Figure 2.

Survival and differentiation of grafted organoids. Organoids were grafted and harvested at the indicated dpi, coronal sections were analyzed using immunofluorescence and confocal microscopy. (a) Double immunofluorescence staining for SOX2 and NeuN (left), and GFP and SMI312 (right). At 14 dpi, the graft expresses both the NPCs marker SOX2 and the mature neuronal marker NeuN but shows low numbers of SMI312-positive processes. At 50 dpi, the graft retains a lower expression domain of SOX2, NeuN+ cells, and the SMI312-positive area increase. White solid lines indicate the apical/ventricular surface. Dotted white lines indicate the radial glia VZ-L regions. Yellow lines indicate the graft-host border. (b) Quantification of the percentage (mean ± s.e.m.) of SOX2+/DAPI+ and of NeuN+/DAPI+ cells in the graft at the indicated grafting time point. P values were calculated using one-way ANOVA with post hoc Tukey’s test between group comparisons (F(2, 6) = 18.81; P = 0.0026 for NeuN, F(2, 6) = 24.3; P < 0.0001 for SOX2), n = 3 independent animals per group. Asterisks indicate pair-wise comparisons with 14 dpi. (c) Comparative quantification of the percentage of SOX2+/DAPI+ and of NeuN+/DAPI+ cells in the graft compared with stage-matched organoids in culture at the indicated grafting or culture time point. Data are presented as mean ± s.e.m., unpaired two-tailed t-test. For SOX2, 53 day vs. 14 dpi (t = 3.059, df = 8, P = 0.0156) and 102 d vs. 50 dpi (t = 1.617, df = 9, P = 0.1369, not significant). For NeuN, 53 d vs. 14 dpi (t = 1.208, df = 8, P < 0.2617, not significant) and 102 d vs. 50 dpi (t = 12.15, df = 9, P < 0.0001). Day 53 (n = 7 organoids), day 102 (n = 8 organoids) from three independent patches. n = 3 animals for 14 dpi and 50 dpi. (d) Immunofluorescence staining for GFP and human-specific GFAP (hGFAP) at the indicated time points showing astrocyte differentiation in the graft with increased abundance over time. Right panel is a higher magnification of the boxed area. (e) Immunostaining for oligodendrocyte marker Olig2 in the graft at 50 and 90 dpi. (f) Human graft contains Iba1+ microglia that do not co-localize with GFP. (g) Double immunofluorescence staining for presynaptic marker Synapsin (Syn) and the postsynaptic marker PSD95 at 50 dpi, showing a co-association between pre- and post-synaptic compartments and the formation of synaptic connections in the graft. The box indicates the region of magnification from the left panel; arrowheads indicate direct contact between a pre- and post-synapses puncta. Image shows a single plane confocal-Z-section. Nuclei were counterstained with DAPI. Scale bars: 100 μm in a, 50 μm in d–f, 5 μm in f (right panel) and g. *P < 0.05,

**P < 0.01, ****P < 0.0001. n.s., not significant.