IMAGING REDOX STATE HETEROGENEITY WITHIN INDIVIDUAL EMBRYONIC STEM CELL COLONIES

Abstract

Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. NADH redox ratio (NADH/(Fp + NADH)), where NADH is the reduced form of nicotinamide adenine dinucleotide and Fp is the oxidized flavoproteins, has been established as a sensitive indicator of mitochondrial redox state. In this paper, we report our redox imaging data on the mitochondrial redox state of mouse ESC (mESC) colonies and the implications thereof. The low-temperature NADH/Fp redox scanner was employed to image mESC colonies grown on a feeder layer of gamma-irradiated mouse embryonic fibroblasts (MEFs) on glass cover slips. The result showed significant heterogeneity in the mitochondrial redox state within individual mESC colonies (size: ∼200-440 μm), exhibiting a core with a more reduced state than the periphery. This more reduced state positively correlates with the expression pattern of Oct4, a well-established marker of pluripotency. Our observation is the first to show the heterogeneity in the mitochondrial redox state within a mESC colony, suggesting that mitochondrial redox state should be further investigated as a potential new biomarker for the stemness of embryonic stem cells.

Keywords: NADH; Redox imaging; flavoproteins; pluripotency; redox ratio.

Fig. 1.

The first and second rows show typical pseudo-color redox images of mouse embryonic stem cell colonies grown on a cover slip, the second row being the enlarged view of the redox images from one of the colonies. The x and y axes represent spatial coordinates in terms of pixels (40 μm). The color bars of the Fp and NADH images indicate the nominal concentrations in μM relative to the corresponding reference standards, and the color bar of NADH redox ratio (NADH/(Fp + NADH)) image indicates the range of the redox ratio. The bottom row delineates the corresponding histograms of the images of the individual colony shown in the middle row, where the x axes represent the NADH redox ratio or nominal concentration. The y axes represent the number of pixels in the colony having a specific value of NADH redox ratio or nominal concentration.

Fig. 2.

An mESC colony immunostained for Oct4 and co-stained with DAPI. From left to right: Oct4 image, pseudo-color Oct4 image, pseudo-color DAPI image, and pseudo-color ratio image of Oct4 to DAPI with the pixel intensity shown by the color bars in arbitrary units. The x and y axes represent spatial coordinates.