**2. Experimental Section**

#### *2.1. Cell Culture and Di*ff*erentiation*

Generation and maintenance of iPSCs (WT I line) is described in Lenzi et al. (2015) [17]. In brief, cells were cultured in Nutristem-XF (Biological Industries, Cromwell, CT, USA) supplemented with 0.1% Penicillin-Streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) in hESC-qualified Matrigel (CORNING, New York, NY, USA) coated plates. Medium was changed every day and cells were passaged every 4–5 days using 1 mg/mL Dispase (Gibco, Waltham, MA, USA). The cortical neurons differentiation protocol has been adapted and modified from Shi et al. (2012) [18]. iPSCs were treated with Accutase (Thermo Fisher Scientific) promoting single cell dissociation and plated in Matrigel coated dishes in Nutristem-XF supplemented with 10 μM Rock Inhibitor (Enzo Life Sciences, Farmingdale, NY, USA) and 0.1% Penicillin-Streptomycin with a seeding density of 65,000 cells per cm2. After three days, medium was changed to N2B27 medium (DMEM-F12, Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham, Sigma Aldrich; Neurobasal Medium, Gibco; 1X N2 supplement, Thermo Fisher Scientific; 1X Glutamax, Thermo Fisher Scientific; 1X NEAA, Thermo Fisher Scientific; 1X B27, Miltenyi Biotech; 1X Penicillin-Streptomycin) supplemented with SMAD inhibitors, 10 μM SB431542 and 500 nM LDN-193189 (both from Cayman Chemical, Ann Arbor, MI, USA). This was considered day 0 (D0). Medium was changed every day. After 10 days, cells were passaged with 1 mg/mL Dispase and re-plated into poly-L-ornithine/laminin (Sigma Aldrich, St. Louis, MO, USA) coated dishes in N2B27 medium. Starting from day 10, medium was changed every other day. At day 20, cells were dissociated using Accutase and plated into poly-L-ornithine/laminin coated dishes with a seeding density of 65,000 cells per cm2 in N2B27 medium supplemented with 10 μM Rock Inhibitor for 24 h. Medium was changed twice a week and 2 μM Cyclopamine (Merck, Kenilworth, NJ, USA) was supplemented to N2B27 medium at day 27 for 4 d. Around day 30, cells were dissociated again with Accutase and re-plated into poly-L-ornithine/laminin coated dishes with a seeding density of 65,000 cells/cm<sup>2</sup> in N2B27 medium supplemented with 10 μM Rock Inhibitor for 24 h. From day 40, N2B27 medium was supplemented with 20 ng/mL BDNF (Sigma Aldrich, St. Louis, MO, USA), 20 ng/mL GDNF (Peprotech, London, UK), 200 ng/mL Ascorbic Acid (Sigma Aldrich), 1 mM cyclic AMP (Sigma Aldrich, St. Louis, MO, USA) and 5 μM DAPT (Adipogen Life Sciences, San Diego, CA, USA).

#### *2.2. Preparation of Gel-Adhesive Glass Substrates and Bioink for 3D Bioprinting*

Standard microscopy glass slides were functionalized following a published protocol [19] with minor changes. Briefly, standard glass slides were exposed to air plasma (3 min, 27 W, 600 mTorr) and quickly soaked in a 5% v/v solution of 3-aminopropyl triethoxysilane (Aptes, Sigma Aldrich, St. Louis, MO, USA) in deionized water for 2 h, washed with deionized water and ethanol and then air dried. Afterwards, slides were soaked in 0.2 M solution of 4-morpholineethanesulfonic acid (MES, PH4.5,

Sigma Aldrich, St. Louis, MO, USA) containing 1% w/v alginate (Fmc Biopolymers, Philadelphia, PA, USA), EDC (0.4% w/v; Sigma Aldrich, St. Louis, MO, USA) and NHS (0.3% w/v; Sigma Aldrich, St. Louis, MO, USA) overnight at room temperature. Finally, slides were washed with water and ethanol, air dried, cut into 5 mm × 5 mm squares using a glass cutting pen, UV-sterilized and stored for later use. Alginate solution was prepared by dissolving alginate powder (GP1740, Fmc Biopolymers, Philadelphia, PA, USA) in 25 mM HEPES buffered saline (HBS). A stock solution of alginate was prepared at a concentration of 4% w/v, sterile-filtered, divided in working aliquots and stored at +4 ◦C for later use. The day of the bioprinting experiment, Matrigel precursor solution was thawed in ice for 2 h and mixed with alginate stock solution at a ratio of 1:1 v/v. Typically, 300 μL of Matrigel/alginate mixture was prepared for each experiment. All solutions were manipulated and kept in ice baths. Differentiating cells were collected from cell culture plates by single cell dissociation mediated by Accutase treatment. Cells were resuspended in the Matrigel/alginate mixture at a 1:1 ratio to obtain the bioink with 2% alginate as final concentration.
