Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase

Area of Science:

• Stem cell biology
• Molecular biology
• Genetics

Background:

• Cellular reprogramming generates induced pluripotent stem cells (iPSCs) but typically involves a small fraction of cells.
• Previous studies analyzed bulk cell populations, hindering single-cell resolution of reprogramming events.

Purpose of the Study:

• To identify single-cell gene expression patterns during cellular reprogramming.
• To determine novel markers that predict successful iPSC generation.
• To elucidate the gene regulatory network governing pluripotency induction.

Main Methods:

• Single-cell gene expression profiling of 48 genes across different reprogramming stages.
• Comparative analysis of gene expression patterns between early and late reprogramming phases.
• Identification of key transcription factors and their hierarchical relationships.

Main Results:

• Early reprogramming stages exhibit significant cell-to-cell gene expression variability, while late stages show more uniform expression.
• Esrrb, Utf1, Lin28, and Dppa2 are superior predictors of iPSC fate compared to Fbxo15, Fgf4, and Oct4.
• A hierarchical gene expression model emerged, with Sox2 as an upstream regulator in the late phase.
• Downstream factors, excluding core pluripotency factors, can activate the pluripotency circuitry.

Conclusions:

• Single-cell analysis provides critical insights into the heterogeneity and dynamics of cellular reprogramming.
• Novel gene expression markers can improve the efficiency and prediction of iPSC generation.
• A hierarchical gene regulatory network, independent of Oct4, Sox2, Klf4, c-Myc, and Nanog, drives pluripotency induction.