Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naı ¨ve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placentaderived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

Limited T cell availability and proliferative exhaustion present major barriers to successful T cell-based immunotherapies and may potentially be overcome through the use of ''rejuvenated'' induced pluripotent stem cells derived from antigen-specific T cells (T-iPSCs). However, strict antigen specificity is essential for safe and efficient T cell immunotherapy. Here, we report that CD8ab T cells from human T-iPSCs lose their antigen specificity through additional rearrangement of the T cell receptor (TCR) a chain gene during the CD4/CD8 double positive stage of in vitro differentiation. CRISPR knockout of a recombinase gene in the T-iPSCs prevented this additional TCR rearrangement. Moreover, when CD8ab T cells were differentiated from monocyte-derived iPSCs that were transduced with an antigen-specific TCR, they showed monoclonal expression of the transduced TCR. TCR-stabilized, regenerated CD8ab T cells effectively inhibit tumor growth in xenograft cancer models. These approaches could contribute to safe and effective regenerative T cell immunotherapies.

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.

No abstract

The effectiveness of chimaeric antigen receptor (CAR) T-cell immunotherapies against solid tumours relies on the accumulation, proliferation and persistency of T cells at the tumour site. Here we show that the proliferation of CD8αβ cytotoxic CAR T cells in solid tumours can be enhanced by deriving and expanding them from a single human induced-pluripotent-stem-cell clone bearing a CAR selected for efficient differentiation. We also show that the proliferation and persistency of the effector cells in the tumours can be further enhanced by genetically knocking out diacylglycerol kinase, which inhibits antigen-receptor signalling, and by transducing the cells with genes encoding for membrane-bound interleukin-15 (IL-15) and its receptor subunit IL-15Rα. In multiple tumour-bearing animal models, the engineered hiPSC-derived CAR T cells led to therapeutic outcomes similar to those of primary CD8 T cells bearing the same CAR. The optimization of effector CAR T cells derived from pluripotent stem cells may aid the development of long-lasting antigen-specific T-cell immunotherapies for the treatment of solid tumours.

Tumor-infiltrating lymphocytes (TIL), which include tumor-specific T lymphocytes with frequency, are used for adoptive cell transfer therapy (ACT) in clinical practice. The optimization of TIL preparation has been investigated to reduce the senescence and increase the abundance of TIL, as both the quality and quantity of the transferred cells have great influence on the outcome of TIL-based ACT (TIL-ACT). Considering the effects of cell reprogramming on senescence, we expected that the anti-tumor effect could be enhanced by TIL regeneration. To confirm this hypothesis, we established tumor-specific TIL-derived iPS cells (TIL-iPSC) with human colorectal cancer specimens. T cells differentiated from TIL-iPSC (TIL-iPS-T) retained not only intrinsic T cell functions and tumor specificity, but also exhibited improved proliferation capacity and additional killing activity. Moreover, less differentiated profiles and prolonged persistency were seen in TIL-iPS-T compared with primary cells. Our findings imply that iPSC technology has great potential for TIL-ACT.

Adoptive T cell transfer is a potentially effective strategy for treating cancer and viral infections. However, previous studies of cancer immunotherapy have shown that T cells expanded in vitro fall into an exhausted state and, consequently, have limited therapeutic effect. One way to overcome this obstacle is to use induced pluripotent stem cells (iPSCs) as a cell source for making effector T cells. In recent years, there have been several reports on generating effector T cells suitable for adoptive immunotherapy. The reported findings suggest that using iPSC technology, it may be possible to stably derive large numbers of juvenile memory T cells targeted to cancers or viruses. In this review, we describe a strategy for applying iPSC technology to immunotherapy and the characteristics of T cells derived from iPSCs. We also discuss how these technologies can be applied clinically in the future.

Chimeric antigen receptor (CAR) is an artificial protein that provides HLA-independent antigen specificity to T cells. CAR-T therapy has shown remarkable clinical responses especially in hematologic malignancies. But this therapy requires cell preparation for each patient and it cause some limitations for applicability of CAR-T therapy. We have reported regeneration of T cells from iPSCs (Cell Stem Cell. 2013). Since this technology can provide unlimited number of T cells, CAR-T therapy using iPSCs is thought to broaden its applicability. To target solid tumors, it is important to avoid immunosuppressive factors from tumor microenvironment and to exert sufficient cytotoxicity. Tumor reactive T cells are known to fall into anergy state by continuous antigen stimulations. To overcome immunosuppression in tumor microenvironment, enhancement of TCR signaling by modification of genes related to TCR signal is a promising strategy. Recent studies revealed that the efficacy of CAR-iPS-T cells are not equivalent to primary CAR-T cells. To enhance the efficacy of CAR-iPS-T cells and to produce resistant CAR-T cells to immunosuppression, we focused on TCR signaling pathway. We found that antigen reactivity of CAR-iPS-T cells was insufficient compared with primary CAR-T cells. To overcome the weakness of TCR signal, we disrupted genes negatively related to TCR signal and successfully enhanced TCR signal. As a result, genome edited CAR-iPS-T cells could persist longer in vivo and displayed enhanced tumor suppressive function comparable with primary CAR-T cells. Genome edited iPSCs can be a unlimited cell source of enhanced CAR-T cells. These findings indicate that regenerated CAR-T cells derived from genome edited iPSCs would be a promising CAR-T therapy which would overcome immunosuppressive tumor microenvironment. Citation Format: Tatsuki Ueda, Shoichi Iriguchi, Yohei Kawai, Atsutaka Minagawa, Hiroyuki Miyoshi, Seitaro Terakura, Yasushi Uemura, Knut Woltjen, Yuzo Kodama, Hiroshi Seno, Yasumichi Hitoshi, Tetsuya Nakatsura, Koji Tamada, Shin Kaneko. Enhanced effector responses of regenerated CAR-T cells derived from genome edited iPSCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1432.