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

Background: Chimeric antigen receptor (CAR) T cell treatment involves in vitro 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. Methods: The Alkaline phosphatase assay and MycoEasy rapid mycoplasma detection kit was implemented for detection of hiPSCs and mycoplasma, respectively. The CD34+ HSPCs were harvested in AggreWellTM 400 using a 37-micron reversible strainer. Likewise, the lymphoid progenitor and CD4+CD8+ 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 in vivo antitumor effects of the CAR-T cells were evaluated using experimental animals (mice). Results: 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 in vivo and in vitro 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. Conclusion: This study developed a culture method that is efficient and clinically useful to make functional CD8+ 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+ T and hiPS-CAR-T cells cells for use in cell-based cancer therapy.

Generation of a NIS-EGFP-Fluc triple-reporter human embryonic stem cell line by PiggyBac transposon system.

One major challenge in stem cell therapy is to longitudinally track cell fate after cells transplantation. Molecular Imaging approaches enabling noninvasive long-term monitoring the transplanted cells are imperative for assessment of the safety and efficiency. Here, we used PiggyBac technology to insert triple reporter genes: NIS, EGFP and Firefly luciferase into a human embryonic stem cell line (hESCs, H9) and obtained a reporter hESCs line (NIS-EGFP-Fluc H9). The triple-reporters allows the transplanted NIS-EGFP-Fluc H9 cells and their derivates to be fluorescence, bioluminescence and even PET/SPECT imaged. This triple-reporter hESCs line provides a valuable imaging platform for cell-based therapeutics clinical translation.

Evaluation of [18F]tetrafluoroborate as a Potential PET Imaging Agent in a Sodium Iodide Symporter-Transfected Cell Line A549 and Endogenous NIS-Expressing Cell Lines MKN45 and K1.

[18F]tetrafluoroborate (TFB) has been introduced as the 18F-labeled PET imaging probe for the human sodium iodide symporter (NIS). Noninvasive NIS imaging using [18F]TFB has received much interest in recent years for evaluating various NIS-expressing tumors. Cancers are a global concern with enormous implications; therefore, improving diagnostic methods for accurate detection of cancer is extremely important. Our aim was to investigate the PET imaging capabilities of [18F]TFB in NIS-transfected lung cell line A549 and endogenous NIS-expressing tumor cells, such as thyroid cancer K1 and gastric cancer MKN45, and broaden its application in the medical field. Western blot and flow cytometry were used to assess the NIS expression level. Radioactivity counts of [18F]TFB, in vitro, in the three tumor cells were substantially higher than those in the KI inhibition group in the uptake experiment. In vivo PET imaging clearly delineated the three tumors based on the specific accumulation of [18F]TFB in a mouse model. Ex vivo biodistribution investigation showed high [18F]TFB absorption in the tumor location, which was consistent with the PET imaging results. These results support the use of NIS-transfected lung cell line A549 and NIS-expressing tumor cells MKN45 and K1, to investigate probing capabilities of [18F]TFB. We also demonstrate, for the first time, the feasibility of [18F]TFB in diagnosing stomach cancer. In conclusion, this study illustrates the promising future of [18F]TFB for tumor diagnosis and NIS reporter imaging.

Synthesis and evaluation of Al18F-NODA complex conjugated 2-(4-aminophenyl)benzothiazole as a potential tumor imaging agent.

The objective of the study was to prepare and evaluate a 18F-radiolabled tracer (Al18F-5), derivated from the antitumor agent 2-(4-aminophenyl)benzothiazole, as a PET probe for tumor imaging. Al18F-5 was successfully prepared with approx. 40% radiochemical yield in aqueous phase. In in vitro cell uptake experiments and competition assay, Al18F-5 displayed good tumor-binding ability and specificity in HeLa cells (24.7 ± 0.9% ID/106 cells, IC50 = 63.8 ± 13.6 nM) and MCF-7 cells (6.8 ± 0.3% ID/106 cells, IC50 = 331.1 ± 33.7 nM). The nonradioactive compound, Al19F-5, visibly marked HeLa cells and MCF-7 cells but did not stain HEB cells in florescent staining, which further indicated the tumor-binding ability of Al18F-5. In in vivo PET imaging, HeLa and MCF-7 tumors were clearly delineated by specific accumulation of Al18F-5 in model mice. In biodistribution study, Al18F-5 exhibited good tumor uptake (4.66 ± 0.13% ID/g and 3.69 ± 0.56% ID/g, respectively), moderate tumor-to-muscle ratio (3.38 and 2.48, respectively) at 1 h post injection, which were in a good consistency with the results of PET imaging. In conclusion, Al18F-5 might be developed as a candidate PET probe for tumor imaging, though additional optimizations are still needed to improve pharmacokinetics in vivo.