Fig. 1

Development of a 3D human neuron/astrocyte co-culture. A Schematic representation of the protocol to generate the 3D human neuron/astrocyte co-culture. Ngn2-hiPSCs are plated on day -2 for neural induction in stem cell (TeSR) medium containing ROCK inhibitor (RI), doxycyclin (dox), NT3 and BDNF. One day after medium (DMEM) change on day -1, Ngn2-neural progenitors are detached on day 0 and mixed with human primary astrocytes in medium and Geltrex for neuronal maturation in medium (NB + B27) containing NT3 and BDNF with weekly media refreshments for 4 weeks. B 100 µm-thick maximum intensity confocal image projection of neurons and astrocytes in 3D co-culture. Immunostaining was performed for neurons (NeuroChrom), astrocytes (GFAP) and presynapses (SYP1). Single channels are shown as a heatmap corresponding to the focal plane of the fluorescence (bottom, red; top, blue). C 160 µm-thick maximum intensity confocal image projection of neurons and astrocytes in 3D co-culture. Immunostaining was performed for neurons (β-3-tubulin, green) and astrocytes (GFAP, magenta). Single channels are shown in greyscale and the merged image includes nuclei (DAPI, blue). A zoom of the boxed area indicated in the merged image is shown