Embryonic stem cells commit to differentiation by symmetric divisions following a variable lag period

Abstract

Mouse embryonic stem (ES) cells are derived from the epiblast of the preimplantation embryo and retain the capacity to give rise to all embryo lineages. ES cells can be released into differentiation from a near-homogeneous maintenance condition. Exit from the ES cell state can be accurately monitored using the Rex1-GFPd2 transgenic reporter, providing a powerful system for examining a mammalian cell fate transition. Here, we performed live-cell imaging and tracking of ES cells during entry into differentiation for 48 hours in defined conditions. We observed a greater cell surface area and a modest shortening of the cell cycle prior to exit and subsequently a reduction in cell size and increase in motility. We did not see any instance of cells regaining ES cell identity, consistent with unidirectional developmental progression. Transition occurred asynchronously across the population but genealogical tracking revealed a high correlation in cell-cycle length and Rex1-GFPd2 expression between daughter cells. A population dynamics model was consistent with symmetric divisions during exit from naive pluripotency. Collapse of ES cell identity occurred acutely in individual cells but after a variable delay. The variation in lag period can extend up to three generations, creating marked population asynchrony.2

Abscission Couples Cell Division to Embryonic Stem Cell Fate

“…However, we did not observe significant differences in REX1-GFP intensity dynamics or downregulation timings between daughter cells, even when division was very asymmetric in size ( Figures 3 C and 3D). In fact, the timing of REX1 downregulation was strongly correlated between sister cells ( Figure 3 E) and the variance of REX1-GFP levels was very low between sisters ( Figure 3 F), consistent with a recent study analyzing REX1 dynamics in single cells during pluripotency exit ( Strawbridge et al., 2020 ). These data indicate that sister cells exit the ES cell state in a highly correlated manner and suggest that size asymmetries at cell division do not influence the timing of naive pluripotency exit.…”

“…To investigate the role of cell division in exit from naive pluripotency, we first tested the effect of inhibiting cell division altogether. We used ES cells expressing a short half-life naive pluripotency reporter REX1-GFPd2 expressed from the endogenous REX1 locus ( Kalkan et al., 2017 ; Strawbridge et al., 2020 ), since REX1 downregulation correlates with naive pluripotency exit ( Kalkan et al., 2017 ; Mulas et al., 2017 ). ES cells were cultured in N2B27 medium supplemented with the MEK inhibitor PD0325901, the GSK-3 inhibitor CHIRON, and leukemia inhibitory factor (2i/LIF culture medium), and naive pluripotency exit was initiated by placing cells in N2B27 medium alone (differentiation medium hereafter) ( Mulas et al., 2019 ).…”

“…In this study, the cells used were: E14 wild type ES cells, E14 ES cells stably expressing H2B-RFP, a kind gift from Jonathan Chubb ( Cannon et al., 2015 ), E14 ES cells stably expressing REX1-GFP and GAP43-mCherry (a kind gift from Carla Mulas and Stanley Strawbridge, Austin Smith lab, Stem Cell Institute, University of Cambridge ( Strawbridge et al., 2020 )), E14 cells stably expressing REX1-GFP ( Kalkan et al., 2017 ), E14 cells expressing the Fucci2a system ( Mort et al., 2014 ) (a kind gift from Ian James Jackson, the University of Edinburgh), HeLa cells expressing H2B-mCherry and Tubulin-GFP ( Dimitracopoulos et al., 2020 ) (a kind gift from Buzz Baum, MRC LMCB, University College London), and suspension HeLa cells ( Chugh et al, 2017 ).…”

Capture of mouse and human stem cells with features of formative pluripotency

“…Although the formative phenotype is produced within 48hrs of ESC withdrawal from 2i, generation of stable FS cell lines requires several passages. The inherent asynchronicity of exit from naïve pluripotency (Strawbridge et al, 2020) together with imperfect in vitro transition conditions result in extensive initial heterogeneity, as also observed for EpiLC (Hayashi et al, 2011;Kalkan et al, 2017) . Passaging enriches for FS cells, similar to EpiSC generation (Guo et al, 2009), but a more streamlined and efficient capture would be advantageous for future research.…”

Mouse embryonic stem cells switch migratory behaviour during early differentiation

“…While mouse ES cells have been reported to display some level of motility in vitro (Strawbridge et al, 2020, Turco et al, 2012, their migration modes and the underlying mechanisms, and how these might change as ES cells start differentiating, have not been investigated. Here, we investigate whether the shape change characterizing early differentiation in mouse ES cells is associated with a change in the cells' motile potential.…”