Novel Functional Changes during Podocyte Differentiation: Increase of Oxidative Resistance and H-Ferritin Expression
Figure 2
Podocyte differentiation under nonpermissive (37°C) condition. At 50–60% confluence, nondifferentiated cells were placed from 33°C to 37°C to induce differentiation which was followed by video microscopy for 14 days. (a) On selected time-lapse phase contrast images change in the shape and size of podocytes is indicated by broken outlines. Black arrows show shorter and rounded projections of differentiating podocytes. White arrows indicate longer and spindle like projections of arborizing podocytes. Mitotic cells, marked with red double sharps, become detached from the surface of the vessel for a while before the daughter cells adhere again to the bottom. Red asterisks denote apoptotic cells. (b) Modified Giemsa staining was performed on parallel cell cultures at different time points. Static visualization of the cell culture supports the findings of dynamic assessment by time-lapse monitoring. Cytoplasmic staining (pink), nuclear staining (blue). Magnification 10x. (c) Light microscopy and immunofluorescent microscopy images of cell cultures demonstrate characteristic features of human podocytes. Left panels represent nondifferentiated podocytes grown under permissive condition; right panels represent differentiated podocytes after 10 days of culturing under nonpermissive condition. (i) Light microscopy images, scale bars: 300 μm, each. Nondifferentiated cells are small in size and show a cobble-stone like morphology, whilst differentiated cells have enlarged cell bodies with large nucleus, bulky cytoplasm, and ramifying processes. (ii) Synaptopodin-Cy3 (red) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Synaptopodin expression is present only in differentiated cells and shows filamentous staining. (iii) F-actin (phalloidin-FITC, green) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Images are representative of three experiments with similar results.
