Fig. 1.

CAR-expressing iPSCs can differentiate into CAR-macrophage cells. a Overview of deriving CAR-iMacs from CAR-iPSCs. b Flow cytometry analysis of iPSC-derived cells at different stages of differentiation with stage-specific markers. c qRT-PCR showing pluripotent marker gene and key macrophage marker gene expression at different stages of CAR-iPSC differentiation. n = 3, error bar: standard error of the mean. d Hierarchical clustering of transcriptomes of CAR-iPSCs, their differentiated cells, and primary and untransduced iPSC-differentiated macrophages in different states. e Principal component analysis (PCA) of the same samples as in d. f Top GO terms enriched in genes up-regulated on day 28 differentiated CAR-iMac cells compared with CAR-iPSCs, and the right panel is an example of GSEA analysis of the GO terms. NES normalized enrichment score. P = 0: P value is a very small number. g UMAP plot showing separation between human iPSCs and CAR-iMac cells. h UMAP plot showing subpopulation clustering of CAR-iMac cells. Ten clustered C0–C9 were identified and labeled as 0–9 with different colors. i Heatmap showing blasting the C0-C9 clusters of cells illustrated in g against a human single-cell atlas database containing single-cell RNA-seq data of hundreds of cell types including macrophages (https://scibet.cancer-pku.cn). j Trajectory analysis of differentiated cells along a pseudotime axis. k Heatmap showing averaged expression of M1 or M2 signature pathway genes in different clusters of cells illustrated in i. l Heatmaps to compare (benchmark) the 10 clusters (C0-C9) of CAR-iMac cells against previously published M1 or M2 polarized macrophages using metabolism genes. Human iPS cells differentiated macrophages polarized by IFN-γ and LPS; IPS_M2: Human iPS cells differentiated macrophages polarized by IL-4; HM_M1: Human PBMC-derived macrophages polarized by IFN-γ and LPS; HM_M2: Human PBMC-derived macrophages polarized by IL-4