Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in

Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in both normal and injured adult mammalian spinal cord. 2006; Vessal et al., 2007) and normal conditions (Shechter et al., 2007, 2010). However, as we gain more insight into the presence and implications of neurogenesis in the adult spinal cord, the question remains as to how these adult spinal cord NSPCs are regulated. A better understanding of the basic biology of these NSPCs will facilitate future attempts to manipulate these cells under pathological conditions. Unlike in the adult spinal cord, the event of neurogenesis in the adult hippocampus has been strongly established (Alvarez-Buylla and Garcia-Verdugo, 2002; Ming and Song, 2005, 2011). Astrocytes from this Lopinavir brain region have been shown to induce neurogenesis of adult hippocampal NSPCs via the Wnt signaling pathway (Track et al., 2002; Lay et al., 2005). Furthermore, diffusible factors from the neurovascular niche are reported to stimulate neurogenesis in the adult subventricular zone (Palmer et al., 2000; Shen et al., 2004, 2008). On the other hand, astrocytes from the adult spinal cord do not promote neurogenesis in culture (Track et al., 2002). Growth factors, such as fibroblast growth factor 2 (FGF2), epidermal growth factor, nerve growth factor, and vascular endothelial growth factor, can Lopinavir elicit a range of cellular responses including cell proliferation, migration, differentiation, and cell death through various classes of receptor tyrosine kinases (RTKs) (Hubbard and Till, 2000). The activation of these different RTKs in Lopinavir turn induces the activation of several signal transduction pathways including the (DIV) when compared to 0 DIV (Physique ?(Physique1C).1C). A thymidine analogue, EdU, was added to the culture to pulse-label those cells undergoing cell division at 4 DIV, 6 h prior to fixation (Physique 1D,i). During the 6-h pulse of EdU-labeling, 25% of the cells were EdU+ (Physique 1D,ii). In addition, co-labeling of EdU and nestin, used here as an adult NSPC marker (Lendahl et al., 1990; Johansson et al., 1999; Namiki and Tator, 1999; Fu et al., 2005), revealed that 32 5% (= 4) of nestin+ cells were EdU+ and that 46 12% (= 4) of EdU+ cells were nestin+ (Physique ?(Figure1E).1E). Immunostaining with nestin at 0, 1, and 4 DIV showed an increase of nestin+ cells over time; with a 12-fold increase in percentage by 4 DIV (Physique ?(Figure1F).1F). Cells were also labeled Lopinavir with another progenitor marker, neural-glial antigen 2 (NG2), as studies have shown that NG2+ cells are proliferative and can give rise to neurons (Belachew et al., 2003; Tamura et al., 2007; Guo et al., 2010). At 1 DIV 28% of the cells were immunopositive for NG2 (Physique 1G,i,ii) and 19% of the nestin+ cells were also NG2+ at 1 DIV (Physique 1G,ii). NG2 and nestin coexpressing cells were also detected at 3 DIV (Physique 1G,iii). The cells that were NG2+ were stellate-like, with short processes Rabbit polyclonal to DDX3 extending from all directions; and the cells that were only nestin+ were mostly rounded/spindle-shaped. The morphology of the cells co-labeled with nestin and NG2 was comparable to that of the nestin+ cells (Physique 1G,iii). Based on the cell morphology, some of the EdU+ cells that were not immunopositive for nestin were likely NG2+ (not shown). Stage-specific embryonic antigen 1 (SSEA-1), a marker for undifferentiated NSPCs (Capela and Temple, 2002; Sabourin et al., 2009), was observed in about one-tenth of the cells in culture at 3 DIV (Physique ?(Physique1H).1H). Taken together, the manifestation of these markers demonstrates the presence of uncommitted NSPCs at 1C4 DIV. Physique 1 Enrichment of adult rat spinal cord neural stem/progenitor cells (NSPCs) in culture. (A) Dissociated adult spinal cord cells, 1 h after isolation. (W) Spinal cord cells after 4 days in FGF2 (i) or in basal medium (ii). (C) Adult spinal cord cells proliferated … Neuronal differentiation in adult spinal cord NSPC cultures Maintaining the expanded populace of adult spinal cord NSPCs in medium made up of FGF2 eventually resulted in neuronal differentiation. At 0 DIV and 1 DIV, about 23 and.