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. 2017 Jun 1;34(11):1981–1995. doi: 10.1089/neu.2016.4602

FIG. 4.

FIG. 4.

Increasing neuronal differentiation of green fluorescent protein (GFP) human neural stem cell grafts (hNSCs) with time in vivo is evident from 8 week images (A–H) and corresponding 16 week images (A’–H’), in A, the image of the whole hemisphere at 8 weeks post-transplantation shows overlap of 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), GFP, doublecortin (DCX), and NeuN fluorescence. The black square in A is shown at a higher magnification in B–H. The DAPI-stained nucleus indicated by a white arrow in B is GFP (C), strongly DCX+ (D) with weak NeuN immunoreactivity (E). Overlay of GFP with DCX (F), DCX with NeuN (G), and GFP with NeuN (H) confirms neuronal differentiation of transplanted hNSC. Corresponding 16 week images (A’–H’) show weak, diffuse DCX (D’) and stronger NeuN (E’) that is confirmed by fluorescence overlay (F’–H’). Scale bar 10 μm. Neuronal marker expression at 16 weeks. Transplant-derived neurons show immunoreactivity to a mature neuronal marker, Calbindin (I–K). DAPI-stained whole hemisphere image shows GFP transplant (I) that is Calbindin positive (Cal+) (J). Overlay renders transplant yellow (K). Scale bar 1 mm. The white square (in I–K) near the corpus callosum is shown at a higher magnification to highlight a single GFP cell bearing neuronal morphology that is Cal+ (I’–K’). Scale bar 10 μm. DAPI and anti-synaptophysin antibody stained whole hemisphere with 16 week transplant shows synpatophysin immunoreactivity in gray matter and transplant but not white matter (L). Quantitation of synaptophysin immunoreactivity (red puncta in L) revealed greater synaptophysin puncta in transplant (green) than in host (red) (M). The region in the white box is shown at a higher magnification as an orthogonal view with two representative synaptophysin puncta, one each for transplant (orange arrow) and host (red arrow) (N). A zoomed-in view shows the presence of GFP signal in transplant (orange arrow) but not host (red arrow) synaptophysin puncta. Quantitation of fluorescence is shown in P. Scale bar 10 μm. The scale bar for A, A’, and I–L is 1 μm.