Figure 2.

Graft survival and proliferation. (A) Schematic representation of transplantation experiments. C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ mice aged 8 to 12 weeks were sacrificed, the neurogenic regions (PVR_V-SVZ, PVR_MB) were dissected, and aNSCs expanded as neurospheres for 14 days. Afterwards, singularized aNSCs were transplanted into the hippocampal neurogenic niche (dentate gyrus) of wild-type mice. Both animal groups received intraperitoneal BrdU injections for 7 consecutive days after transplantation. Mice were sacrificed on day 7. (B) A representative fluorescence image of PVR_V-SVZ aNSC grafts correctly located within the DG shows that GFP⁺ cells (green, B₁) are located in the SGZ and its adjacent regions. Daily BrdU injections labeled actively proliferating grafted cells (red, B₂). Overview of the different subregions in the hippocampus, namely the granular zone, subgranular zone and hilus. Proliferating graft cells can be identified as GFP⁺/BrdU⁺ labeling (arrows; B₃). Scale bars, 10 µm. (C) Quantitative immunohistochemistry revealed a low graft survival in both grafts, but significantly more GFP⁺-grafted cells in PVR_V-SVZ as compared to PVR_MB aNSC grafts one week post transplantation. (D) Subregional analyses of transplant location within the DG showed lower GFP⁺ cell numbers in GZ as compared to SGZ and hilus, with no differences between the two transplant origins (two-way mixed ANOVA with hippocampal region [SGZ, GS, hilus] and transplanted aNSC type [PVR_V-SVZ vs. PVR_MB] as independent variables; see Supplementary Table S1 for the statistical results). (E,F) The daily BrdU labeling of proliferating cells revealed a higher proliferation capacity of grafted aNSCs derived from PVR_V-SVZ as compared to that of PVR_MB aNSCs (E). The absolute GFP⁺/BrdU⁺ cell counts within DG were 24.9 ± 9.9 cells per DG in PVR_V-SVZ grafts versus 4.2 ± 9.9 cells per DG in PVR_MB grafts (p = 0.249). (F) Subregional analyses of the transplant location displayed no relevant differences between the DG subregions with a tendency to lower proliferation rates in the hilus (two-way mixed ANOVA with hippocampal region [SGZ, GZ, hilus] and transplanted aNSC type [PVR_V-SVZ vs. PVR_MB] as independent variables; see Supplementary Table S2 for the statistical results). p-values are from an unpaired two-sided t-test (C,E) or post-hoc t-tests with Bonferroni adjustment ((D,F); PVR_V-SVZ n = 8; PVR_MB n = 22). Abbreviations: EGF—epidermal growth factor; FGF-2—fibroblast growth factor-2; PVR_V-SVZ—periventricular region of the ventricular-subventricular zone of the lateral wall of the lateral ventricles; PVR_MB—periventricular region of the midbrain; aNSCs—adult neural stem cells; BrdU—5′-bromo-2′-desoxyuridine; DG—dentate gyrus of the hippocampus; SGZ—subgranular zone; GZ—granular zone; GFP—green fluorescent protein.