File:Novel-Functional-Changes-during-Podocyte-Differentiation-Increase-of-Oxidative-Resistance-and-H-976394.f1.ogv

English: Method: Time-Lapse Videomicroscopy. Long-term time-lapse microscopy analysis was performed on the culture under nonpermissive condition from day 0 to day 14 to follow the process of differentiation. An inverse microscope, placed in a CO2 incubator, was equipped with high-sensitivity digital cameras and connected to an image-acquisition computer system. Frames were recorded every minute and the whole video sequence was converted to database form. The time of exposure was indicated in the right lower corner of each frame. Exposures were converted to video films by speeding up the projection to 30 exposures/seconds. Results: Podocyte Growth and Differentiation under Nonpermissive (37°C) Condition. We placed nondifferentiated human podocytes expressing the thermosensitive A58 T antigen at 50–60% confluency from 33°C to 37°C to induce differentiation. We followed changes in cell number and morphology by video microscopy for 14 days. we observed that the initial exponential growth phase lasted for 9 days. After cell number reached its peak between days 9 and 10,we saw a slight decrease in cell number followed by stabilization of the culture (days 10–14) when we could hardly detect any mitotic event.When the temperature was shifted to 37°C the morphology of nondifferentiated podocytes gradually transformed into that of differentiated cells. At permissive temperature (33°C) nondifferentiated podocytes showed typical epithelial cobblestone morphology. In contrast, differentiated podocytes are characterized by enlarged cell bodies with an irregular shape and the formation of processes involving shorter and rounded as well as longer and spindle-like projections. Formations of spindle-like projections were often preceded by partial retraction of cytoplasmatic protrusions. Many differentiated cells developed cellular hypertrophy, some of the cells died, and others started to arborize. We detected numerous binucleated but nonproliferating cells. After day 9 we observed a reduction of cell number, but themotility of cells remained high even after differentiation, and the culture proved capability of migration until all bare fields of the culturing vessel were completely covered.