Investigating neural differentiation of mouse P19 embryonic stem cells in a time-dependent manner by bioinformatic, microscopic and transcriptional analyses.

BACKGROUND: As an available cell line, mouse pluripotent P19 has been widely employed for neuronal differentiation studies. In this research, by applying the in vitro differentiation of this cell line into neuron-like cells through retinoic acid (RA) treatment, the roles of some genes including DNMT3B, ICAM1, IRX3, JAK2, LHX1, SOX9, TBX3 and THY1 in neural differentiation was investigated. METHODS AND RESULTS: Bioinformatics, microscopic, and transcriptional studies were conducted in a time-dependent manner after RA-induced neural differentiation. According to bioinformatics studies, we determined the engagement of the metabolic and developmental super-pathways and pathways in neural cell differentiation, particularly focusing on the considered genes. According to our qRT-PCR analyses, JAK2, SOX9, TBX3, LHX1 and IRX3 genes were found to be significantly overexpressed in a time-dependent manner (p < 0.05). In addition, the significant downregulation of THY1, DNMT3B and ICAM1 genes was observed during the experiment (p < 0.05). The optical microscopic investigation showed that the specialized extensions of the neuron-like cells were revealed on day 8 after RA treatment. CONCLUSION: Accordingly, the neural differentiation of P19 cell line and the role of the considered genes during the differentiation were proved. However, our results warrant further in vivo studies.

Comparative analysis of protein-protein interaction networks in neural differentiation mechanisms.

Neural differentiation as a major process during neural cell therapy is one of the main issues that is not fully characterized. This study focuses on the major deconstruction of the transcriptional networks that regulate cell fate determination during neural differentiation under the influence of RA signalling. In our studies, we used four different microarray datasets containing a total of 15,660 genes to determine which genes were differentially expressed during neural differentiation from pluripotent stem cells (P19), among the 17 samples from four different datasets that were integrated via meta-analysis approaches. Of the 15,660 gene expression in our data integration, 443 DEGs are induced during neural differentiation. Upstream dissection of these 443 DEGs revealed a network of protein-protein interactions (PPIs) from TFs and kinases, as well as intermediate proteins between them, which are indicated by three (POU51, NANOG, and FOXO1) down-expression genes and one PAX6 up-expression gene playing roles in up-stream of these 443 induced DEGs during neural differentiation. The constructed network from the PPIs database revealed that four novel sub-networks play major roles in neuron differentiation in cluster 3, retinol metabolism in cluster 4, Rap1 signalling pathways in cluster 2, and axonogenesis in cluster 6. These four clusters have revealed very useful information about how neural characterization will be created from pluripotent stem cells. This research reveals a plethora of information on the neural differentiation process, including cell commitment and neural differentiation, and lays the groundwork for future research into particular pathways involving protein-protein interactions in neurogenesis.