Characterization of Porcine Ventral Mesencephalic Precursor Cells following Long-Term Propagation in 3D Culture
Figure 6
Differentiation of long-term propagated embryonic porcine neural tissue spheres (NTS). Differentiation of VM NTS (51 and 170 days of age) induced by mitogen withdrawal did after 25 days induce a high number of β Tubulin III positive cells in the central core of the differentiated NTS (a), but no TH-positive cells were identified. Instead, the differentiated cells primarily expressed the astroglial marker GFAP and displayed a characteristic astroglial morphology. To verify the differentiation potential of the protocols used, 51-day-old NTS cultures derived from the embryonic porcine subventricular zone (SVZ) were included in the differentiation study as a positive control. Differentiation of SVZ NTS for 25 days by mitogen withdrawal (b) or by addition of the TH-inducing factors a-FGF, forskolin, TPA, and db-cAMP (c), known to stimulate TH expression in mammalian forebrain cultures, both resulted in β Tubulin III- and TH-positive cells. The β Tubulin III-positive cells had a characteristic neuronal morphology and were located in clusters/clones in the zone of migrating cells and in the central core of the differentiated SVZ NTS. The TH-positive cells were more numerous and appeared more morphologically immature by having shorter processes (c) compared to the cultures differentiated under mitogen-free conditions (b). Arrows mark soma of TH-positive cells. Scale bars = 200 μm and 100 μm in microphotographs of β Tubulin III expression at low and high magnification, respectively. 100 μm in microphotographs of GFAP and TH expression.
