GoKawiilEthics statement
All experiments involving human cell lines were approved by the Harvard University IRB and ESCRO committees. All animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Harvard University. All experiments were performed in accordance with relevant guidelines and regulations, and in accordance with informed consent obtained from the donors of the originating cells or tissues.
Mouse experiments and housing
To develop the mouse-endogenous chimeroid system, we used wild-type CD-1 and C57BL/6 mice (Charles River Laboratories). We used embryos of both sexes, collected at E11.5. The sex of each embryo was not determined prior to the experiment. Sample size was not predetermined using specific methods. Mice were maintained under standard housing conditions in a temperature- and humidity-controlled facility under a 12 h–12 h light–dark cycle (light, 07:00 to 19:00) with ad libitum access to food and water. The ambient temperature was maintained at 20–24 °C and the relative humidity at 30–70%.
Human pluripotent stem cell culture
All human PS cell lines were maintained as previously detailed7,9. In brief, MTESR1 medium (StemCell Technologies), mTESR+ medium (StemCell Technologies) or StemFlex medium (Gibco), all with 1% of added penicillin–streptomycin Solution (Corning), were employed for culture of stem cells in cell culture dishes (Falcon) precoated with 1% Geltrex (Gibco), at 37 °C in 5% CO 2 . All human PS cells were maintained below passage 55 and tested negative for mycoplasma (assayed using the MycoAlert PLUS Mycoplasma Detection Kit, Lonza).
Characterization of the PS cell lines
The psychiatric control Mito210 male iPS cell line was provided by B. Cohen (McLean Hospital); the PGP1 male iPS cell line was provided by G. Church; the GM08330 male iPS cell line was provided by M. Talkowski (MGH) and was originally derived from fibroblasts obtained from the Coriell Institute for Medical Research6,7,30. The H1 male human embryonic stem cell line (also known as WA01) was purchased from WiCell; and the 11a iPS cell line was obtained from the Harvard Stem Cell Institute. The female CW50037 iPS cell line was from the California Institute for Regenerative Medicine (CIRM) iPS cell collection. All lines were authenticated as follows. The PGP1 iPS cell line was authenticated by short-tandem-repeat (STR) analysis (performed by TRIPath). The 11a cell line was authenticated by karyotyping as previously described40. The Mito210 iPS cell line was authenticated with genotyping analysis (Fluidigm FPV5 chip) performed by the Broad Institute Genomics Platform. The CW50037 iPS cell line was authenticated using single-nucleotide polymorphism (SNP) genotyping (Illumina Global Screening Array (GSA) from Illumina; processed at the Genomics Platform, Broad Institute) for cell line identification and detection of chromosomal abnormalities. The H1 and GM08330 lines were authenticated by STR analysis (performed by WiCell). The GM08330 parental line has a previously reported30 interstitial duplication in the long (q) arm of chromosome 20; all other lines were karyotypically normal. No commonly misidentified lines were used in this study.
Cortical organoid and chimeroid differentiation
Dorsally patterned cortical organoids were generated following a protocol previously published by our group7. In brief, on day 0, feeder-free cultured human PS cells, 75–85% confluent, were enzymatically dissociated to single cells with Accutase (Gibco), and 9,000 cells per well were reaggregated in ultra-low-cell-adhesion 96-well plates with V-bottom conical wells (sBio PrimeSurface plate; Sumitomo Bakelite) using the same pluripotent cell medium in which they were previously maintained. At day 1, 80 μl of medium was replaced with cortical differentiation medium (CDM) I, containing Glasgow-MEM (Gibco), 20% knockout serum replacement (Gibco), 0.1 mM minimum essential medium non-essential amino acids (MEM-NEAA) (Gibco), 1 mM pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), with 1% of penicillin–streptomycin solution (Corning). From day 0 to day 6, ROCK inhibitor Y-27632 (Millipore) was added to the medium at a final concentration of 20 μM. Patterning small molecules, WNT inhibitor IWR1 (Calbiochem) and TGFβ inhibitor SB431542 (Stem Cell Technologies), were added from day 0 to day 18, at a concentration of 3 μM and 5 μM, respectively.
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