Induction of stemness gene by chemical stimulus in human PBMNCs

Abstract

Background Discovery of Induced Pluripotent Stem cells (iPS) has opened new possibilities of patient-specific disease treatment and figured out molecular state of development. However, there has been various limitations to the use of clinical application due to the potential risks resulting from genetic modification and the difficulties in obtaining suitable primary cells. For overcome these problems, researchers try to find safe alternative methods to generating iPS without genome insertion and transgene. and non-invasive methods to separate primary cells. Although the validity of simple conversion of somatic cells into iPS using chemical stimulus only is being debated until now, trials and studies to find ideal chemicals to induce reprogramming were still being continued. To assess feasibility of simple and safety method, we have treated low-pH (acidic) chemicals which can be external stimuli in mononuclear cell isolated from human peripheral blood. We have investigated the expression of OCT3/4 gene as embryonic stem cell marker which determined pluripotency of cell to evaluate dedifferentiation effect of low-pH chemicals on PBMC.

Methods and results A peripheral blood mononuclear cells (PBMNCs) were isolated from human whole blood using ficoll density gradient method. Isolated PBMNCs were immersed in 500μl mild acidic solution (pH5.2) and nitroglycerin solution for 30 minutes. Then, the stimulated PBMNCs were centrifuged for washing out remaining solution and plated on non-adhesive culture plate with basic FGF (10 ng/ml) daily for 10 days. After 10 days incubation, the PBMNCs were harvested and the expression of stem cell markers was analysed using Reverse transcription polymerase chain reaction (RT-PCR), Immunostaining analysis, and FACS. Various pH conditions and cell number were tested. The pH 5.2 and 2.5x106 cells/ml were optimal conditions for Oct3/4 expression. Conclusion Nitroglycerin stimulus can induce expression of stem cell marker OCT3/4, which is a crucial transcription factor for reprogramming in PBMNCss. This suggest that nitroglycerin could be one of ideal chemicals for enhancing reprogramming effects.