Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells

Abstract

Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression dynamics of retinoic acid driven mESC differentiation from pluripotency to lineage commitment, using an unbiased single-cell transcriptomics approach. We find that the exit from pluripotency marks the start of a lineage transition as well as a transient phase of increased susceptibility to lineage specifying signals. Our study reveals several transcriptional signatures of this phase, including a sharp increase of gene expression variability and sequential expression of two classes of transcriptional regulators. In summary, we provide a comprehensive analysis of the exit from pluripotency and lineage commitment at the single cell level, a potential stepping stone to improved lineage manipulation through timing of differentiation cues.

Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

“…As cells with longer cell cycles tend to increase their dwell time in G1 phase relative to other phases, we expected slow-dividing cells to be enriched for G1 cells. Consistently, the reddest population ( Figure 4G-J, Red) was enriched for G1 phase cells, while the bluest ( Figure 4G (Drukker et al, 2012;Semrau et al, 2017). Cell cycle rate heterogeneity was not only seen in the RA-treated samples, but also for cells kept in pluripotency maintenance conditions ( Figure 4G-J), corroborating reports that cellular heterogeneity exists under pluripotency maintenance conditions (Furusawa et al, 2006;Stewart et al, 2006) and is often associated with the cell cycle (Furusawa et al, 2006;Gonzales et al, 2015).…”

Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency

“…Progression through pluripotency is marked by sequential downregulation of naïve factors and upregulation of peri‐implantation epiblast markers, followed by appearance of lineage specification markers 16, 17, 61, 62. We examined levels of naïve pluripotency transcription factors in RSK 1*23 cells during transition.…”

Network Inference with Granger Causality Ensembles on Single-Cell Transcriptomic Data

“…We further test SCINGE on a second dataset that tracks retinoic aciddriven differentiation from mouse embryonic stem cells to extraembryonic endoderm and neuroectoderm cells over 96 hours [44]. We infer a trajectory for the biological process using Monocle 2 and select 1886 cells from cell states 1 and 2 ( Figure S1 and Section 3.3.2).…”

“…Many expected GO terms and regulators are represented in Table 1 and Figure 4. However, classic neuroectoderm regulators like Sox1, Nes, and Pax6 [44] are missing because they are excluded from the limited shortlist of genes in the SCINGE input. We only run SCINGE on the top 626 significantly differentially expressed genes along the differentiation trajectory detected by Monocle 2.…”

“…The second dataset was obtained from Semrau et al [44], where SCRBseq data was obtained at nine collection times during 96 hours from mouse embryonic stem cells differentiating into neuroectoderm and extraembryonic endoderm-like cells. We order the cells using Monocle 2 [22], with the ordering genes chosen by Monocle 2 in an unsupervised manner by identifying genes that are differentially expressed in response to the introduction of the growth medium.…”