Timed changes in expression of self-renewal and lineage-associated genes across differentiation trajectories. (A) Experimental design used to track the sequential transcript outputs of in vitro–stimulated P-mix CB cells by single-cell CITE-seq analysis. For details, see supplemental Methods. (B) Hierarchical clustering of cell cycle states based on scaled gene expression values. Columns represent 16 269 individual cells colored by time point and cell cycle state. Rows represent genes expressed in specific cell cycle phases. (C) The percentages of cells in different cell cycle phases at each time point assessed. (D) UMAP presentation of transcriptome data (after cell cycle regression) combined from 12 181 single cells from the day 7, 10, and 13 time points. Cells are divided into A and B clusters based on the first UMAP dimension. (E-F) UMAP projection of 10 385 cells in the A cluster. Cells are colored by surface phenotype inferred from antibody-derived oligonucleotide tag signals (E) or assigned cell cycle phase (F). (G) Percentage of different cell cycle phases in cluster A cells separated by different phenotypic subsets. (H) Unsupervised modeling of lineage trajectories embedded in the UMAP manifold. (I) Distribution of TF activities in the UMAP space. Expression of each TF module is indicated by colored dots. Gray lines represent a density level of 0.006 of all cells in cluster A. (J) Top 30 most active TF motifs in each lineage trajectory. Columns represent individual cells ordered by pseudotime. Rows represent TFs that are detected in >20 target genes. Colors on the heat map denote presence (colored by lineage) or absence (blank) of the TF.