The tetracycline regulatory system has been widely used to control the transgene expression. With this powerful tool, it might be possible to effectively control the functional activity of chimeric antigen receptor (CAR) T cells and manage the severe side effects after infusion. In this study, we developed novel inducible CD19CAR (iCAR19) T cells by incorporating a one-vector Tet-on system into the CD19CAR construct. The iCAR19 T cells showed dox-dependent cell proliferation, cytokine production, CAR expression, and strong CD19-specific cytotoxicity. After 48 h of dox induction, the relative CAR expression of induced cells was five times greater than that of uninduced cells. Twenty-four hours after dox removal, CAR expression significantly decreased by more than 60%. In cytotoxicity assays, dox-treated cells induced significantly higher specific lysis against target cells. These results suggested that the activity of iCAR19 T cells was successfully controlled by our Tet-on system, offering an enhanced safety profile while maintaining a robust anti-tumor effect. Besides, all manufacture processes of the lentiviral vectors and the T cells were conducted according to the Good Manufacturing Practice (GMP) standards for subsequent clinical translation.

Background: Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods.Results: We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration.Conclusions: We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also forin vitroapplications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

Low-frequency magnetic fields exert a significant inhibitory effect on tumor growth and have been developed as a therapeutic modality. However, the effect of a low-frequency magnetic field on the interaction between cells is still poorly understood. This study aimed to preliminarily evaluate the direct effect of magnetic field ditectely on cultured cells and indirect effect mediated by cell-environment (conditioned medium). 293 T cells, Hepg2 cells, A549 cells have been cultured at 37 ± 0.18 °C in presence of an extremely low-frequency magnetic field of 20 Hz, 5-mT. The adherent tumor cells were more sensitive to magnetic field inhibition in the original environment (conditioned medium) with adherence inhibition rate for Hepg2 and A549 estimated at 18% and 30% respectively. The inhibition effect was suppressed when the suspended cells separated or clump density at a low density. The nontumor cell lines showed no inhibitory effect on exposure to a low-frequency magnetic field. The intracellular ion fluorescence (IIF) showed that the magnetic field significantly altered the membrane potential, indicating hyperpolarization of the adherent cells (ΔIIF 293 T cells: − 25%, ΔIIF Hepg2 cells: − 20% and ΔIIF A549 cells: − 13%) and depolarization of the suspended cells (ΔIIF Raji cells: + 9%). In addition, the conditioned media collected after magnetic field exposure acted on unexposed tumor cells and caused inhibition. Our findings might provide a basis for the mechanism of magnetic field interaction between cells and cell environment in the future.

Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

Extracellular vesicles (EVs) play a critical role in intercellular communication and have shown great potential in disease treatment and tissue repair. However, large-scale production and clinical application of EVs still face challenges such as donor heterogeneity, batch-to-batch variability, and high production costs. This study aimed to establish a stable and reproducible EV production platform using hTERT-immortalized placental mesenchymal stromal cells (PC-MSCs). We demonstrated that stable expression of the TERT gene promotes cell proliferation in vitro while retaining surface marker expression patterns and differentiation capabilities similar to those of primary cells. The EVs produced by immortalized cells maintain similar particle size distributions, protein markers, and protein expression profiles to those of their parental cells, and their therapeutic efficacy was validated in a bleomycin (BLM)-induced pulmonary fibrosis (PF) rat model. In summary, we systematically described the effect of immortalization via ectopic hTERT gene expression on EVs produced from PC-MSCs. These findings provide a new theoretical basis for the sustainable production and scale-up of EVs, as well as a new solution for the treatment of PF.