Lineage tracing of T cell differentiation from T-iPSC by 2D feeder-free culture and 3D organoid culture

Abstract

Introduction: T cells induced from induced pluripotent stem cells(iPSCs) derived from antigen-specific T cells (T-iPS-T cells) are an attractive tool for T cell immunotherapy. The induction of cytotoxic T-iPS-T cells is well established in feeder-free condition for the aim of off-the-shelf production, however, the induction of helper T-iPS-T cells remains challenging.

Methods: We analyzed T-iPS-T cells matured in 3D organoid culture at different steps in the culture process at the single-cell level. T-iPS-T cell datasets were merged with an available human thymocyte dataset based in single-cell RNA sequencing (scRNA-seq). Particularly, we searched for genes crucial for generation CD4+ T-iPS-T cells by comparing T-iPS-T cells established in 2D feeder-free or 3D organoid culture.

Results: The scRNA-seq data indicated that T-iPS-T cells are similar to T cells transitioning to human thymocytes, with SELENOW, GIMAP4, 7, SATB1, SALMF1, IL7R, SYTL2, S100A11, STAT1, IFITM1, LZTFL1 and SOX4 identified as candidate genes for the 2D feeder-free induction of CD4+ T-iPS-T cells.

Discussion: This study provides single cell transcriptome datasets of iPS-T cells and leads to further analysis for CD4+ T cell generation from T-iPSCs.

Keywords: 3D organoid; CD4; T cell differentiation; iPSC; scRNA seq.

Figure 1

Generation of CD4SP and CD8SP T-iPS-T cells by organoid culture. (A) Schemes of CD4 SP and CD8SP T-iPS-T cell differentiation. EBs were generated from a single-cell dissociated T-iPSC proliferated in feeder- and serum-free condition. The emerging iHPCs were differentiated on 2D and 3D conditions. The T cell maturation in the 2D condition required OKT3 stimulation, however, no additional stimulation is introduced in the 3D condition. (B) Flow cytometry analysis of T-iPS-T cells differentiated under 7 days OKT3 stimulation based on the expression levels of CD4 and CD8a. Results represent 3 independent experiments. (C) Flow cytometry analysis of hematopoietic differentiation from a T-iPSC clone, TkT3V1-7, based on the expression levels of CD4 and CD8a at 12 weeks after ATO induction showing clearly independent CD4⁺CD8^- and CD4^-CD8⁺ populations. The gating strategies involved the delineation of live cells expressing CD3 and abTCR. Results represent more than 3 independent experiments. (D) Flow cytometric analysis of T-iPS-T cells on organoid culture at 6 weeks based on CD4 and CD8a expressions. Distinct CD4⁺CD8^- and CD4^-CD8⁺ populations were already emerging in this 6 weeks culture. Results represent more than 3 independent experiments.

Figure 2

scRNA-seq analysis of T-iPS-T cells in organoid culture. (A) Flow cytometry plots of organoid cultures showing CD4 and CD8a expressions showing gradual maturation process of T cells. The maturation initiated from presenting CD4⁺CD8^- population. The population gradually disappeared by 3 weeks and became CD4⁺CD8⁺. The CD4⁺CD8^- population emerged again by 6 weeks. The gating strategies involved the delineation of live cells expressing CD3 and abTCR. Results represent more than 3 independent experiments. (B) Weekly UMAP plots with RCA analysis of 3D organoid cultures. At week 2, differentiating cells were heterogeneous and included erythroid, myeloid and lymphoid cells. However, as the culture progressed, the cellular heterogeneity decreased, and a large fraction of cells was assigned to lymphoids or lymphoblasts by week 6. (C) UMAP plot of cumulative data across all samples including all time points of organoid samples color-coded by clusters. This merged plot contained erythroid, myeloid, stem-like clusters other than lymphoid cluster. The clusters were named depending on the RCA score. (D) Histograms showing the number of cells in each week. Cluster 0, 1, 2 (highlighted with red) belonged to lymphoid cells and 6 week sample contained mainly lymphoid cells. Most of the non-lymphoid clusters belonged to samples from earlier differentiation cultures.

Figure 3

Comparison of the scRNA-seq and the CITE-seq analysis of T-iPS-T cells in organoid culture. (A) Scaled expressions of CD3e, CD4 and CD8A mRNA (top) and these surface proteins, labelled with oligonucleotide-labeled antibodies, (bottom) of the merged dataset shown on UMAP plots generated as in Figure 2C . CD3e was presented most of the lymphoid population. CD4 and CD8 were partially presented in lymphoid population in this analysis, however, on the cite-seq analysis, CD3, CD4 and CD8A were presented on the surface of the cells of this lymphoid cluster. (B) mRNA expression (top) of ATO cultures compared with CITE-seq analysis (bottom), showing CD4 and CD8A expression levels. The CITE-seq analysis showed similar tendency to FACS results ( Figure 2A ).

Figure 4

scRNA-seq analysis of extracted T cell lineage-committed population of the 3D organoid culture T-iPS-T cells. (A) UMAP plot of selected organoid-cultured cells committed to T cell lineage based on CITE-seq results color-coded by cultured week. (B) UMAPs of the gene expressions of CD4, CD8A, and CD8B (top) and protein surface expressions of CD4 and CD8A through CITE-seq(bottom). (C) UMAP plots as in (A) depicted with individual weeks. The DP population was highlighted with blue circle and the SP population was highlighted with red circle.

Figure 5

Pseudotime analysis of a merged plot of organoid culture T-iPS-T cells and thymocytes. (A) Trajectory analysis of all organoid cultured T-iPS-T cell data merged with published human thymocyte datasets. The red circle was T cell lineage cells depending on CD4, CD8A and CD8B expression of thymocyte datasets. (B) Feature plots of CD4, CD8A and CD8B in merged dataset of 3D organoid cultured T-iPS-T data and published human thymocyte datasets (top) and in 3D organoid cultured T-iPS-T data (bottom). (C) Feature plots of CD4 and CD8A through CITE-seq. The results were showed on merged plots (top). The human thymocyte datasets were subtracted in bottom figures. (D) T cell lineage cells which highlighted with red circle in (A). The trajectory branches were divided with color. (immature cells (red), CD4SP (green), CD8SP (blue)). (E) Distinct UMAP plots of T-iPS-T cells shown by week projected onto UMAP generated as in (A). T-iPS-T cells at 2 weeks mainly projected onto DP clusters defined by human thymocytes and merged onto SP clusters as the differentiation progressed.

Figure 6

Analysis of developing T-iPS-T with human thymocytes along with trajectory. (A) Heatmap of DEG modules along the pseudotime. (B) Top 10 significant genes of each module cluster.

Figure 7

Transcriptomic profiles of differentiated cells from 2D and organoid culture by scRNA-seq analysis. (A) Merged UMAP of organoid-cultured cells, 2D-cultured cells and published human thymocytes. (B) Scaled expressions of CD4, CD8A and CD8B on UMAP generated as in (A). (C) Scaled expression of CD4 in organoid-cultured cells, thymocytes and 2D-cultured cells. The blue circle shows the population presented with CD4⁺ SP cells in thymocytes and organoid-cultured cells.