HiPSCs from 3 typically developing individuals (CTR_M1; CTR_M2 and CTR_M3) were generated as previously described as part of EU-AIMS and STEMBANCC EU IMIs [12 , 15 (link)]. Ethical approval for the generation of hiPSCs was approved under the PiNDS study (REC: 13/LO/1218). HiPSCs were grown and maintained in E8 medium (Life Technologies) with E8 supplement (Life Technologies). HiPSCs were passaged by first dissociating attached cells from the plate, by incubating them in Versene (Thermo Fisher) for 4–5 min in 37 °C incubator. Versene helps gently lift edges of iPSC colonies, after which it was replaced with E8 and cells were lifted using a cell lifter. Free-floating cells were re-plated onto fresh geltrex-coated (Thermo Fisher) plates. For maintenance, cells were re-plated at ~ 70% confluency. For starting a new experiment, cells were re-plated at ~ 100% confluency.
E8 supplement
Manufactured by Thermo Fisher Scientific
Sourced in United States
The E8 supplement is a cell culture media supplement designed to support the growth and viability of cells in vitro. It provides a standardized mixture of essential nutrients, vitamins, and other growth factors required for optimal cell performance.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
B27 supplement, by Thermo Fisher Scientific (5 mentions)
E8 medium, by Thermo Fisher Scientific (4 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (4 mentions)
Neurobasal medium, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Geltrex coated, by Thermo Fisher Scientific (3 mentions)
Essential 8 medium, by Thermo Fisher Scientific (3 mentions)
Matrigel, by Corning (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (3 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (3 mentions)
N2 supplement, by Thermo Fisher Scientific (3 mentions)
Vitronectin, by Thermo Fisher Scientific (2 mentions)
Knockout dmem medium, by Thermo Fisher Scientific (2 mentions)
Pd0325901, by Merck Group (2 mentions)
Essential 8 flex medium, by Thermo Fisher Scientific (2 mentions)
Glutamax supplement, by Thermo Fisher Scientific (2 mentions)
Actinomycin d, by Merck Group (2 mentions)
15 protocols using e8 supplement
1
Generation and Maintenance of Human iPSCs
2
Generation and Maintenance of Human iPSCs
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HiPSCs from 3 typically developing individuals (CTR_M1; CTR_M2 and CTR_M3) were generated as previously described as part of EU-AIMS and STEMBANCC EU IMIs [cocks, deans, shum et al]. Ethical approval for the generation of hiPSCs were approved under the PiNDS study (REC: 13/LO/1218). HiPSCs were grown and maintained in E8 medium (Life Technologies) with E8 supplement (Life Technologies). HiPSCs were passaged by first dissociating attached cells from the plate, by incubating them in Versene (Thermo Fisher 15040066) for 4-5 minutes in 37°C incubator. Versene helps gently lift edges of iPSC colonies, after which it was replaced with E8 and cells were lifted using a cell lifter. Free-floating cells were replated onto fresh geltrex-coated (Thermo Fisher 12760013) plates. For maintenance, cells were re-plated at ~70% confluency. For starting a new experiment, cells were re-plated at ~100% confluency.
3
Culturing Common Marmoset Embryonic Stem Cells
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The common marmoset (C. jacchus) embryonic stem cell line cj36711 (link),12 (link) established at the Wisconsin National Primate Research Center (kindly provided by Dr. T. G. Golos) was cultured in E8 medium (Cat# A15169-01; Thermo Fisher Scientific, Waltham, MA), with E8 supplement (1X; Cat# A15171-01; Thermo Fisher Scientific); GlutaMAX (1X; Cat# 35050-061; Life Technologies, Grand Island, NY); lipid concentrate (1:100; Cat# 11905-031; Life Technologies); recombinant human nodal (100 ng/mL; Cat# 3218-ND; R&D Systems, Minneapolis, MN); and glutathione (1.94 μg/mL; Cat# G4251; Sigma Aldrich, St. Louis, MO). Cells were grown on matrigel-coated plates (0.083 μg/well; Cat# 354230; BD Biosciences, San Jose, CA) and passaged using Accutase (Cat# 7920; Stem Cell Technologies, Vancouver, Canada) when reaching 80% confluency.
4
Culturing Pluripotent H9 Cells
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H9 cells were cultured in 6-well plates coated with Matrigel (1 : 70 Matrigel (Corning): DMEM/F-12 Glutamax (Life Technologies)) in Essential 8 medium with E8 supplement (all Thermo Fisher). Cells were maintained at 37°C/5% CO2. Cultures were fed every day. Cells were subcultured every 4–6 days using Dispase (Life Technologies) or ReLeSR (Stem Cell Technologies) until colonies reached maximum size. Spontaneously differentiating cells were manually removed.
5
Profiling Transcriptional Dynamics in Cell Lines
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HCT116 (CCL-247), HeLa (CCL-2), HEK293T (CRL-3216), LN-18 (CRL-2610), U-2 OS (HTB-96) cell lines were obtained from ATCC and cultured according to recommended protocols. Human iPSC WTC-11 (Coriell Institute) cells were cultured on Vitronectin (Thermo Fisher Scientific) coated 6-well plates or glass coverslips (for smRNA FISH purposes) in Essential 8 Flex medium (Thermo Fisher Scientific) with E8 supplement (Thermo Fisher Scientific), Rock inhibitor and 2.5% penicillin-streptomycin. iPS cells we passaged with EDTA in dPBS. Mouse embryonic stem cells (Harvard Stem cell institute) were cultured on top of gelatin (0.1%, EMD Millipore) coated plates or glass coverslips (for smRNA FISH purposes). Embryonic stem cell media was prepared as follows: KnockOut DMEM medium (Thermo Fisher) supplemented with ESC FCS (Millipore Sigma), non-essential amino acids (Thermo Fisher), GlutaMAX supplement (Thermo Fisher), penicillin-streptomycin (Thermo Fisher), 50 mM 2-mercaptoethanol, LIF, CHIR99021 and PD0325901 (Sigma-Aldrich).
Actinomycin D (Sigma-Aldrich) was used at final concentration of 5 μg/mL in full growth media. Cell pellets and coverslips were harvested at 0, 40 min, 2.5 h and 4.5 h after adding Actinomycin D, and processed for RNA extraction and smRNA FISH as described below.
Actinomycin D (Sigma-Aldrich) was used at final concentration of 5 μg/mL in full growth media. Cell pellets and coverslips were harvested at 0, 40 min, 2.5 h and 4.5 h after adding Actinomycin D, and processed for RNA extraction and smRNA FISH as described below.
6
Actinomycin D Treatment of Stem Cells
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Cell lines were obtained from ATCC and cultured according to recommended protocols. Human iPS WT-11 cells were cultured on Vitronectin (Thermo Fisher Scientific) coated 6-well plates or glass coverslips (for smRNA FISH purposes) in Essential 8 Flex medium (Thermo Fisher Scientific) with E8 supplement (Thermo Fisher Scientific), Rock inhibitor and 2.5% penicillinstreptomycin. iPS cells we passaged with EDTA in dPBS. Mouse embryonic stem cells were cultured on top of gelatin (0.1%, EMD Millipore) coated plates or glass coverslips (for smRNA FISH purposes). Embryonic stem cell media was prepared as follows: KnockOut DMEM medium (Thermo Fisher) supplemented with ESC FCS (Millipore Sigma), non-essential amino acids (Thermo Fisher), GlutaMAX supplement (Thermo Fisher), penicillin-streptomycin (Thermo Fisher), 50 mM 2-mercaptoethanol LIF and 2i (and CHIR99021, Sigma-Aldrich and PD0325901, Sigma-Aldrich).
Actinomycin D (Sigma-Aldrich) was used at final concentration of 5 μg/mL in full growth media.
Cell pellets and coverslips were harvested at 0, 40 min, 2.5 h and 4.5 h after adding Actinomycin D, and processed for RNA extraction and smRNA FISH as described below.
Actinomycin D (Sigma-Aldrich) was used at final concentration of 5 μg/mL in full growth media.
Cell pellets and coverslips were harvested at 0, 40 min, 2.5 h and 4.5 h after adding Actinomycin D, and processed for RNA extraction and smRNA FISH as described below.
7
Reprogramming Human Fibroblasts to Pluripotent Stem Cells
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Human fibroblasts from patients (Sainio et al., 2018 (link)) and healthy controls (Trokovic et al., 2015 (link)) were reprogrammed into pluripotent stem cells with overexpression of MYC, KLF4, SOX2, and OCT4 by the Biomedicum Stem Cell Center (University of Helsinki, Finland) as in Trokovic et al. (2015 (link)). Stem cells were grown in feeder-free conditions with Matrigel (Corning) and E8 with E8 supplement (Gibco) in normoxia at 37°C. At 90% confluency, cells were passaged with 0.5 mM EDTA (Invitrogen) in PBS.
8
Directed Differentiation of Human iPSCs to Cardiomyocytes
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Human WT iPSC (WiCell, Madison, WI, USA), passage 18–25, were maintained on growth factor-reduced Matrigel (Corning Corp., Corning, NY, USA.) coated plates in E8 medium (Gibco, Gaithersburg, MD, USA) with E8 supplement (Gibco, Gaithersburg, MD, USA). Patient derived human iPS cells were reprogrammed from fibroblasts with Oct4, Sox2, Lin28, and Nanog. A directed differentiation protocol to derive cardiomyocytes using serum-free, chemically defined media supplemented with CHIR99021, IWP2, Activin A, and KY0211 in stage specific manner, as previously described [37 (link)]. This protocol yielded contractile clusters by days 9 to 12 post-differentiation. Monolayers ranging between 20–50 days of maturity were plated on Matrigel coated dishes and maintained with RPMI B27+ until use.
9
Fibroblast and Induced Pluripotent Stem Cell Media Formulations
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Fibroblast medium: DMEM (ThermoFisher), 10% fetal bovine serum (FBS, Hyclone), 1% non-essential amino acids (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 55 μM β-mercaptoethanol (ThermoFisher) and 1 mM sodium pyruvate (ThermoFisher). Naive medium (t2iLGoY)19 (link): 50:50 mixture of DMEM/F12 (ThermoFisher) and neurobasal medium (ThermoFisher), supplemented with 2 mM l -glutamine (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 0.5% N2 supplement (ThermoFisher), 1% B27 supplement (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 10 ng ml−1 human leukaemia inhibitory factor (made in-house), 250 μM l -ascorbic acid (Sigma), 10 μg ml−1 recombinant human insulin (Sigma), 1 μM PD0325901 (Miltenyi Biotec), 1 μM CHIR99021 (Miltenyi Biotec), 2.5 μM Gö6983 (Tocris), 10 μM Y-27632 (Abcam). Primed hiPS cell medium (KSR/FGF2): DMEM/F12 (ThermoFisher), 20% knockout serum replacement (KSR) (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 1% non-essential amino acids (ThermoFisher), 50 ng ml−1 recombinant human FGF2 (Miltenyi Biotec), 1% penicillin-streptomycin (ThermoFisher). Primed hiPS cell medium (Essential 8 (E8)): 10 ml of E8 supplement (Gibco) to 500 ml medium basal (Gibco), supplemented with 1% penicillin-streptomycin (Gibco).
10
Cerebellar Organoid Electroporation Protocol
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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.
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