Figure 4.

Differentiation capacity of grafted aNSCs depending on the graft origin. (A) Representative triple fluorescence immunostaining of a PVR_V-SVZ graft within the DG (the arrows illustrate a GFP⁺/BrdU⁺/NeuroD1⁺-grafted NSC; A₄). GFP identifies the transplanted cells (green, A₁), BrdU (red, A₂) indicates the cells proliferating after transplantation, and NeuroD1 serves as a marker for immature neurons (blue, A₃). Scale bars, 10 µm. (B,C) The relative NeuroD1⁺ cell counts with the DG normalized to total GFP⁺-grafted cell counts did not differ between the two grafts (PVR_V-SVZ vs. PVR_MB). (C) The daily BrdU labeling of proliferating cells revealed a higher proliferation capacity of grafted NeuoD1⁺ cells derived from PVR_V-SVZ as compared to that of PVR_MB aNSCs. The p-values are from unpaired two-sided t-tests (PVR_V-SVZ n = 8; PVR_MB n = 7). (D) Representative triple fluorescence immunostaining of a PVR_V-SVZ graft within the DG (the arrows illustrate a GFP⁺/BrdU⁺/NeuN⁺-grafted cell; D₄). GFP identifies the transplanted cells, (green, D₁), BrdU (red, D₂) indicates the cells which proliferated after transplantation, and NeuN serves as a marker for neurons (blue, D₃). Scale bars, 10 µm. (E–H) The relative NeuN⁺ cell counts with the DG normalized to total GFP⁺-grafted cell counts demonstrated an exclusive neuronal differentiation in PVR_V-SVZ grafts. (F) The BrdU labeling index shows that ~37% of NeuN⁺ derived from previously proliferating cells. Notably, most grafted NeuN⁺ neurons are located within the GZ (G; arrow in D₄), with no differences in the BrdU labeling index (H). The p-values are from unpaired (E,F) or paired (G,H) two-sided t-tests (PVR_V-SVZ n = 7; PVR_MB n = 8). (I,J) Relative NG2⁺ polydendrocyte counts with the DG normalized to total GFP⁺-grafted cell counts varied markedly and did not differ between the two grafts (PVR_V-SVZ vs. PVR_MB). The relative amounts of BrdU⁺ polydendrocytes were similar in both grafts (J). The p-values are from unpaired two-sided t-tests (PVR_V-SVZ n = 6; PVR_MB n = 5). Abbreviations: PVR_V-SVZ—periventricular region of the ventricular-subventricular zone of the lateral wall of the lateral wall of the lateral ventricles; PVR_MB—periventricular region of the midbrain; aNSC—adult neural stem cell; SGZ—subgranular zone; GZ—granular zone; GFP—green fluorescent protein; BrdU—5′-bromo-2′-desoxyuridine; NeuroD1—neuronal differentiation 1 protein; NeuN—neuronal nuclei protein; DG—dentate gyrus.