Abstract
Abstract: :
Purpose: To characterize the size, morphology, and growth properties of neurospheres from progenitor cells residing in adult mouse, rat, and human ciliary body. Methods: Cells from the ciliary bodies of FVB.Cg–Tg(GFPU)5Nagy mice, Wistar rats, and human cadaver eyes were isolated and cultured in media containing EGF, FGF2, and heparin (30 mice; 12 rats; 2 human eyes). Some cells proliferated to form groups of cells (neurospheres, NS). The size of primary mouse, rat, and human NS was measured for 7–9 days then NS were dissociated and recultured for another 7–9 days. Mouse, rat, and human cells were seeded at dilutions from 100 to 4000 cells/well and cultured for 5 days, after which NS in each well were counted. Primary NS between 5–7 days of growth were harvested, mounted, and the size of individual cells was measured. Number of cells/NS was averaged from the dissociation of 10 NS. Whole and dissociated mouse and rat spheres were induced to differentiate by the withdrawal of EGF and the addition of fetal calf serum in the media. Results: NS formation was evident after 2 days in culture. Isolated mouse, rat, and human ciliary cells had an average cell diameter of 25 micrometers, and this size was similar among daughter cells in NS of mouse and rat. Dilution experiments showed that approximately one in 323 mouse, 220 rat, and 204 human ciliary cells developed into a NS. For rat and human ciliary body, one cell in 200 developed into a NS. After one week in culture, the mean NS size was 162 ± 14 (mouse), 56 ± 9 (rat), and 144 ± 8 µm (human). NS growth was rapid during the first 5 days after seeding. The cells/NS after 1 week in culture averaged 1183 (mouse, n =3), 5360 (rat, n=1), and 685 (human, n=2) cells. After dissociation and reculturing, secondary NS developed similarly, but by the third passage, NS growth waned. Withdrawal of EGF from the media resulted in NS differentiation as seen by changes in cell morphology. Conclusions: Among adult ciliary body cells in rodents and humans, 0.1–1% retain proliferative capacity with specific stimulation and produce thousands of daughter cells for up to 3 cycles of NS development and dissociation.