*3.1. 3D Bioprinting of Di*ff*erentiating Human iPSC-Derived Neurons and Glia*

An outline of the 3D bioprinting method developed in this work is shown in Figure 1A. We used a customized extrusion bioprinter developed in-house (Figure S1). This platform consists of a custom extrusion 3D bioprinter integrating a microfluidic printing head constituted of two independent needles arranged in a coaxial configuration. The deposition strategy is based on the use of calcium-alginate gel as templating agent for the printing of blended extracellular matrices and cells. This provides a precise control on the relative position of cells within the 3D construct, down to the micrometer scale with high reproducibility [16], independently on the 3D embedding matrix of election. Reportedly, bioink composition is crucial to ensure long-term iPSCs viability and maintenance of 3D structures [3,6]. Pilot experiments revealed that Matrigel is the best candidate for in vitro differentiation of neuronal cells in 3D, when compared with transglutaminase/gelatin or photo-crosslinked gelatin methacryloyl gels. For 3D printing experiments, different ratios of Matrigel/alginate and post-printing treatments have been tested. We obtained the best results using a solution containing 2% w/v alginate and 0.5× Matrigel (~50% dilution from stock), printed using 0.33 mM CaCl2 crosslinking solution, and subsequently exposing the printed construct to alginate-lyase enzyme at a concentration of 0.2 μg/mL in cell culture media for 12 h, starting the exposure 3 h after the printing protocol.

The cellular components of the bioink, neuronal and glial precursors, were derived by differentiation of human iPSCs by a multistep protocol in conventional bidimensional (2D) culture conditions (Figure 1B). Efficient induction of a neural cortical fate was obtained by initial dual SMAD inhibition and subsequent block of Hedgehog signaling with cyclopamine [16]. Representative images of differentiating cells are shown in Figure S2. During this standard differentiation process, human iPSCs exited from pluripotency (loss of *NANOG* expression) and gradually acquired a neural character, as shown by the progressive expression of neural progenitor cells (NPCs; *PAX6*, *NCAD*), neuronal precursors (*TBR2*, *FOXG1*) and neurons (*TBR1*, *TUJ1*, *NeuN*, *MAP2*) markers (Figure S2A). Further characterization by immunostaining analysis showed progressive acquisition of a neuronal morphology and expression of neurofilament proteins (Figure S2B,C). The astrocyte marker *GFAP* was also expressed at late time points (Figure S2B,C).
