Neurogenic Potential of Spinal Cord Organotypic Culture

There are several neurogenic niches in the adult mammalian central nervous system. In the central nervous system, neural stem cells (NSC) localize not only to the periventricular area, but are also diffusely distributed in the parenchyma. Here, we assessed neurogenic potential of organotypic cultures prepared from adult mouse spinal cord. Slices were placed on Millipore inserts for organotypic culture and incubated in neurobasal media supplemented with B27 and N2 for up to 9 weeks. After 3-4 weeks, the cell's aggregates formed in the slices. The aggregate's cells were BrdU-uptake, nestin and alkaline phosphatase positive. At the later stage of incubation, we observed Oct3/4 in the inner mass of the neurospheres as well as expression of Dppa1, which is an Oct-4 downstream target gene and a marker for pluripotency. To check differentiation, the formed neurospheres were isolated and cultured for several days in differentiation media. The obtained data demonstrated the cells from isolated neurospheres differentiate into astrocytes and MAP2-positive neurons. Immunostaining for HB9 and Lim2 revealed subsequent differentiation of MAP2-positive cells into motor neurons and interneurons, respectively. We hypothesized neuronal loss and/or long-term culturing of spinal cord slices may trigger a reset of the internal cell program and promote proliferation and further differentiation of NSC.