Single-Cell Gene Expression Profiles Define Self-Renewing, Pluripotent, and Lineage Primed States of Human Pluripotent Stem Cells

• Single-cell transcript profiles characteristic of distinct substates of pluripotency
• Prospective isolation of substate with high self-renewal, no lineage priming
• Self-renewing subpopulation marked by expression of specific ligands and receptors

SummaryPluripotent stem cells display significant heterogeneity in gene expression, but whether this diversity is an inherent feature of the pluripotent state remains unknown. Single-cell gene expression analysis in cell subsets defined by surface antigen expression revealed that human embryonic stem cell cultures exist as a continuum of cell states, even under defined conditions that drive self-renewal. The majority of the population expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity, consistently high transcripts for all pluripotency-related genes studied, and no lineage priming. This subpopulation was characterized by its expression of a particular set of intercellular signaling molecules whose genes shared common regulatory features. Our data support a model of an inherently metastable self-renewing population that gives rise to a continuum of intermediate pluripotent states, which ultimately become primed for lineage specification.