To characterize organoids with each type of morphology, we performed DNA microarray analysis to identify genes expressed predominantly in each type of organoids on day35 of cortical organoid differentiation protocol (SFBEq).
Overall design: To generate cortical organoids, we employed the differentiation method previously described by Kitahara et al., Stem Cell Reports, 2020. Human induced pluripotent stem cells are cultured in DMEM/F-12 GlutaMAX supplemented with 20% (v/v) KnockOut Serum Replacement (KSR), 5 µM SB431542, and 3 µM IWR1e, using low adheshion 96 well plates.Defining the day on which floating culture of cell aggregates was started as day0, half the medium was changed once every three days until day 15. On day 18, the aggregated cells were transferred to 90-mm non-coated dishes and further cultured in DMEM/F-12 GlutaMAX supplemented with 1% (v/v) N-2 Supplement, 1% (v/v) Chemically Defined Lipid Concentrate (CDLC), 0.25 µg/ml Amphotericin B , 100 U/ml penicillin, and 100 µg/ml streptomycin until day 35. Organoids were cultured on an orbital shaker and maintained at 50 rpm in 20% O2 and 5% CO2 to eliminate the need for cell culture in 40% O2. Complete medium changes were performed once every 3 or 4 days from day 18 to day 35. On day35, partial structures in organoids were classified into 7 groups according to their morphologies. (1) Rosettes, organoids with rosette structures throughout; (2) Potato-like”, those with low transparency and no clear internal structures; (3) Balloon-like, those with balloon-like cystic structures; (4) Cotton-like, organoids with fibrous epithelial-like structures; (5) Transparent, transparent organoids and cyst-like internal structures; (6) Jelly-like, organoids with a transparent periphery and no clear internal structures; and (7) Pigmented, organoids with pigmentation.The tissues with distinct morphological features were isolated by dissection and subjected to microarray analysis. For each morphological groups, 3 samples are analyzed.
Less...