Geltrex coated dishes

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Geltrex-coated dishes are a type of cell culture substrate designed for the growth and maintenance of various cell types. The dishes are pre-coated with Geltrex, a soluble form of the extracellular matrix, providing a natural and biologically relevant surface for cell attachment and proliferation.

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10 protocols using geltrex coated dishes

Induced Pluripotent Stem Cell Generation

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Primary human
fibroblasts were
maintained in mouse embryonic fibroblast (MEF) containing Dulbecco’s
modified Eagle’s medium (DMEM) glutamax (Life Technologies),
supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin
(Life Technologies). Viral transduction was performed as described
previously.^{76 (link)} Briefly, a lentiviral vector
expressing OCT4, KLF4, SOX2, c-MYC, and a mixture containing MEF medium
and 4 mg/mL hexadimethrine bromide (Sigma) was used. Cells were incubated
for 24 h in this mixture, followed by MEF medium for 5 days. Hereafter,
cells were transferred to irradiated MEFs in human embryonic stem
cell (huES) medium containing DMEM-F12 (Life Technologies), knockout
serum replacement (Life Technologies), penicillin/streptomycin, l-glutamine (Life Technologies), non-essential amino acids (Life
Technologies), β-mercaptoethanol (Merck Millipore), and 20 ng/mL
recombinant human fibroblast growth factor-basic (bFGF; Life Technologies).
Potential iPSC colonies were selected on the basis of their embryonic
stem cell-like morphology. Feeder-free iPSCs were cultured on Geltrex-coated
dishes (Life Technologies) in mTeSR1 medium (Stem Cell Technologies)
and passaged enzymatically with Accutase (Innovative Life Technologies).
All cell lines were tested for mycoplasma contamination every other
week.

Varderidou-Minasian S., Verheijen B.M., Harschnitz O., Kling S., Karst H., van der Pol W.L., Pasterkamp R.J, & Altelaar M. (2021). Spinal Muscular Atrophy Patient iPSC-Derived Motor Neurons Display Altered Proteomes at Early Stages of Differentiation. ACS Omega, 6(51), 35375-35388.

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Generation and Characterization of hiPSCs

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PBMCs or fibroblasts obtained either from human fetal liver tissues or human adult skin were isolated after collagenase dissociation and reprogrammed into iPSCs with the integration-free based Sendai virus (Cytotune 2.0 kit catalog #A16517 from Life Technologies). Fibroblasts were used at low population doubling (<5) to insure high efficiency of reprogramming. Emerging hiPSC colonies were manually picked and cultured under feeder-free conditions in Essential 8 medium on Geltrex-coated dishes (Life Technologies). hiPSC clones were maintained in Essential 8 Flex medium (Life Technologies) in feeder-free conditions and passaged at least 15 times to increase stable pluripotency. hiPSC generation and characterization were performed in the iPSC cell reprogramming core facility of CHU Sainte-Justine. hiPSC colonies were stained with antibodies for anti-human SSEA-4, Sox2, OCT4, and TRA1-60 followed by incubation with appropriate ALEXA-conjugated secondary antibodies using the pluripotent Stem Cell 4-Marker Immunocytochemistry Kit following the manufacturer's instructions (catalog #A24881 from Life Technologies). Karyotypes were produced by G-banding and analyzed by the CHU Sainte-Justine Cytogenetic Department.

Benabdallah B., Désaulniers-Langevin C., Colas C., Li Y., Rousseau G., Guimond J.V., Haddad E, & Beauséjour C. (2019). Natural Killer Cells Prevent the Formation of Teratomas Derived From Human Induced Pluripotent Stem Cells. Frontiers in Immunology, 10, 2580.

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Differentiation of iPSCs to Neurons

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HESCs (line I3) were maintained and differentiated to rosette-forming neural stem cells (NSCs) according to standard protocols and differentiated to neurons as described previously (Additional file 9) [14 (link)]. iPSCs were obtained and validated according to established protocols. In brief, NSCs were transduced with lentiviral TetON-system in order to generate a stable inducible cell line harboring the reprogramming factors OCT4 and KLF4 (pLVXTP-Tet-On (Clontech), FUW-OCT4, and FUW-KLF4 (Addgene)). Cells were induced to reprogram by the addition of 1 μg/ml doxycycline (Sigma-Aldrich) and cultured on irradiated mouse embryonic fibroblasts in Knockout Dulbecco’s modified Eagle’s medium containing 20% serum replacement, 1% non-essential amino acids, 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol, and 4 ng/ml FGF2. doxycycline was withdrawn upon colony formation, and doxycycline-independent colonies were mechanically isolated and further propagated. iPSC lines were differentiated to NSC and cultured as described [14 (link)]. Terminal differentiation of NSC was performed on Geltrex-coated dishes (Life technologies) in DMEM/F12 and Neurobasal (B27 supplement 1:50, penicillin/streptomycin 1:100, cAMP 300 ng/ml, and 1.6 g/l glucose) mixed at a 1:1 ratio. Neurons were cultured for 2, 4, and 6 weeks, and media were changed every second day (Additional file 9).

de Boni L., Gasparoni G., Haubenreich C., Tierling S., Schmitt I., Peitz M., Koch P., Walter J., Wüllner U, & Brüstle O. (2018). DNA methylation alterations in iPSC- and hESC-derived neurons: potential implications for neurological disease modeling. Clinical Epigenetics, 10, 13.

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Generating Polyclonal GFP-Labeled hiPSCs

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HiPSCs (Cellartis hIPS4 cell line) cells were transfected using the Neon transfection System (Life Technologies, Cat. No. MPK10025). Briefly, a mix of aPX1-pm-eGFP construct and piggyback transposase (Yusa et al., 2011 (link)), with a final concentration of 1–2 μg/μl, was added to a solution of 10⁷ cells and a single pulse of 1100 V with 30 ms duration was delivered via the Neon transfection System. Transfected cells were then plated back on Geltrex coated dishes (10 μg/cm², Life Technologies) and cultured in DEF-based medium supplemented with bFGF (30 ng/ml, Peprotech) and Noggin (10 ng/ml, Peprotech). In this way, a polyclonal line ChiPS4-pm-GFP was created, and this was used in the experiments reported here.

Dady A., Davidson L., Halley P.A, & Storey K.G. (2022). Human spinal cord in vitro differentiation pace is initially maintained in heterologous embryonic environments. eLife, 11, e67283.

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Feeder-free hESC Maintenance Protocol

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Cell lines used in this study were derived from the RUES2 cell line, part of the NIH Human Embryonic Stem Cell Registry (RUES2-NIHhESC-09-0013). hESCs were maintained in mouse embryonic fibroblast conditioned HUES Media in feeder-free conditions (conditioned media—CM), supplemented with bFGF 20 ng/mL, and replaced daily. hESCs were passaged at 70%–80% confluency every 3–4 days into Geltrex-coated dishes (Life Technologies) using Gentle Cell Dissociation Reagent (STEMCELL Technologies). Cells were tested for Mycoplasma spp. at 2-monthly intervals.

Laundos T.L., Li S., Cheang E., De Santis R., Piccolo F.M, & Brivanlou A.H. (2023). Huntingtin CAG-expansion mutation results in a dominant negative effect. Frontiers in Cell and Developmental Biology, 11, 1252521.

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Febrile Liver Fibroblasts Reprogrammed to hiPSCs

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Fibroblasts were first isolated either from human fetal liver tissues or human skin after collagenase dissociation. Single cell fibroblasts cultures were then reprogrammed into hiPSCs using integration‐free Sendai virus (Cytotune 2.0 kit catalog # A16517 from Life Technologies). Fibroblasts were used at low population doubling (between 5 and 10) to increase efficiency of reprogramming. Emerging colonies from transduced cells were manually picked and cultured under feeder‐free conditions in Essential 8 and Essential 8 Flex medium on Geltrex‐coated dishes (Life Technologies). hiPSC clones were passaged at least 15 times to increase stable pluripotency. hiPSC generation and characterization were done in the iPSC‐cell reprogramming core facility of CHU Sainte‐Justine. hiPSC colonies were stained with the antibodies for anti‐human SSEA‐4, Sox2, OCT4, and TRA1‐60 overnight at 4°C using the pluripotent Stem Cell 4‐Marker Immunocytochemistry Kit (catalog # A24881 from Life Technologies), followed by incubation with an ALEXA secondary antibodies for 30 minutes at room temperature. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Karyotypes were produced by G‐banding and analyzed by the CHU Ste‐Justine cytogenetic department.

Benabdallah B., Désaulniers‐Langevin C., Goyer M., Colas C., Maltais C., Li Y., Guimond J.V., Tremblay J.P., Haddad E, & Beauséjour C. (2020). Myogenic progenitor cells derived from human induced pluripotent stem cell are immune‐tolerated in humanized mice. Stem Cells Translational Medicine, 10(2), 267-277.

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Human Pluripotent Stem Cell Maintenance

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Human pluripotent stem cell line H9 [46, XX, (Wicell)] was used in this study (24 (link)). hPSCs were maintained on Matrigel® (BD Biosciences) coated dishes with StemPro (Thermo Fisher Scientific) or Essential 8 medium (Thermo Fisher Scientific). During maintenance, the cells were split with 0.5 mM EDTA (Thermo Fisher Scientific, MA) and plated in 1:3–1:8 ratios. Medium was changed daily and before the differentiation, cells were plated on Geltrex-coated dishes (Thermo Fisher Scientific) and grown until >90% confluency.

Yellapragada V., Liu X., Lund C., Känsäkoski J., Pulli K., Vuoristo S., Lundin K., Tuuri T., Varjosalo M, & Raivio T. (2019). MKRN3 Interacts With Several Proteins Implicated in Puberty Timing but Does Not Influence GNRH1 Expression. Frontiers in Endocrinology, 10, 48.

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Feeder-Free and Stroma-Supported iPSC Culture

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iPSCs were cultured either in feeder-free or in classical stroma-supported murine embryonic fibroblast (MEF) feeder conditions. Feeder-free cultures were performed in Geltrex coated dishes (ThermoFisher Scientific, Illkirch, France, A1413201) and fed daily with Essential 8 flex Medium (ThermoFisher Scientific, A2858501). iPSCs were passaged twice a week in aggregates with EDTA dissociation. Alternatively, they were maintained on mitomycin-C-inactivated Mouse Embryonic Fibroblasts (MEF) feeder cells with DMEM KnockOut (Gibco, Illkirch, France, 10829018) supplemented with 10% Knock-Out Serum Replacement (Gibco, Illkirch, France, 10828010), Glutamax (1X) (Gibco, Illkirch, France, 35050061), Penicillin/Streptomycin (100 U/mL) (Gibco, Illkirch, France, 15140122), β-mercaptoethanol, and basic FGF (0.1 mg/mL) (Miltenyi Biotec, Paris, France, 130-093-840). For passaging iPSCs cultured on MEFs, cells were incubated with collagenase (10 mg/mL) (Fisher Scientific, Illkirch, France, 10780004) for 5–7 min at 37 °C, 5% CO₂, and the appropriate amount of aggregates was used to seed new MEF-coated dishes.

Imeri J., Desterke C., Marcoux P., Telliam G., Sanekli S., Barreau S., Erbilgin Y., Latsis T., Hugues P., Sorel N., Cayssials E., Chomel J.C., Bennaceur-Griscelli A, & Turhan A.G. (2023). Modeling Blast Crisis Using Mutagenized Chronic Myeloid Leukemia-Derived Induced Pluripotent Stem Cells (iPSCs). Cells, 12(4), 598.

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Generating NOGGIN-Conditioned Medium

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Collecting NOGGIN-conditioned medium: RUES2-ePB-TRE::V5-NOGGIN cells were cultured on Geltrex-coated dishes (Thermo Fisher; A1413302) at a density of 2.5 × 10⁵ cells cm⁻². The cells were stimulated with 0.2 μg ml⁻¹ doxycyline for in 2 ml E8 36 h. NOGGIN-conditioned E8 medium was then centrifuged to remove cell debris, and supplemented with 1X Knockout-SR, 1X MEM NEAA, 1X GlutaMAX, 1X Insulin/Transferrin/Selenium, 1X Pyruvate, 1X B27 without vitamin A, 1X 2-Mercaptoethanol, and 20 ng ml⁻¹ bFGF.
NOGGIN-conditioned medium was added to either the apical or basal compartment of transwell filter cassette before being collected after 36 h for western blotting as described above.
Epithelium membrane integrity was assessed via fluorescent-conjugated 40-kDA Dextran-TxRed/40-kDA Dextran-FITC permeability assay and immuno visualization of an apical meshwork of ZO-1.

Phan-Everson T., Etoc F., Li S., Khodursky S., Yoney A., Brivanlou A.H, & Siggia E.D. (2021). Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport. Developmental cell, 56(13), 1930-1944.e5.

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Maintenance of Feeder-Free hiPSC Lines

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Established hiPSC lines were maintained in feeder‐free culture conditions in either mTeSR1 (STEMCELL Technologies) or iPS‐Brew (Miltenyi Biotec, Bergisch Gladbach, Germany) on Geltrex coated dishes (Thermo Fisher Scientific, Waltham, MA) as described previously.⁽⁹⁾ In‐house‐derived hiPSC line Sv20 derived from peripheral blood mononuclear cells and the K3 hiPSC line produced from foreskin fibroblasts were used for the studies, which were characterized previously in detail.⁽⁵, ⁹⁾

Abbey D., Elwyn S., Hand N.J., Musunuru K, & Rader D.J. (2020). Self‐Organizing Human Induced Pluripotent Stem Cell Hepatocyte 3D Organoids Inform the Biology of the Pleiotropic TRIB1 Gene. Hepatology Communications, 4(9), 1316-1331.

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