Oct4Promoter Activity in Stem Cells Obtained through Somatic Reprogramming

Abstract

Multiple methods exist that can reprogram differentiated cells to a pluripotent state similar to that of embryonic stem cells (ESCs). These include somatic cell nuclear transfer (SCNT), fusion-mediated reprogramming (FMR) of somatic cells with ESCs, and the production of induced pluripotent stem cells (iPSCs). All of these methods yield cells in which the endogenous Oct4 gene is reactivated. We were interested in comparing the activity of the Oct4 promoter in three different classes of pluripotent cells, including normal ESCs, FMR cells (FMRCs), and iPSCs. We prepared cells of all three types that harbor a transgene composed of the mouse Oct4 promoter driving green fluorescent protein (Oct4-GFP). All cell derivations started with a characterized transgenic Oct4-GFP mouse, and from this we derived ESCs, FMRCs, and iPSCs with the Oct4-GFP transgene present in an identical genomic integration site in all three cell types. Using flow cytometry we assessed Oct4 promoter expression, cell cycle behavior, and differentiation kinetics. We found similar levels of GFP expression in all three cell types and no significant alterations in pluripotency or differentiation. Our results suggest that the pluripotent condition is a potent "local attractor" state, because it can be achieved through three vastly different avenues.

Identifying the Target Traumatic Brain Injury Population for Hyperbaric Oxygen Therapy

“…The cells are then fixed and permeabilized in fix/perm buffer. For staining in microtiter plates, the plate is spun for 3 min at 100× g at 8 • C. Then, the plate is processed to resuspend the pellet immediately in 100 µL of primary antibodies for OCT4, Nanog, and SSEA markers diluted with the staining buffer [128][129][130]. The plate is then incubated for 30 min at 4 • C with the primary antibodies.…”

Construction of a Dual‐Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation

“…Upon differentiation of PSCs, Oct4 expression was gradually reduced and finally silenced along with epigenetic modifications [6]. The silenced Oct4 in differentiated somatic cells can be reactivated by several reprogramming processes such as fusion-induced reprogramming, somatic cell nuclear transfer (SCNT), or generation of induced pluripotent stem cells (iPSCs) [7, 8], suggesting the importance of Oct4 in maintenance and self-renewal of pluripotent cells. An Oct4 reporter system, constructed by integrating the Oct4 promoter into GFP, can be used as an efficient marker to mimic the endogenous Oct4 gene expression in mouse [9].…”