We then differentiated the hippocampal NPCs into DG granule neurons by using Wnt3a and BDNF, two soluble factors that have been shown to be essential for the generation and maturation of DG granule neurons. Our results showed that whereas the NPCs were able to give rise to neurons as indicated by transiently increased levels of NEUROD1 and DCX as well as a steadily increased level of MAP2AB, PROX1 levels were significantly higher in cultures treated with Wnt3a and BDNF (Figure 2E). Furthermore, immunostaining analysis with vGLUT, a glutamatergic neuronal marker, and GABA, a GABAergic neuronal marker, showed that our protocol produced predominantly glutamatergic neurons (>85%) (Figures 2G and 2G′), with low occurrence of GABAergic neurons (<15%) (Figure 2F). Quantitative analysis for colabeling of PROX1 and MAP2AB revealed a significantly higher number of PROX1+ neurons in Wnt3a/BDNF-treated cultures (∼70%) compared to the control cultures (∼20%) (Figure 2D).
