Generating Forebrain Organoid Fusions

All hPSC experiments were conducted following prior approval from the University of California Los Angeles (UCLA) Embryonic Stem Cell Research Oversight Committee (ESCRO) and Institutional Review Board. Cortex (Cx) and ganglionic eminence (GE) organoids were generated from the H9 hESC line⁵⁸ or Rett hiPSCs³⁹ as described previously^{17 (link)} and outlined in schematic form in Fig 1a. Fusion was performed with minor modifications as previously reported^{19 (link)}. Cx and GE Organoids were cut at day 56 and two halves (e.g. Cx+GE or Cx+Cx) were combined in a microcentrifuge tube containing 300 μl of N2B27 media^{17 (link)} and placed in a hyperoxic incubator containing 5% CO₂ and 40% O₂ for 72 hours. Fused structures were then carefully transferred to 24-well oxygen permeable dishes (Lumox, Sarstedt) and maintained in a hyperoxic environment with media changes every other day until their use. Neuron migration experiments were conducted by infection of either a Cx or GE organoid with 5 μl of ~1.98×10¹³ ml⁻¹ AAV1-tdTomato (pENN.AAV.CAG.tdTomato.WPRE.SV40, a gift of Dr. James M. Wilson, University of Pennsylvania Vector Core AV-1-PV3365) on day 56 and fusion was performed as described 3 days after infection.