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. 2019 Jan 17;12(2):274–289. doi: 10.1016/j.stemcr.2018.12.015

Figure 1.

Figure 1

Co-culture of NT-3-NSCs and TRKC-NSCs in 3D GS In Vitro to Establish an NSC-Derived Neural Network Scaffold with Functional Synaptic Structure

(A) A neurosphere from GFP-transgenic rat pups comprised NESTIN+ NSCs.

(B) PLGA tubes with diameter of 3 mm and length of 2 mm (left), a piece of GS (middle), and appearance of the GS scaffold (right).

(C) NSCs infected with Adv-NT-3 were NT-3+ staining (arrow).

(D) NSCs infected with Adv-TRKC showed TRKC+ staining (arrow).

(E) Scanning electron microscopy showed that NSC-derived cells in the GS scaffold formed contacts with one another. A neurite of an NSC-derived neuron-like cell was in contact with another neuron-like cell body (red arrow).

(F) Bar chart showing the percentages of MAP2+, APC+, GFAP+, and NESTIN+ cells in the NSCs, NT-3-NSCs, TRKC-NSCs, and NT-3-NSCs + TRKC-NSCs groups (n = 5/group).

(G and H) MAP2+ (purple, arrows), GFP+ (green) NSC-derived neurons were labeled with (G) SYN (red) and (H) PSD95 (red) in the NT-3-NSCs + TRKC-NSCs group.

(I) Western blot analysis of SYN and PSD95 (n = 5/group).

(J–L) Transmission electron microscopy showing profiles of some synaptic structures between the NSC-derived neurons in the NT-3-NSCs + TRKC-NSCs group. (K and L) High magnifications of two boxed areas in (J) showing pre-synaptic components with spherical agranular vesicles (arrows), post-synaptic membrane (arrowheads), and synaptic cleft.

(M) EM showed that several neurite terminals (numbered from 1 to 4) aggregate together to form axodendritic-like or axoaxonal-like synapses (arrows).

(N) Representative traces of mEPSCs.

(O) Bar charts showing the frequency and amplitude of mEPSCs in 4 groups (n = 10/group).

One-way ANOVA with least significant difference (LSD) t; ∗p < 0.05 indicates significant difference from the NSC group; #p < 0.05 indicates significant difference from the NT-3 NSCs; &p < 0.05 indicates significant difference from the TRKC NSC group. Scale bars: 40 μm in (A), (C), and (D); 10 μm in (E); 5 μm in (G) and (H); 100 nm in (J); 200 nm in (K–M).