Figure 2.

Phenotypic and Growth Characterization of Neurites in Bidirectional μTENNs

(A–F) Confocal reconstructions of 1-cm GFP+ ESC-derived constructs cultured for 24 (A) or 60 DIV (C) and stained for Tuj1 (magenta) and Hoechst (blue). Magnified images of the neurite tracts qualitatively show that neurites populate the center of the micro-column with a lesser density at 24 DIV (B) compared with 60 DIV (D). Quantification of the mean GFP (E) and Tuj1 (F) intensities in the different regions of interest in A and C confirm that neurites at the center of the μTENN reach a more homogeneous distribution at the later time point.

(G–I) (G) Representative confocal image of a bidirectional iPSC-derived organoid μTENN grown in a 0.5-cm micro-column for 30 DIV and stained for neuronal somata/dendrites (MAP2, green), axons (tau, red), and nuclei (Hoechst, blue). Higher magnification of the organoid cell mass (H) and the neurite region (I) show the morphology of the neurons and the purely axonal nature of the intervening neurites, respectively.

(J) Z stack reconstructions of an ∼200-μm-thick cross section of the axonal tract (same μTENN as in G) demonstrate axons distributed within the micro-column lumen as well as at the collagen-agarose border.

Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ****p < 0.0001; Tukey's multiple comparisons test). Scale bars: 500 μm (A and C), 100 μm (B, D, H, and I), and 200 μm (G).