Polyamine supplement

WA09 hESCs and AICS-5 iPSCs were purchased from the WiCell Institute and H20961 iPCs were provided by our collaborators at The Weizmann Institute of Science. Both the cell lines were expanded and maintained in mTeSR™1 and mTeSR™ Plus (Stem Cell Technologies), passaged with Versene, and cultured on plates coated with growth factor reduced Matrigel (Corning). Small molecule cocktail (CEPT) (Chen et al., 2021) consisting of 50 nm Chroman-1 (Medchem Express), 0.7 μM trans-ISRIB (Tocris), 5 μM Emricasan (Selleckchem), Polyamine Supplement (Sigma) diluted 1:1000 and Antioxidant Supplement (Sigma) diluted 1:1000 was added during passaging and sorting to promote cell viability. All cell lines were grown in a humidified incubator at 37°C, 5% CO₂.
Media for NCSC differentiation and maintenance was prepared in the following way:

KSR Medium (1L): 820 mL Knockout DMEM, 150 mL Knockout Serum Replacement medium, 10 mL L-glutamine (200 mM), 1 mL β-mercaptoethanol.

N2 Base Medium (1L): DMEM f-12 medium, 25 mg/L Insulin, 10 μM apo-Transferrin, 30 nM Sodium Selenite, 100 μM Putrescine, 20 nM Progesterone

NCSC Specification medium (1L): Base N2 medium (prepared as above), 200 μM Sodium L-ascorbate, 20 ng/mL BDNF, 100 ng/mL FGF-8b, 20 ng/mL Shh, 10 μM Y-27632

NCSC Maintenance Medium (1L): Base N2 medium (prepared as above), 10 ng/mL FBF basic, 20 ng/mL EGF.

Between D90 and D120, TCF7L2-tdTomato⁺ hThOs were paired with VGLUT1-tdTomato⁺ hCOs of similar age. Each pair was transferred to a well of a low-attachment, 24-well plate in 500 mL Fusion media (BrainPhys supplemented with 1× N2, 50× B27 without vitamin A, 1× glutamax, 1× NEAA, 1× antibiotic-antimycotic, 200 µM ascorbic acid, 100 µM cAMP, 1% ES-FBS, 10 µM DAPT, 20 ng/mL BDNF, 20 ng/mL GDNF, and the CEPT cocktail (50 nM Chroman 1 [HY-15392, MedChem Express], 5 µM emricasan (S7775, Selleckchem), 0.7 µM trans-ISRIB (#5284, Tocris), and polyamine supplement (#P8483, Sigma-Aldrich^{105 (link)}) supplemented with 0.5% v/v GFR-Matrigel. The plate was left tilted and undisturbed in the incubator. After 3 days, 60% of the media in each well was replaced with fusion media. This was done slowly, while keeping the plate tilted with minimal disturbance to the “fused” organoid pair in each well. The same was done on D6 and D9. Subsequently, 80% of the media was replaced every 3 days. On D4, the plate was transferred to an orbital shaker at 80 rpm. The shaker speed was increased to 90 rpm on D5, 100 rpm on D6, and starting D7, the assembloids were kept at 110 rpm. Between 5 and 10 weeks postfusion, assembloids were harvested for electrophysiological experiments.