Essential 8 basal medium

Human ESCs (H9, passages 60–80, WiCell Agreement No. 16-W0060) and human iPSCs (IMR90-4, passage 40–50, WiCell Agreement No.17-W0063) were maintained in a feeder-free environment on vitronectin (Life Technologies, Maryland, USA)-coated plates21. (link), 22. (link). A standard protocol using Essential 8 basal medium (Life Technologies, Carlsbad, CA, USA) was previously reported on http://www.wicell.org. hPSC colonies were passaged by using EDTA (Lonza Inc., MA, USA) when they became 70% confluent.

Human iPSCs (ATCC-DYS0100) were maintained in self renewal on a layer of Geltrex (Gibco, A14133-01), in Essential 8 Basal Medium (Gibco, A15169-01) supplemented with E8 Supplement (50X, Gibco A15171-01) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco, 15140-122). All cells were mycoplasma free. iPSCs were dissociated with 0.5 mM EDTA (pH 8.0; Invitrogen, 15575-038) for 3-min incubation to maintain cell clusters. Cerebellar organoids were cultured as previously described (31 (link), 55 (link)) and were electroporated at 35 days of differentiation protocol with pPBase, pPB CAG LSL Venus, pPB CAG LSL Gfi1:FLAG-IRES-GFP, pPB CAG LSL MYC, and either pS100b-cre or pSox2-cre resuspended in buffer 5 (under patent) (9 (link)). For the 24-hour analysis, organoids were electroporated at 35 days of differentiation protocol with pPB CAG LSL Venus and either pS100b-cre or pSox2-cre. Organoids were transferred inside the electroporation cuvettes (2 mm; VWR, ECN 732-1136), and electroporation was performed with the Gene Pulser XcellTM. Twenty-four hours after electroporation or 39 days after electroporation, they were fixed in 4% PFA, cryoprotected in 30% sucrose, and embedded in Frozen Section Compound (Leica, 3801480). Organoids were cryosectioned at 40 μm with Thermo Fisher Scientific HM525 NX cryostat.

Microglia were differentiated as previously described. Briefly, hiPSCs expressing six inducible transcription factors (MAFB, CEBPα, IRF, PU1, CEBPβ, IRF5) were grown in Essential 8^™ Basal Medium (Gibco/Thermo Fisher Scientific, Cat. No. A15169–01), 10 μM ROCK inhibitor, and 2 μg/ml Doxycycline on Matrigel and 10 cm Poly-D-Lysine coated plates at a density of 1.5 million cells per dish. After 2 days, cells were grown in a differentiation media consisting of Advanced DMEM/F12 (Gibco/Thermo Fisher Scientific, Cat. No. 35050–061), 1X GlutaMAX, 2ug/ml doxycycline, 100 ng/mL Human IL34 (Peprotech; Cat. No. 200–34) and 10 ng/mL Human GM-CSF (Peprotech; Cat. No. 300–03). Two days later, media was exchanged for iTF-Microglia media consisting of Advanced DMEM/F12, 1X GlutaMAX, 2 μg/mL doxycycline, 100 ng/mL Human IL-34, 10 ng/mL Human GM-CSF, 50 ng/mL Human M-CSF (Peprotech; Cat. No. 300–25) and 50 ng/mL Human TGFB1 (Peprotech; Cat. No. 100–21C). On day 8, cells were dissociated with TypLE express (Gibco/Thermo Fisher Scientific, Cat. No. 12605–028) and seeded into iAssembloids.