Figure 2. Slower morphological maturation of human iNs.

iNs were lipofected 4 days prior fixation with a plasmid-expressing cytosolic GFP and fixed at different time points. (A, B) Examples of monopolar (left), bipolar (middle) and multipolar (right) iNs from chimpanzee (SandraA, A) and human (409B2, B). Cell morphology is highlighted by GFP expression (green); the nuclei are stained with DAPI (cyan). Scale bars are 20 µm. (C, D) Monopolar (black), bipolar (dark gray), and multipolar (gray) iNs in ape (C) and human (D) expressed as % of total. (E, F) Chimpanzee (E, SandraA, orange) and human (F, 409B2, green) multipolar iNs development over time. Zoom-ins show the cell body. Scale bar is 1 mm. (G, H) Total neurite length, expressed in µm for multipolar and bipolar iNs. (G) Apes multipolar iNs show a higher total neurite length at d7 (p=0.0098) and d14 (p=0.0215) compared to human iNs. The scale is logarithmic. (H) Apes bipolar iNs show a higher total neurite length at d7 (p=0.0278) and d14 (p=0.0003). The scale is logarithmic. (I) Relative length of the longest neurite (axon) in apes and human multipolar iNs. Data representation as normalized data against the mean per batch of the ape data in logarithmic scale. Ape iNs show a higher axon length at d14 (p=0.0294) compared to human iNs, according to total neurite length. Human iNs show a higher axon length at d35 (p=0.0094) compared to ape iNs. (J) Relative longest neurite (axon) length in bipolar iNs. Ape iNs show a higher axon length at d14 (p=0.0426). Data representation as normalized data against the mean per batch of the ape data, the scale is logarithmic. (K, L) Total number of Sholl intersections for multipolar (E) and bipolar (F) iNs. Apes iNs show a higher number of Sholl intersections at d14 for both multipolar iNs (p=0.0469) and bipolar (p=0.0008) iNs. The scale is logarithmic. (M, N) Total dendrite length, expressed in microns for multipolar (M) and bipolar (N) iNs. Ape iNs show a higher total dendrite length at d14 for both multipolar (p=0.0009) and bipolar iNs (p=0.04471). The scale is logarithmic. For all graphs, black rhombs represent the mean and black lines the median. Significance score: p<0.05*, p<0.01**, p<0.001***, Mann–Whitney U test.

Figure 2—figure supplement 1. Characterization of axons in iNs.

iNs were lipofected with a plasmid-expressing cytosolic GFP (GFP, green), fixed at d35, and stained with an axonal marker (Pan-Neu, magenta). (A) Chimpanzee iN stained with a pan-neurofilament antibody. The corresponding Imaris tracing is on the right. The arrowheads indicate that the longest of the neurites -as judged by the complete tracing in Imaris- is also positive for axonal marker (white arrowhead). Scale bar is 30 µm. (B) Low-magnification maximum-intensity projection of GFP-labeled cells (green) stained with pan-neurofilament antibody (magenta). The Imaris tracing on the right corresponds to the white box. Magenta tracing corresponds to the longest neurite. Of note, ape and human iNs generate long axons, compared to dendrites. As the longest neurite (axon) encompasses a large part of the total neurite length. Scale bar is 300 µm. (C) Quantifications show that (1) for 100% of the stained cells, the longest neurite is positive for axonal marker (upper pie chart) and (2) the majority of the cell (96% of total) features one axon. 4% of the cells were found to have two axons (bottom pie chart). Of note, in our system the % of cells with two axons is lower than what reported for iNs autaptic culture (Rhee et al., 2019). This is possibly due to the NGN2-iNs system in conjunction with the culture conditions.

Figure 2—figure supplement 2. Dynamics of structural maturation in bipolar and multipolar iNs.

iNs were lipofected 4 days prior fixation with a plasmid-expressing cytosolic GFP and fixed at different time points to quantify their structural features. (A, B) Chimpanzee (A, SandraA, orange) and human (B, 409B2, green) bipolar iNs development over time. Zoom-ins show the cell body. Scale bar is 1 mm. (C) Relative frequency of monopolar (black), bipolar (dark gray), and multipolar (gray) iNs for all the cell lines used in this study: chimpanzee (SandraA and JoC) and bonobo (BmRNA) and human (409B2, SC102A, and H9).