organism: Homo sapiens
description: Protocols: Micro-dissected fetal brain tissues were snap-frozen and stored at -80°C until RNA extraction. hiPSCs lines: Fibroblasts of CTL1 (phenotypically normal male without intellectual disability or other physician-diagnosed neuropsychiatric diagnosis) were received from BC Children's Hospital, Vancouver. CTL2 (male) hiPSC line was received from the Wellcome Trust Sanger Institute. CTL3 (male) hiPSC line was received from the university of Sheffield. Fibroblasts of CTL04 (female) were received from Genomic and Genetic Disorders Biobank at the IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (Italy, affiliated to the Telethon Network of Genetic Biobanks). hiPSC were cultured in TeSR-E8 medium (Stemcell technologies, 05990), with daily media change, at 37 °C, 5 % CO2 and 3 % O2 in standard incubators. hiPSC were grown on matrigel-coated dishes (Corning, 354248) and passaged using ReLeSRTM (Stemcell technologies, 05872). hiPSCs reprogramming: Skin fibroblasts from four healthy individuals were reprogrammed using non integrating self-replicating mRNAs as previously described (Yoshioka et al., 2013) (Stemgent, 00-0071) (CTL1, CTL3 and CTL4) or Sendai virus (CTL2) (CytoTune-iPS 2.0 Sendai Reprogramming Kit; Thermo Fisher Scientific, A16517). CBO were generated using an adaptation of the previously described protocol published by Pasca et al in 2015 (26005811), introducing orbital shaking on day 12 of differentiation as described in (31607568). Briefly, hiPSC were grown on feeders for 3-4 days in a medium composed of 80% DMEM/F12 medium (1:1), 20% Knockout serum (Thermo Fisher Scientific, 10828028), 1% Non-essential amino acids (NEAA, Lonza BE13- 14E), 0.1 mM cell culture grade 2-mercaptoethanol solution (Thermo Fisher Scientific, 31350010), 2 mM L-Glutamine (Thermo Fisher Scientific, 25030081), P/S 100 U/mL, and FGF2 at a final concentration of 20 ng/mL (Thermo Fisher Scientific, PHG0021). Daily media change was performed. Embryoid bodies (EB) were generated by detaching hiPSC with dispase for 40 minutes and plating on ultralow attachment 10 cm plates (Corning, 3262) in the first differentiating medium composed of 80% DMEM/F12 medium (1:1), 20% Knockout serum, 1% NEAA, 0.1 mM cell culture grade 2-mercaptoethanol solution, 2 mM L-Glutamine, P/S, 100 U/mL, 7,5 μM Dorsomorphin (MedChem express, HY-13418A), 10 μM TGFβ inhibitor SB431542 (MedChem express, HY-10431), and ROCK inhibitor 5 μM. EB were grown in normal oxygen incubators. EB were left undisturbed for 1 day and at 48h media change was performed with differentiation medium 1 without ROCK inhibitor. Dorsomorphin and TGFβ inhibitor are used to perform Dual-SMAD inhibition, pushing neuroectoderm specification. Dual-SMAD inhibition was performed for a total of 5 days, with daily media change. On day 6 the second differentiation medium was added until day 25 with daily media change for the first 12 days, and then every other day. The second differentiation medium was composed of neurobasal medium (Thermo Fisher Scientific, 12348017) supplemented with 1X B-27 supplement without vitamin A (Thermo Fisher Scientific 12587001), 2 mM L-Glutamine, P/S, 100 U/mL, 20 ng/mL FGF2and 20 ng/mL EGF (Thermo Fisher Scientific, PHG0313). Human FGF2 and EGF were used to amplify the pool of neural progenitors. On day 12, CBO were moved to ultra-low attachment 10 cm dishes and grown on shakers to enhance oxygen and nutrient supply. On day 26, FGF2 and EGF were replaced with 20 ng/mL brain-derived neurotrophic factor (BDNF, Peprotech 450-02) and 20 ng/mLneurotrophin-3 (NT3, Peprotech 450-03) to promote differentiation of neural progenitors towards the glutamatergic fate. From day 43 onwards, BDNF and NT3 were removed and from day 50 the medium was supplemented with Amphotericinβ to prevent mould formation. CBO were generated using an adaptation of the previously described protocol published by Pasca et al in 2015 (26005811), introducing orbital shaking on day 12 of differentiation as described in (31607568). Briefly, hiPSC were grown on feeders for 3-4 days in a medium composed of 80% DMEM/F12 medium (1:1), 20% Knockout serum (Thermo Fisher Scientific, 10828028), 1% Non-essential amino acids (NEAA, Lonza BE13- 14E), 0.1 mM cell culture grade 2-mercaptoethanol solution (Thermo Fisher Scientific, 31350010), 2 mM L-Glutamine (Thermo Fisher Scientific, 25030081), P/S 100 U/mL, and FGF2 at a final concentration of 20 ng/mL (Thermo Fisher Scientific, PHG0021). Daily media change was performed. Embryoid bodies (EB) were generated by detaching hiPSC with dispase for 40 minutes and plating on ultralow attachment 10 cm plates (Corning, 3262) in the first differentiating medium composed of 80% DMEM/F12 medium (1:1), 20% Knockout serum, 1% NEAA, 0.1 mM cell culture grade 2-mercaptoethanol solution, 2 mM L-Glutamine, P/S, 100 U/mL, 7,5 μM Dorsomorphin (MedChem express, HY-13418A), 10 μM TGFβ inhibitor SB431542 (MedChem express, HY-10431), and ROCK inhibitor 5 μM. EB were grown in normal oxygen incubators. EB were left undisturbed for 1 day and at 48h media change was performed with differentiation medium 1 without ROCK inhibitor. Dorsomorphin and TGFβ inhibitor are used to perform Dual-SMAD inhibition, pushing neuroectoderm specification. Dual-SMAD inhibition was performed for a total of 5 days, with daily media change. On day 6 the second differentiation medium was added until day 25 with daily media change for the first 12 days, and then every other day. The second differentiation medium was composed of neurobasal medium (Thermo Fisher Scientific, 12348017) supplemented with 1X B-27 supplement without vitamin A (Thermo Fisher Scientific 12587001), 2 mM L-Glutamine, P/S, 100 U/mL, 20 ng/mL FGF2and 20 ng/mL EGF (Thermo Fisher Scientific, PHG0313). Human FGF2 and EGF were used to amplify the pool of neural progenitors. On day 12, CBO were moved to ultra-low attachment 10 cm dishes and grown on shakers to enhance oxygen and nutrient supply. On day 26, FGF2 and EGF were replaced with 20 ng/mL brain-derived neurotrophic factor (BDNF, Peprotech 450-02) and 20 ng/mLneurotrophin-3 (NT3, Peprotech 450-03) to promote differentiation of neural progenitors towards the glutamatergic fate. From day 43 onwards, BDNF and NT3 were removed and from day 50 the medium was supplemented with Amphotericinβ to prevent mould formation. Total RNA was extracted from snap-frozen pellets of CBO at day 25, 50, 100, 150, 200, fetal brain tissues using the RNeasy Mini Kit (Qiagen, 74104). Purified RNA was quantified using a NanoDrop spectrophotometer and RNA quality was checked with an Agilent 2100 Bioanalyzer using the RNA nano kit (Agilent, 5067-1512). Library preparation for RNA sequencing was performed according to TruSeq Total RNA sample preparation protocol (Illumina, RS-122-2202), starting from 250 ng - 1 μg of total RNA. cDNA library quality was assessed at on Agilent 2100 Bioanalyzer, using the high sensitivity DNA kit (Agilent 5067-4626).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA12118571
attributes.tag: INSDC center alias
attributes.value: High Definition Disease Modelling Lab, Stem Cell and Organoid Epigenetics, IEO, European Institute of Oncology, IRCCS, Milan, Italy. Department of Oncology and Hemato-oncology, University of Milan, Milan, Italy.
attributes.tag: INSDC center name
attributes.value: High Definition Disease Modelling Lab, Stem Cell and Organoid Epigenetics, IEO, European Institute of Oncology, IRCCS, Milan, Italy. Department of Oncology and Hemato-oncology, University of Milan, Milan, Italy.
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:57:42Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:57:42Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11239:3391B_DAY205_REP1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: hiPSCs derived cortical brain organoid
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: normal
attributes.tag: growth condition
attributes.value: organoid
attributes.tag: individual
attributes.value: 3391B (CTL4)
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: zone of skin
attributes.tag: progenitor cell type
attributes.value: human induced pluripotent stem cell
attributes.tag: replicate
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-11239:3391B_DAY205_REP1
attributes.tag: sex
attributes.value: female
attributes.tag: time
attributes.value: 200