AUTHOR=Chen Yanyan , Huang Pufeng , Niu Mengda , Tian Chuanhuizi , Zhang Tingting , Peng Zhiping 

TITLE=Regeneration of T cells from human-induced pluripotent stem cells for CAR-T cell medicated immunotherapy

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1159507

DOI=10.3389/fbioe.2023.1159507

ISSN=2296-4185

ABSTRACT=<p><bold>Background:</bold> Chimeric antigen receptor (CAR) T cell treatment involves <italic>in vitro</italic> production of T cells from patient blood with synthetic receptors specific to a cancer antigen. They circumvent the major histocompatibility complex to recognize the tumor antigen, reducing hematologic malignancy remission rates by 80%. Considering the efficacy of CAR-T treatment, the present work aimed at generating functional clusters of differentiation (CD)8 + T cells from human induced pluripotent stem cells (hiPSC) and to generate hiPS-CAR-T cells with high antigen-specific cytotoxicity.</p><p><bold>Methods:</bold> The Alkaline phosphatase assay and MycoEasy rapid <italic>mycoplasma</italic> detection kit was implemented for detection of hiPSCs and <italic>mycoplasma</italic>, respectively. The CD34<sup>+</sup> HSPCs were harvested in AggreWellTM 400 using a 37-micron reversible strainer. Likewise, the lymphoid progenitor and CD4<sup>+</sup>CD8<sup>+</sup> DP T cells were also harvested. The Cell Counting Kit-8 (CCK-8) assay was used to mark cytotoxicity and ELISA was used to detect IFN-γ secretion. Further, flow cytometry and transwell chambers were used to assess cell cycle, and migration and invasion. Finally, the <italic>in vivo</italic> antitumor effects of the CAR-T cells were evaluated using experimental animals (mice).</p><p><bold>Results:</bold> Results revealed that a serum-free, feeder layer-free differentiation system significantly yielded hiPSC-based T cell immunotherapy with interleukin-2, interleukin-15, and activators at the differentiation stage to promote the maturation of these cells into human induced pluripotent stem (hiPS)-T cells. The infection of hiPSCs with the CD19 CAR lentivirus resulted in the production of the hiPSC-CAR-T cells. We validated the function of hiPS-CAR-T cells <italic>in vivo</italic> and <italic>in vitro</italic> experimentation which revealed no significant differences in cell morphology and function between hiPSC-derived hiPS-CAR-T cells and peripheral blood-derived CAR-T cells.</p><p><bold>Conclusion:</bold> This study developed a culture method that is efficient and clinically useful to make functional CD8<sup>+</sup> T cells from hiPSC and to get hiPS-CAR-T cells with high antigen-specific cytotoxicity that are not very different from CAR T cells found in peripheral blood. As a result, our findings may open the way for the clinical use of hiPSC to create functional CD8<sup>+</sup> T and hiPS-CAR-T cells cells for use in cell-based cancer therapy.</p>