PGK-GFP Lentivirus

Using a lentiviral-mediated labeling approach, researchers investigated whether the adult hippocampus retains persistent self-renewing neural stem cells (NSCs). A single injection of a lentiviral vector expressing green fluorescent protein (LV PGK-GFP) into the subgranular zone (SGZ) of the adult hippocampus allowed for efficient, stable, and long-term labeling of self-renewing NSCs and their progeny. Interestingly, a subset of labeled cells showed the ability to proliferate multiple times and give rise to Sox2+ cells, clearly demonstrating that NSCs have the capacity to self-renew over a long period of time (up to 6 months). Furthermore, using GFP+ cells isolated from the SGZ of mice that had received LV PGK-GFP injections 3 months earlier, researchers demonstrated that some GFP+ cells exhibited basic properties of NSCs, such as self-renewal and multipotency. Lentiviral (LV)-mediated labeling studies revealed that hippocampal NSCs are not responsible for the burst of astrocyte formation, suggesting that signals released by impaired perforant pathways do not affect NSC fate decisions. Therefore, the gene delivery system using LVs is a unique approach to understand the complex nature of NSCs and may have a translational impact in gene therapy by effectively targeting NSCs.

To test whether LV PGK-GFP was successfully transduced into NSCs with long-term proliferative capacity, the researchers introduced BrdU 6 months after LV PGK-GFP injection. The hippocampus was then examined 24 hours after the last BrdU injection to identify proliferating cells. The researchers found that 6.25±1.8% of GFP+ cells in the SGZ of the DG were positive for BrdU. This observation suggests that the initially targeted cells or their progeny maintained their proliferative capacity over a 6-month period (Figure 1A). 10±1.4% of these GFP/BrdU double-labeled cells expressed the SOX2 marker (Figure 1B), which is known to represent self-renewing and multipotent NSCs. Doublecortin (DCX), a transient marker that marks neuroblasts and immature neurons, was used to measure the continuous neurogenesis of GFP+ cells. Indeed, they observed both GFP/DCX double-labeled cells (26.8±2% of GFP+ cells; n = 3 Figure 1C) and GFP/BrdU/DCX cells triple-labeled cells (60±2.3% n = 3; Figure 1D). Interestingly, these results together suggest that NSCs are continuously generated from targeted GFP+ cells and are actively generating neuroblasts even 6 months after LV injection.

Figure 1. A long-lasting NSC population in the adult hippocampus. (Suh H, et al. 2018)