Figure 5. Ependymal cell-driven neurogenesis in the central canal zone after neural stem cell transplantation as demonstrated with confocal microscopy and comparison of doublecortin-positive neuron counts in area X after live or dead neural stem cell transplantation.

(A & B) Some migrating ependyma-derived cells (red, DiI positive) differentiate into cells with neuronal phenotypes identified by labeling with the migratory neuronal precursor marker DCX (green) in area X (A, arrow) and outside of area X (B, arrow). Note the colocalization of green and red in the cytoplasm (resulting in a yellow composite color). The central canal is indicated by an asterisk in (A). (C) This confocal picture shows that, in the central canal, a neural stem cell (NSC)-derived cell (HNu positive) that had migrated from the transplantation site is DCX positive (arrowhead). A few host cells (HNu negative) are also DCX positive (arrows), a pattern revealing their phenotype as migrating neuronal precursors. (D) Retrograde trans-synaptic tracer pseudorabies virus (PRV) staining in spinal area X of nude rats shows that some NSC-derived cells (HNu positive) are PRV positive (arrowhead). Some ependymal cells are also PRV immunoreactive (arrows). PRV particles can be transferred either directly from primarily labeled motor neurons or indirectly from PRV-positive nerve cells innervating motor neurons, a reasoning that leads to the conclusion that PRV-positive cells in area X are mature neurons. (E) Comparison by Student’s t-test of DCX-positive host cell counts in area X shows that SOD1 G93A rats with live NSC grafts (‘SOD1 with grafts’) or nude rats with live NSC grafts (‘Nude with grafts’) have significantly higher numbers of DCX-positive cells compared with SOD1 G93A rats with dead NSC grafts (‘SOD1 control’) or nude rats with dead NSC grafts (‘Nude control’).

Scale bars: (A) 10 μm; (B) 10 μm; (C & D) 25 μm.

DAPI: 4’,6-diamidino-2-phenylindole; DCX: Doublecortin; HNu: Human nuclear protein antibody; SOD1: Superoxide dismutase 1.