Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone

Abstract

We developed an optimized Dipheylthiocarbazone or Dithizone (DTZ) with improved physical and chemical properties to characterize human islets and insulin-producing cells differentiated from embryonic stem cells. Application of the newly formulated iDTZ (i stands for islet) over a range of temperatures, time intervals and cell and tissue types found it to be robust for identifying these cells. Through high transition zinc binding, the iDTZ compound concentrated in insulin-producing cells and proved effective at delineating zinc levels in vitro.

Figure 1

Solutions of iDTZ show improved stability under various temperatures and lengths of time. Solutions were prepared as described using standard DTZ (A) and iDTZ (B) and exposed to a range of temperatures and storage intervals (−80°, −20°, 4 °C, and 22 °C and 1, 7, and 30 days).

Figure 2

Solutions of iDTZ show improved islet staining under various conditions employing an automated quantification system. Human islets stained with iDTZ or standard DTZ solution visualized at the indicate time points (a). Human islets stained with fresh iDTZ solution and islets stained with iDTZ solution stored for 6 months at −20 °C (b) and visualized 48 hours post-staining. Human islet cryo-sections were stained with iDTZ solution (c). Representative pictures from three experiments are shown. All the pictures were obtained using an Olympus microscope.

Figure 3

Human islets and insulin-producing cells differentiated from embryonic stem cells are rapidly identified by iDTZ. Stage 6 and 7 cell clusters were stained positively by iDTZ as compared to other stages (1–5) (a). Human islets stained with iDTZ (b). Immuno-fluorescent staining of stage 7 differentiated human embryonic stem cells showing insulin (red), and DAPI staining of the nucleus (blue), (c). The scale bar represents 50 μm.

Figure 4

Zinc levels can be measured using iDTZ. There was a highly significant correlation between zinc concentration and the absorbance readings at 570 nm (R² = 0.9985, p < 0.0001).