Figure 7

Human iPSC-derived NESC differentiate into neurons expressing Purkinje cell markers and the new neurons establish inhibitory and excitatory synapses in mice cerebella. CNT (CNT NESC) and MJD CLA (MJD NESC) iPSC-derived NESC expressing GFP (green) transplantation, mice cerebella were evaluated for cerebellar neurons differentiation and synapses establishment. Representative immunofluorescence confocal microscopy images showing that the grafted cells differentiated into (A) PCP4- and (B-D) Calbindin-positive (red) cells, markers of Purkinje cells, and in (E–G, J and K) Parvalbumin- and (H and I) GABA-positive (red) cells, showed by the colocalization between GFP (green) and the cell markers (red). D) Representative immunofluorescence confocal microscopy image of human nuclei antigen (HuNu) colocalization with Calbindin. Cerebellar sections analyzed A, C-U) six and B) two months after cell transplantation. Scale bars: (A-C) 50 μm, (D) 20 μm, and (E-K) 10 μm. The lateral panels in (A) and (C) exhibit individual color channels of the cells indicated by the white arrows in the graft, scale bars: 20 μm. White arrows show cells positive for the cell markers. (L, N, Q, and S) Representative immunofluorescence confocal microscopy images showing (L and N) colocalization of puncta positive for presynaptic Synaptophysin (red) and postsynaptic Gephyrin (green) displaying inhibitory synapses in neurites (GFP, white) derived from (L) CNT NESC and (N) MJD NESC transplanted in cerebella. Scale bars: 5 μm, upper small inserts: 1 μm. M and O) 3D representations of Z-stack confocal images, using Imaris software, of presynaptic Synaptophysin (red) and postsynaptic Gephyrin (green) immunolabeling showing inhibitory synapses in neurites (GFP, white) derived from CNT and MJD NESC grafted cells, respectively. Scale bars: (M) 2 μm, O) 0.5 μm; the upper small inserts show a higher magnification of the synapses indicated by the white arrows. (P) Inhibitory synapses number per neurite length normalized for CNT NESC; CNT NESC n = 22 neurites from 3 grafted mice, MJD CLA NESC n = 14 neurites from 3 grafted mice. (Q and S) Colocalization of puncta positive for presynaptic VGluT1 (green) and postsynaptic PSD95 (red) showing excitatory synapses in neurites (GFP, white) derived from (Q) CNT NESC and (S) MJD NESC transplanted in cerebella. Scale bars: 5 μm, upper small inserts: 1 μm; the upper small inserts show a higher magnification of the synapses indicated by the white arrows. (R and T) 3D representations of Z-stack confocal images, using Imaris software, of presynaptic VGluT1 (green) and postsynaptic PSD95 (red) immunolabeling showing excitatory synapses in neurites (GFP, white) derived from CNT and MJD NESC grafted cells, respectively; scale bars: 0.5 μm. U) Excitatory synapses number per neurite length normalized for CNT NESC; CNT NESC n = 17 neurites from 3 grafted mice, MJD CLA NESC n = 14 neurites from 3 grafted mice. DAPI: blue, Data are expressed as mean ± SEM, *p < 0.05, Unpaired t-test with Welch's correction.