Cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells

Direct lineage conversion is a promising approach for disease modeling and regenerative medicine. Cell divisions play a key role in reprogramming of somatic cells to pluripotency, however their role in direct lineage conversion is not clear. Here we used transdifferentiation of fibroblasts into neuronal cells by forced expression of defined transcription factors as a model system to study the role of cellular division in the direct conversion process. We have shown that conversion occurs in the presence of the cell cycle inhibitors aphidicolin or mimosine. Moreover, overexpression of the cell cycle activator cMyc negatively influences the process of direct conversion. Overall, our results suggest that cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III β-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration