Forebrain organoids were generated as previously described60 (link). On Day 0, iPSC colonies were detached by ReLeSR and aggregated to form EBs by Aggrewell (STEMCELL Technologies, #34811). The following day, EBs were resuspended and transferred to 6-well plate (Corning Costar) rotating at 110 rpm, containing DMEM/F12, 20% KnockOut Serum Replacement, 1X Non-essential Amino Acids, 1X GlutaMax, 1 μM LDN193189, 5 μM SB-431542 and 2 μg/mL heparin (STEMCELL Technologies). On Day 6, half of the medium was replaced with induction medium consisting of DMEM/F12, 1X N2 Supplement, 1X Non-essential Amino Acids, 1X GlutaMax, 1 μM CHIR99021 and 1 μM SB-431542. On Day 7, organoids were embedded in Matrigel and cultured stationarily in Low-attachment plate (Corning Costar) in the induction medium. On Day 14, organoids were mechanically dissociated from Matrigel by manual pipetting in a 10 mL pipette tip. Organoids were transferred to 6-well plate rotating at 110 rpm, containing differentiation medium consisting of DMEM:F12/Neurobasal, 1X N2 and 1X B27 Supplements, 1 × 2-Mercaptoenthanol, 1X Non-essential Amino Acids, and 2.5 mg/ml human Insulin. From Day 35 to Day 60, 1% Matrigel was supplemented in differentiation medium.
Gracia-Diaz C., Zhou Y., Yang Q., Maroofian R., Espana-Bonilla P., Lee C.H., Zhang S., Padilla N., Fueyo R., Waxman E.A., Lei S., Otrimski G., Li D., Sheppard S.E., Mark P., Harr M.H., Hakonarson H., Rodan L., Jackson A., Vasudevan P., Powel C., Mohammed S., Maddirevula S., Alzaidan H., Faqeih E.A., Efthymiou S., Turchetti V., Rahman F., Maqbool S., Salpietro V., Ibrahim S.H., di Rosa G., Houlden H., Alharbi M.N., Al-Sannaa N.A., Bauer P., Zifarelli G., Estaras C., Hurst A.C., Thompson M.L., Chassevent A., Smith-Hicks C.L., de la Cruz X., Holtz A.M., Elloumi H.Z., Hajianpour M.J., Rieubland C., Braun D., Banka S., French D.L., Heller E.A., Saade M., Song H., Ming G.L., Alkuraya F.S., Agrawal P.B., Reinberg D., Bhoj E.J., Martínez-Balbás M.A, & Akizu N. (2023). Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders. Nature Communications, 14, 4109.
Other organizations :
Children's Hospital of Philadelphia, University of Pennsylvania, University College London, National Hospital for Neurology and Neurosurgery, Consejo Superior de Investigaciones Científicas, Institut de Biologia Molecular de Barcelona, Seoul National University, Translational Therapeutics (United States), Universitat Autònoma de Barcelona, Vall d'Hebron Institut de Recerca, Helen DeVos Children's Hospital, Boston Children's Hospital, University of Manchester, University Hospitals of Leicester NHS Trust, Leicester Royal Infirmary, Guy's Hospital, King Faisal Specialist Hospital & Research Centre, University of Health Sciences Lahore, Aga Khan University Hospital, University of Messina, Buraydah Colleges, Saudi Aramco Medical Services Organization, Centogene (Germany), Temple University, University of Alabama at Birmingham, HudsonAlpha Institute for Biotechnology, Kennedy Krieger Institute, Albany Medical Center Hospital, University Hospital of Bern, University of Bern, St Mary's Hospital, Manchester University NHS Foundation Trust, Genomics England, Alfaisal University, University of Miami Hospital, Jackson Memorial Hospital, NYU Langone Health
Aggregation of detached iPSC colonies to form EBs using Aggrewell
Culture of EBs in rotating 6-well plates containing DMEM/F12, 20% KnockOut Serum Replacement, 1X Non-essential Amino Acids, 1X GlutaMax, 1 μM LDN193189, 5 μM SB-431542 and 2 μg/mL heparin
Replacement of half the medium with induction medium consisting of DMEM/F12, 1X N2 Supplement, 1X Non-essential Amino Acids, 1X GlutaMax, 1 μM CHIR99021 and 1 μM SB-431542 on Day 6
Embedding of organoids in Matrigel and culturing them stationarily in Low-attachment plate in the induction medium on Day 7
Mechanical dissociation of organoids from Matrigel by manual pipetting on Day 14
Transfer of dissociated organoids to 6-well plate rotating at 110 rpm, containing differentiation medium consisting of DMEM:F12/Neurobasal, 1X N2 and 1X B27 Supplements, 1 × 2-Mercaptoenthanol, 1X Non-essential Amino Acids, and 2.5 mg/ml human Insulin
Supplementation of 1% Matrigel in differentiation medium from Day 35 to Day 60
dependent variables
Not explicitly mentioned
control variables
Rotation speed of 6-well plates at 110 rpm
Concentration of LDN193189 (1 μM), SB-431542 (5 μM and 1 μM), CHIR99021 (1 μM), and human Insulin (2.5 mg/ml) in the culture media
positive controls
Not mentioned
negative controls
Not mentioned
Annotations
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