Rejuvenation of human mesenchymal stem cells using a nonintegrative and conditionally removable Sendai virus vector.

Human mesenchymal stem cells (hMSCs) with extended lifespan and differentiation potential that can recapitulate in vivo characteristics could significantly contribute to basic research, drug development, and cell therapy. Specifically, they could ensure a stable supply of specific cellular resources, and possibly extracellular vesicles. Here, we established a technology for extending the lifespan while maintaining differentiation potential, termed "rejuvenation," of hMSCs (rej-hMSCs) using nonintegrative and conditionally removable temperature-sensitive Sendai virus (SeV) vectors. Various immortalizing factors (i.e., Bmi-1, hTERT, SV40T, and/or HPV E6/E7) were first introduced by the SeV vector into the cells. A combination of three SeVs with Bmi-1, hTERT, or SV40T conferred markedly improved cell proliferation and cloning ability while maintaining differentiation potential and a normal karyotype. An extended lifespan was also demonstrated in other cell types. The rejuvenation of long-passaged or aged hMSCs was also confirmed. SeV vectors were rapidly removed as a function of cell doubling by increasing the temperature from 35 °C to 37 °C or higher, while proliferative ability was maintained. Following FACS sorting, the complete removal of SeV vectors was confirmed by qPCR analyses. Therefore, our cell rejuvenation technology could contribute to research and clinical applications by enabling the supply of modified cells without damaging host chromosomes.

Developing a workflow for the isolation of hybridoma cells producing fully human antigen-specific antibodies using a surface IgG detection method.

The antigen-mediated B cell isolation method, based on the detection of surface IgG (sIgG), has increased the efficiency of therapeutic antibody (Ab) discovery. However, the reduction in sIgG expression on B cells during plasma cell differentiation presents challenges as it enables Ab production from only a small subset of B cells (e.g., memory B cells). The present study aimed to addressed this problem by developing a workflow to isolate human-IgG-secreting hybridoma cells produced by cell fusion, the majority of which express sIgG. We showed that our sIgG-based antigen-coated bead separation method efficiently enriched hybridoma cells expressing antigen-specific Abs with a yield of 83.5% (from the cell fusion pool) and a positive rate of 73.2%. Furthermore, because the separation could be performed after only a short (1-2-day) culture period following cell fusion, diverse hybridoma clones could be obtained, minimizing clonal selection and the incidence of duplicates. Given that the expression of membrane-bound IgG and sIgG are regulated by different splicing mechanisms, we speculate that the cell fusion step potentially attenuated the suppression of human sIgG expression. Overall, our proposed method is expected to markedly improve the efficiency of therapeutic Ab candidate production, which will have important clinical implications.