Figure 2. Hmga2 promotes the self-renewal of fetal and young adult CNS stem cells.

A) Typical neurospheres that formed after 9 days in non-adherent cultures from E14.5 telencephalon cells cultured at very low cell density (~1cell/μl). B) The percentage of cells from E11.5 telencephalon, E14.5 telencephalon, P0 VZ, and P49-56 SVZ of Hmga2^−/− mice and littermate controls that gave rise to multipotent neurospheres in culture, the diameter of these neurospheres, and their self-renewal potential (number and percentage of cells from individual primary neurospheres that gave rise to multipotent secondary neurospheres upon subcloning). Hmga2 deficiency significantly reduced self-renewal at all of these stages and stem cell frequency at P0 and P49-56 (mean±SD for 5–7 independent experiments per stage, *P<0.01, **P<0.05). C) P0 VZ cells were dissociated and plated in adherent cultures at clonal density (0.33cells/μl). Wild-type (+/+) stem cell colonies contained significantly more cells than Hmga2^−/−(−/−) colonies (3 independent experiments). The frequency of dying cells within multilineage colonies after 10 days in culture was examined based on activated caspase-3 staining (D), or the frequency of condensed, fragmented nuclei identified by 4′,6-diamino-2-phenylindole dihydrochloride (DAPI) staining (E). Only rare cells underwent cell death in Hmga2^+/+ and Hmga2^−/− colonies (3 independent experiments). F) A significantly lower percentage of cells within Hmga2^−/− multilineage colonies (22±11%, mean±SD) incorporated a 20 minute pulse of BrdU as compared to Hmga2^+/+ colonies (42±10%; 3 experiments; *P<0.01). G) P0 Hmga2^−/− VZ cells were infected with either GFP-only control lentivirus or with Hmga2+GFP lentivirus. Hmga2 over-expression significantly increased the size and self-renewal of Hmga2^−/− neurospheres (3 experiments: P<0.01, **P<0.05). All T-tests were paired.