Tang Shin Yi

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dbstshy@nus.edu.sg


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    Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells
    (BioMed Central Ltd, 2021-11-21) Tang, Shin Yi; Zha, Shijun; Du, Zhicheng; Zeng, Jieming; Zhu, Detu; Luo, Yumei; Wang, Shu; BIOLOGICAL SCIENCES
    Background: Redirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging. Methods: In the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a “safe harbour” for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells. Results: We detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells. Conclusion: Looking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells. © 2021, The Author(s).
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    Derivation of mimetic ?? T cells endowed with cancer recognition receptors from reprogrammed ?? T cell
    (Public Library of Science, 2019) Zeng, J.; Tang, S.Y.; Wang, S.; BIOLOGICAL SCIENCES
    Using induced pluripotent stem cells (iPSCs) to derive chimeric antigen receptor-modified T (CAR-T) cells has great industrial potential. A previous study used ?? T cell-derived CAR-modified iPSCs to produce CAR-T cells. However, these ?? T cells are restricted to autologous use and only recognize single cancer antigen. To make CAR-T alternative for allogeneic use, we reprogrammed ?? T cell into iPSCs (?? T-iPSCs) to circumvent the risk of graft-versus-host disease. To target multiple cancer-associated antigens, we used an “NK cell-promoting” protocol to differentiate ?? T-iPSCs and to induce expression of natural killer receptors (NKRs). Through such two-step strategy, mimetic ?? T cells endowed with an array of NKRs and thus designated as “?? natural killer T (?? NKT) cells” were derived. With no/low-level expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) and immune checkpoint receptors, ?? NKT cells may provide a potent “off-the-shelf” cytotoxic cell source to recognize multiple ubiquitous antigens in a broad spectrum of cancers. © 2019 Zeng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.