Loss of pluripotency in human embryonic stem cells directly correlates with an increase in nuclear zinc

Abstract

The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.

Figure 1. Spontaneous differentiation correlates with increased nuclear zinc content at hESC colony borders.

A. DIC (10x) image of hESC colony showing a change in morphology at colony border. B. Oct4 labeling lost at colony border but maintained in center of colony indicates that pluripotency is maintained centrally. C. Graph depicting an inverse relationship between quantified Oct4 signal and zinc content from XFM analysis across the colony. Results are plotted as mean ± SEM.

Figure 2. RA induced differentiation correlates with increased nuclear zinc content at the center of hESC colonies.

A. Oct4 (pink) lost at center of hESC colonies treated with RA. Cells are counterstained with Hoechst (blue). B. Graphs depict quantified Oct4 intensity vs. total zinc content from XRF of control (i) and RA treated (ii) hESCs across a typical colony. Results are plotted as mean ± SEM.

Figure 3. Activin A induced differentiated hESCs have increased zinc content.

A. Control (i) and Activin A treated (ii) hESCs stained with Oct 4 (pink) and counterstained with Hoechst (blue). B. Sox17 expression (yellow) in Activin A treated cells. Cells are counterstained with Hoechst (blue). C. Graph depicting zinc content of Activin A treated cells vs. control. Results are graphed as mean ± SEM.

Figure 4. FluoZin-3 staining is unchanged with induction of differentiation.

FluoZin-3 is green, Hoechst 33342 is blue. A. Control H9 cells. B. RA treated cells. C. Activin A treated cells. All images are representative of roughly 20 fields of view from three experimental replicates.

Figure 5. Zinc excluded from condensed chromosomes of mitotic cell.

A. Raster scans mapping Kα line fluorescence of phosphorus (red) and zinc (green) of an interphase (i) and mitotic (ii, iii) cells. B. Overlay demonstrating overlap of zinc and phosphorus in the interphase cell and mutual exclusion in the anaphase cells. The first row of images was obtained with x-ray beam spot size of 0.4×0.3 um with step sizes of 0.2 um. Second row was obtained with 0.6×0.8 um spot size and a 0.5 um step size.