P53 is a protein and transcription factor involved in a myriad of physiological events. Its role as a crucial tumor suppressor has been well-established, however its contribution to other processes such as embryonal development has only recently been revealed. Interestingly, p53 has been shown to regulate mesoendodermal differentiation via direct activation of the Wnt pathway and indirect influence of Smad signaling molecules. Such unexpected discoveries suggest that p53 might have other important regulatory roles during human embryonic development. This prompted us to utilize the expertise in our laboratory to explore the protein’s behavior during neuroectodermal differentiation in vitro, using human embryonic stem cell (hESC)-derived cerebral organoids. Organoids represent a 3D in vitro model which produces structures reminiscent of the human brain, and it has recently been implemented in our laboratory. To study the role of p53, we used the CRISPR/Cas9 system to delete p53 from hESCs and induced the cells to form cerebral organoids. Our preliminary finding showed that the absence of p53 strongly enhances the neural development in vitro and generates cerebral organoids with better neuronal morphology and higher expression of neuronal markers. We therefore aim to explore this phenomenon further. Firstly, using molecular techniques such as qPCR, we will quantify the expression of various neural markers, such as Sox2, β-3-tubulin, Doublecortin and others. In parallel, we will describe possible differences between the growth rate and cell cycle mechanics of wild-type and p53KO (knockout) cells. Secondly, we will functionally visualize the electric potential of the neurons within the cerebral organoids. And lastly, we will initiate experiments to identify molecular pathways responsible for the different p53KO phenotype. Collectively, our findings will aid in completing the repertoire of the enigmatic protein’s actions, as well as potentially unveil completely new pathways taking place during human neural development.

Publikace