Limitations of fluorescent timer protein maturation kinetics to isolate transcriptionally synchronized human neural...

Differentiation of human pluripotent stem cells (hPSCs) into subtype-specific neurons holds substantial potential for disease modeling in vitro. For successful differentiation, a detailed understanding of the transcriptional networks regulating cell fate decisions is critical. The heterochronic nature of neurodevelopment, during which distinct cells in the brain and during in vitro differentiation acquire their fates in an unsynchronized manner, hinders pooled transcriptional comparisons. One approach is to "translate" chronologic time into linear developmental and maturational time. Simple binary promotor-driven fluorescent proteins (FPs) to pool similar cells are unable to achieve this goal, due to asynchronous promotor onset in individual cells. We tested five fluorescent timer (FT) molecules expressed from the endogenous paired box 6 (PAX6) promoter in 293T and human hPSCs. Each of these FT systems faithfully reported chronologic time in 293T cells, but none of the FT constructs followed the same fluorescence kinetics in human neural progenitor cells.