Fig. 9: scRNA-seq of hTOs demonstrates multilineage differentiation of dendritic cells and mature T lymphocytes. | Nature Communications

Fig. 9: scRNA-seq of hTOs demonstrates multilineage differentiation of dendritic cells and mature T lymphocytes.

From: Combinatory differentiation of human induced pluripotent stem cells generates functional thymic epithelium driving dendritic cell and CD4/CD8 T cell development

Fig. 9: scRNA-seq of hTOs demonstrates multilineage differentiation of dendritic cells and mature T lymphocytes.

a The CD45+ EPCAMlo population in D28 hTOs was sorted by flow cytometry, and its transcriptome was sequenced using ultra-high-sensitive bulk RNA-seq. b GO term analysis of differentially expressed genes in CD45+EPCAMlo samples (n = 2), shows enrichment of biological processes associated with antigen processing and presentation. c Cell deconvolution of CD45+EPCAMlo from D28 hTOs (n = 2, Replicate (Rep)1 and Rep2) against the human thymic cell atlas from Park et al. d scRNA-seq analysis of the hematopoietic compartment of D28 hTOs (human Thymic Organoids). The UMAP projection reveals three main cell clusters (T(P): proliferating T cells, T(Q): quiescent T cells and DC: dendritic cells), along with an additional stromal cluster. Cell origin, either from the iPSc line (Individual 1) or the primary ETPs (Individual 2) was assessed by SNP comparison using scSplit (v1.0.8). Cluster identity was assigned based on differentially expressed marker genes. e Expression of DC, stromal, and T cell marker genes in the D28 hTO (human Thymic Organoids) clusters defined in d (Left). CD3 and CD7 expression identify T lymphocytes, LY86 and PLEK identify dendritic cells (DCs) and KRT8/18 mark a rare stromal population. Expression of NKT cell marker genes in the same clusters (Right). No enrichment of NKT cells was detected. f For refined cell cluster annotation, D28 hTO (human Thymic Organoids) scRNA-seq data were projected onto the human thymic cell atlas from Park et al. using Seurat label transfer. The reference UMAP (Left) and the projection of D28 hTO cells (Right) are shown; with reference cells in gray. g Violin plots of normalized expression of CD34, FLT3, a TCR-signaling/response module (LAT, ZAP70, LCK, CD5), and a CD3 module (CD3D, CD3E, CD3G, CD247) in hTO cells mapped to DN, SP, DC1, and DC2 clusters. Each dot represents one cell; n indicates the number of cells per cluster. Statistical comparisons (DN vs SP), TCR signaling/response: p = 0.02494 and CD3 genes: p = 0.04041 (Welch’s one-sided t-test).

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