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. 2020 Oct 28;8:594090. doi: 10.3389/fcell.2020.594090

FIGURE 1.

FIGURE 1

Generation of simplified brain organoids (simBOs) from human primitive neural stem cells (pNSC). (A) Graphical schematics for the generation of pNSCs into simBOs. (B) Phase contrast images of simBOs at days 0, 10, and 20 of spontaneous differentiation. Scale bars represent 200 μm. (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for neural progenitor (PAX6) and neuronal (TUBB3, DCX, MAP2, and, NEUN) markers in simBOs at days 0, 10, and 20 of spontaneous differentiation. Expression value was normalized to those of day 0 (d0). Data represent mean ± standard error (SE) from three technical replicates. Statistical significance of Turkey’s multiple comparisons ***p < 0.001 after one-way ANOVA. (D) Immunofluorescence analysis of simBOs with proliferating neural progenitor markers (PAX6 and Ki67), neuronal markers (TUJ1, MAP2, and NEUN), and apoptotic cell marker (cleaved caspase III; cCASP3) at indicated times. Scale bars represent 100 μm. (E) Immunofluorescence analysis of simBOs with a mature neuronal marker (MAP2) and a synaptic marker (SYN1) in simBOs after day 10 of spontaneous differentiation. Scale bar represents 5 μm. (F) Immunofluorescence analysis of simBOs with mature neuronal marker (MAP2) and astroglial cell marker (GFAP) after day 10 of spontaneous differentiation. Scale bar represents 20 μm. (G) Quantitative graph of the percentage (%) of positive pixels of each marker relative to Hoechst. Data represent mean ± standard error (SE) (n = 19). No significant differences were observed.