ALL STEM CELLS

Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates; however, the regulatory circuits specifying these states and enabling transitions between them are not well understood. Here we set out to characterize transcriptional heterogeneity in mouse PSCs by single-cell expression profiling under different chemical and genetic perturbations. Signalling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signalling pathways and chromatin regulators. Notably, either removal of mature microRNAs or pharmacological blockage of signalling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal and a distinct chromatin state, an effect mediated by opposing microRNA families acting on the Myc/Lin28/let-7 axis. These data provide insight into the nature of transcriptional heterogeneity in PSCs.

PSCs are defined by their unique capacity to differentiate into all the cell types of an organism, while self-renewing in culture. How PSCs reconcile pluripotency and self-renewal and decide among fate choices is a topic of intense interest, with relevance for regenerative medicine and developmental biology. Genomic maps of the regulatory circuitry underlying pluripotency reveal a network of sequence-specific autoregulatory transcription factors targeting self-renewal genes that are active in PSCs,
as well as repressed lineage-specific developmental regulators that exist in a poised state and are capable of driving cells towards differentiated fates1–5. These core transcription factors are thought to interact with chromatin modifiers, non-coding RNAs and external signalling pathways tomaintain pluripotency. This self-sustaining transcriptional program becomes reactivated during reprogramming of somatic cells to pluripotency5.
The discoveries that levels of Nanog and other key PSC regulators fluctuate over time, that PSCs exist in multiple interconvertible states, and that distinct subpopulations of PSCs vary in their capacity to selfrenew or differentiate, hint at the dynamism of the PSC transcriptional program6–13, which may be fundamental to pluripotency14–23. Here, we apply single-cell analytics to PSCs subjected to a range of perturbations to systematically dissect the factors underlying PSC heterogeneity. By doing so,wemap the structure of gene expression variability in PSCs and identify regulatory circuits governing transitions between pluripotent cell states.

Conclusion
The diverse range of conditions under which pluripotency can be induced ormaintained has been accompanied by reports ofmolecular and functional variation. Herewe analysed the dynamic transcriptional landscape of pluripotent stem cells subject to a number of chemical and genetic perturbations.Applying single-cell analytics,wegleaned a number of essential insights. We found that different classes of genes manifest high or low expression variability in PSCs, with housekeeping and metabolic gene sets showing consistent expression across individual cells, while genes involved in signalling pathways and developmentwere considerably more variable. Moreover, expression states of variable regulatory factorswere coupled together, implying the presence of a regulated biological network. Analysis of chemical and genetic perturbations led to the discovery that depletion ofmiRNAsmimicked the transcriptional ground state of pluripotency routinely induced by culture in 2i1LIF, conditions that block the dominant ERK and GSK3 signalling pathways that converge on the Myc/Lin28/let-7 axis. Our data shed light on the transcriptional dynamics of the pluripotent state at the single-cell level, and demonstrate how regulation of gene expression variation relates directly to the transition between pluripotency and differentiation.
Transcriptional heterogeneity is increasingly being recognized as a key component of many biological processes46–48. It will be of interest to map stable and flexible regulatory nodes in networks governing other progenitor and differentiated cell types to discern common principles underlying network architecture and gene expression variability.