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

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.

A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.

Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of "off-the-shelf" T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce "off-the-shelf" and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology.