation through up regulating expression of NeuroD1. Thus our results have proved that P19-GFP-rFoxA2 cells possess partial characteristics of neural cells and FoxA2 plays a critical role to regulate the neural differentiation of pluripotent P19 cells.