The role of cell cycle, growth, and metabolism in species-specific differentiation timing

Abstract

Different mammalian species progress through similar stages during embryonic development and adult life but the pace of these transitions is species-specific. While classically, developmental timing was viewed merely as a consequence of varying body sizes between species, the use of pluripotent stem cells (PSCs) has shown that species-specific developmental timing is maintained during in vitro differentiation. Since then, uncovering cell-intrinsic mechanisms that regulate the timescales of development has become a rising topic of interest. This project aimed to identify such cell-intrinsic mechanisms using in vitro neural progenitor differentiation of mouse, monkey and human PSCs. To facilitate inter-species comparisons, all cells were cultured and differentiated under harmonized conditions. Under these circumstances, mouse cells differentiated more than twice as fast as human cells, recapitulating species-specific differentiation timing. As differentiation and growth need to be tightly coordinated during development, I compared cell cycle durations across the species. Cell cycle durations followed a species-specific trend, with the human cell cycle being 1.47-fold and the monkey cell cycle 1.42- fold longer than the mouse cell cycle. To test if differentiation depends on proliferation, I performed cell cycle and growth manipulations by either a retinoblastoma knock-out line or inhibiting the mammalian target of rapamycin (mTOR). Strikingly, mTOR inhibition caused a drastic extension of cell cycle durations, yet single-cell transcriptomics revealed no systematic delay during early neural differentiation. This showed that differentiation can be uncoupled from growth and proliferation, suggesting alternative mechanisms. One candidate identified was the UDP-glucose pyrophosphorylase 2 (UGP2), required for glycogen synthesis. High UGP2 correlated with slow differentiation. Consequently, glycogen content was highest in human cells, intermediate in monkey and lowest in mouse. Thus, glycogen content is a species-specific cellular property that was unknown to this date. Neural differentiation of UGP2 knock-out cells revealed premature expression of the forebrain marker FOXG1, indicating that UGP2 could contribute to setting differentiation timing. It remains to be tested, how loss of UGP2 globally affects differentiation and by which mechanisms UGP2 acts. Taken together, these findings show that differentiation can be uncoupled from growth and cell cycling and implicate UGP2 and glycogen metabolism in the regulation of timing. This constitutes a novel mechanism by which cells could determine their differentiation speed.