mTeSR1-grown hPSCs (H1, H7, and H9 [WiCell] and ESI035 [ESI BIO]) were seeded for differentiation as single cells using Accutase (Millipore) onto Geltrex-coated plates (Thermo Fisher), at a density of 40,000 cells per well of a 12-well plate. hPSCs were differentiated into anterior primitive streak and, subsequently, into definitive endoderm by treatment with definitive endoderm Induction Medium A for 24 hr, followed by definitive endoderm Induction Medium B for 24 hr (Thermo Fisher) (Loh et al., 2014 (link), 2016 (link)). For detailed methods, see Supplemental Information .
Geltrex coated plate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Geltrex-coated plates are a type of cell culture plate pre-coated with Geltrex, a basement membrane extract derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Geltrex provides a suitable extracellular matrix for the attachment and growth of various cell types, including stem cells and primary cells.
Automatically generated - may contain errors
Lab products found in correlation
Mtesr1 medium, by STEMCELL (6 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Accutase, by STEMCELL (3 mentions)
Essential 8 medium, by Thermo Fisher Scientific (3 mentions)
Accutase, by Merck Group (2 mentions)
Definitive endoderm induction medium a, by Thermo Fisher Scientific (2 mentions)
Definitive endoderm induction medium b for 24 hr, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Mtesr1 media, by STEMCELL (2 mentions)
Tesr e8 medium, by STEMCELL (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Chir99021, by Merck Group (2 mentions)
Stempro accutase, by Thermo Fisher Scientific (2 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
Abt 263, by Santa Cruz Biotechnology (1 mentions)
30 protocols using geltrex coated plate
1
Directed Differentiation of hPSCs to Definitive Endoderm
2
Directed Differentiation of hPSCs to Definitive Endoderm
3
Glioblastoma Stem Cell Characterization
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The GSC-ECLs used in this study were previously isolated from human biopsies following relevant guidelines and national regulations. These cell lines have been characterized as described in previous reports9 (link),10 (link),43 (link),44 (link). Prior to their isolation, the use of these cell lines for research purposes have been approved by the Biomedical Research Ethics Committee “Comité de Ética en Investigaciones Biomédicas de la Fundación para la Lucha contra Enfermedades Neurológicas de la Infancia (FLENI)”. Written informed consent was received from each patient whose tumor tissue was used. NP cells were derived from human embryonic stem cells (WA09, provided by University of Wisconsin—Dr. J. Thomson. hPSCReg ID: WAe009-A)51 (link). Both GSC-ECLs and NPs were grown in a serum-free medium consisting of Neurobasal supplemented with N2, B27, 20 ng/ml epidermal growth factor (EGF), 20 ng/ml basic fibroblast growth factor (bFGF) and plated onto Geltrex-coated plates (10 μg/ml) (all from Thermo Scientific, Rockford, IL, USA). BH3-mimetics and chemotherapeutic agents: ABT-263 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), WEHI-539 (Genentech USA Inc, San Francisco, CA, USA), S63845 (Cayman Chemical, Ann Arbor, MI, USA), TMZ, VCR, and CCNU (Sigma, St. Louis, MO, USA).
4
Parkinson's Disease Fibroblast-Derived iPSC Protocol
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Fibroblasts of a patient with PD harboring the LK2GS mutation (ND38262, ND14317) and healthy control subjects (AG02261) were purchased from the Coriell Institute (Camden, NJ, USA) and reprogrammed into iPSC [36 (link),37 (link)]. In addition, ND14317 was corrected to generate the KIOMi002-A WT iPSC line to prepare an isogenic WT/G2019S pair [38 (link)]. iPSCs were differentiated into pNSCs and cultured as follows [31 (link)]. Healthy control-derived (WT) pNSCs and PD patient-derived (GS) pNSCs were plated on Geltrex™-coated plates with a pNSC medium: 50% Advanced DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA), 50% Neurobasal medium (Thermo Fisher Scientific, Waltham, MA, USA), N-2 Supplement (100X, Thermo Fisher Scientific, Waltham, MA, USA), B-27 Supplement (50X, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM GlutaMax™ (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), 3 uM CHIR 99021 (Tocris Bioscience, Bristol, UK), 2 uM SB 431542 (Tocris Bioscience, Bristol, UK), 10 ng/mL human LIF (PeproTech, Inc., Rocky Hill, NJ, USA), and 5 ug/mL BSA (Sigma-Aldrich, St. Louis, MO, USA). The medium was replaced every alternate day. WT- and LK2GS-pNSCs were dissociated with Accutase cell detachment solution (Millipore Sigma, Burlington, MA, USA) every five–seven days.
5
Inducible Neurogenesis from Human iPSCs
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The human iPSC cell line WTC11 carrying a doxycycline-inducible Ngn2 transgene (Miyaoka et al. 2014 (link); Wang et al. 2017 (link)) were cultivated as previously described (Bell et al. 2021 (link); Buchholz et al. 2022 ). Briefly, iPSCs were cultured on GelTrex™-coated plates (1X, ThermoFisher Scientific) in StemMACS™ iPS-Brew XF (Miltenyi). When reaching confluence, the cultures were passaged with Versene™ passaging solution (ThermoFisher Scientific) and seeded in thiazovivine-supplemented (Axon Medchem) iPS-Brew for 1 day. Cells were grown at 37 °C and 5% CO2 in a humidified incubator.
6
Derivation and Characterization of Human iPSCs
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Euploid and X-monosomic hiPSC were derived from female and male donors, and characterized for pluripotency markers, as well as DNA methylation and X-chromosome inactivation in female euploid hiPSCs via continued XIST expression in RNA-seq as previously described12 (link),57 (link). Human iPSCs were cultured in feeder-free conditions on GelTrex-coated plates (ThermoFisher Scientific) in mTeSR-1 media (Stem Cell Technologies) in 5% CO2 at 37°C. iPSCs were passaged with 0.5M EDTA at least weekly in small aggregates.
7
Organoid SARS-CoV-2 Infection Assay
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Organoids were plated on geltrex-coated plates (12760013, Thermo Fisher) with 100,000 cells per well, and infected at an MOI of 1. At 2 h after addition of the inoculum, the supernatant was removed, cells were washed with PBS and fresh HAO medium was added. Supernatants were harvested for a plaque assay at 24 and 48 h.
8
Maintenance of hiPSC Line BC1-eGFP
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The BC1–eGFP (enhanced green fluorescent protein) (45 (link)) hiPSC line was obtained from L. Cheng (Johns Hopkins University, Baltimore, MD, USA), with verified normal karyotype and was contamination free. hiPSCs were maintained on Geltrex-coated plates (Thermo Fisher Scientific, A1413302) with TeSR-E8 medium (STEMCELL Technologies, 05940) in a 37°C, 5% CO2 humidified incubator. Cells were passaged with ACCUTASE (STEMCELL Technologies, 07920) every 4 to 5 days at ~70% confluence, and autodifferentiated cells were marked and mechanically removed before passaging. We added 10 μM Y-27632 (STEMCELL Technologies, 72304) into the TeSR-E8 medium on the first day after passaging.
9
Inducible gene expression in human embryonic stem cells
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Human embryonic stem cells HES3 were generously donated by Edouard Stanley’s Lab. Cells were maintained in an undifferentiated state under feeder-free conditions on GelTrex-coated plates (15 µg/mL) (Thermo Fisher Scientific, USA). Cells were fed daily with mTeSR medium (STEM CELL Technologies, Canada) and maintained at 37 °C in a humidified atmosphere with 5% CO2 in air. When cells reached approximately 70–80% of confluence, colonies were dissociated using TrypLE 1x (Thermo Fisher Scientific, USA) and cells were seeded on new culture dishes previously coated with Geltrex in mTeSR medium (STEMCELL Technologies, Canada) containing ROCK Inhibitor (Y-27632, Tocris, UK).
For experiments, cells were seeded in mTeSR medium. Once cells attached to the plate (typically after 2 h), medium was changed and doxycycline (Dox) (Doxycycline hyclate, Sigma Aldrich, USA) was added. Cells were incubated with Dox during 48, 72 and 96 h and Dox was renewed daily with medium change. At each of the indicated time points, material was collected to be analyzed later.
For experiments, cells were seeded in mTeSR medium. Once cells attached to the plate (typically after 2 h), medium was changed and doxycycline (Dox) (Doxycycline hyclate, Sigma Aldrich, USA) was added. Cells were incubated with Dox during 48, 72 and 96 h and Dox was renewed daily with medium change. At each of the indicated time points, material was collected to be analyzed later.
10
Differentiation of iPSCs to Neural Progenitors
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On day –1, iPSCs were dissociated with gentle cell dissociation reagent (Stem Cell, 100-0485) and plated on Geltrex-coated plates (Thermo Fisher, A1413302) as a single-cell suspension with StemFlex media and Y-27632. On day 0, media were changed to StemFlex plus 0.5 μg/mL doxycycline (MilliporeSigma, D9891). On day 1, media were refreshed with StemFlex plus doxycycline. On day 2, cells were washed with 1× DPBS (Gibco, 14190-144), treated with accutase to create a single-cell suspension, and replated onto Matrigel and cultured with 3 N media (4 mL pen/strep, Gibco, 15140; 250 mL DMEM/F12, Gibco, 11320-033; Neurobasal Gibco, 21103-049; 125 μL 10 mg/mL insulin, MilliporeSigma, I9278; 2.5 mL Non Essential Amino Acids, Gibco, 11140-050; 2.5 mL N 2 supplement, Gibco, 17502-048; 5 mL B27 supplement, Gibco, 17504-044; 2 μL b-mercaptoethanol stock [12 M], MilliporeSigma, M7522; and 2.5 mL Glutamax Gibco, 35050-061 containing Y-27632 and doxycycline). Media were replaced with 3 N plus doxycycline on day 3. On day 4 and on, one-half of media were replaced daily with 3 N + doxycycline.
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