Fig. 6: Late phase of hTO differentiation. | Nature Communications

Fig. 6: Late phase of hTO differentiation.

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

Fig. 6: Late phase of hTO differentiation.

a Bright-field images of hTOs (human Thymic Organoids) at D11 and D25 showing outgrowths into the hydrogel (n = 6 from independent differentiations and ETP donors) (Created in BioRender. Guillonneau, C. (2025) https://BioRender.com/7oyshki). b 3D immunofluorescence confocal imaging of the medullary epithelial compartment (KRT5) and the hematopoietic compartment (CD45) in D28 hTOs. c Flow cytometry of D28 organoids. Black squares indicate the TEC (CD45−EPCAM+) and thymocyte (CD45+EPCAM-) fractions, along with an undefined (CD45+EPCAMlo) population. d FPKM expression of genes encoding HLA-DR, HLA-DM, HLA-DO, and the co-stimulatory molecule CD86 from bulk RNA-seq of sorted HLA-DRlo and HLA-DRhi TECs from D28 hTOs (n = 1). Source data are provided as a Source Data file. e Deconvolution of HLA-DRhi and HLA-DRlo TECs against the Bautista human TEC atlas. Primary human mTEChi, mTEClo and cTEC samples were included as controls. Proportions were estimated using MuSiC (v0.2.0). f qPCR quantification of AIRE expression in D23 hTO TEP/ETP co-cultures (3D) and in conventional 2D monolayer co-cultures. Relative quantification to GAPDH. Error bars show mean ± SD (n = 6, from independent differentiations and ETP donors). **p = 0.001332 (two-sided Wilcoxon rank sum exact test). Source data are provided as a Source Data file. g Proportion of CD205+ and CD205− cells within the CD45−EPCAM+ fraction of D7, D14 and D21 hTOs (human Thymic Organoids) (Left), their respective percentage of HLA-DR cells (Middle), and mean fluorescence intensity (MFI) of HLA-DR and HLA-DP (Right). Source data are provided as a Source Data file.

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