Expression of SSEA4 and TRA1-60 as Marker of Induced Pluripotent Stem Cells by Small Molecule Compound VC6TFZ on Peripheral Blood Mononuclear Cell

Abstract

IntroductionIt is possible to induce pluripotent stem cells from somatic cells, offering an infinite cell resource with the potential for disease research and use in regenerative medicine. Due to ease of accessibility, minimum invasive treatment, and can be kept frozen, peripheral blood mononuclear cells (PBMC) were an attractive source cell. VC6TFZ, a small molecule compound, has been successfully reprogrammed from mouse fibroblast induced pluripotent stem cells (iPSCs). However, it has not been confirmed in humans. ObjectiveThe aim of this research is to determine whether the small molecule compound VC6TFZ can induced pluripotency of PBMC to generate iPSCs detected with expression of SSEA4 and TRA1-60. MethodsUsing the centrifugation gradient density process, mononuclear cells were separated from peripheral venous blood. Mononuclear cells were cultured for 6 days in the expansion medium. The cells were divided into four groups; group 1 (P1), which was not exposed to small molecules (control group) and groups 2-4 (P2-P4), the experimental groups, subjected to various dosages of the small molecule compound VC6TFZ (VPA, CHIR, Tranylcypromine, FSK, Dznep, and TTNPB). The induction of pluripotency using small molecule compound VC6TFZ was completed within 14 days, then for 7 days the medium shifted to 2i medium. iPSCs identification in based on colony morphology and pluripotent gene expression, SSEA4 and TRA1-60 marker, using immunocytochemistry. ResultsColonies appeared on reprogramming process in day 7th. These colonies had round, large, and cobble stone morphology like ESC. Gene expression of SSEA4 and TRA 1-60 increased statisticaly significant than control group (SSEA4 were P2 p=0.007; P3 p=0.001; P4 p=0.009 and TRA 1-60 were P2 p=0.002; P3 p=0.001; P4 p=0.001). ConclusionSmall molecule compound VC6TFZ could induced pluripotency of human PBMC to generate iPSCs. Pluripotxency marker gene expression, SSEA 4 and TRA 1-60, in the experimental group was statistically significantly higher than in the control group.