200 × 104 hUC-iPSCs were transfected with 3 μg Puc57-DsRed-Neo plasmid or Puc57-AAVS1-EF1a-DsRed-PA-NEO and 2 μg CRISPR-CAS9 AAVS1-DsRed plasmids by electrotransfection. Cells were seeded into the 6 well culture plate pre-coated by Matrigel in 3 mL mTeSR1 with 1.2 μM Thiazovivin (Selleck, S1459). After 24 h, the medium was changed to mTeSR1 with 120 μg/mL G418 for 2–3 day. The DsRed cells were sorted by flow cytometry (MoFlo Astrisos) to 96 well culture plate and 6 well culture plate to obtain the monoclonal pluripotent stem cells.
Thiazovivin
Manufactured by Selleck Chemicals
Sourced in United States, Canada, Netherlands
Thiazovivin is a small-molecule compound that acts as a selective inhibitor of the protein tyrosine phosphatase SHP2. It is commonly used in cell culture and research applications involving the study of cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (5 mentions)
Mtesr1, by STEMCELL (4 mentions)
Chir99021, by Selleck Chemicals (3 mentions)
Puromycin, by Thermo Fisher Scientific (3 mentions)
Versene, by Thermo Fisher Scientific (2 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (2 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (2 mentions)
Accutase, by Merck Group (2 mentions)
Basic fibroblast growth factor (bfgf), by Sino Biological (2 mentions)
Vascular endothelial growth factor (vegf), by Sino Biological (2 mentions)
Thrombopoietin, by Sino Biological (2 mentions)
Activin a, by Sino Biological (2 mentions)
Ly294002, by Selleck Chemicals (2 mentions)
Mycoalert kit, by Lonza (2 mentions)
Mtesr1 medium, by STEMCELL (2 mentions)
Nucleofector 2b device, by Lonza (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Mtesr1, by Selleck Chemicals (2 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
18 protocols using thiazovivin
1
Generation of DsRed-expressing human iPSCs
2
Directed Cardiomyogenic Differentiation of hESCs and hiPSCs
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Culture and directed cardiomyogenic differentiation of the hESC line HES3 NKX2-5eGFP was performed as described previously.39 (link),40 (link) hiPSC line Phoenix (hHSC_Iso4_ADCF_SeV-iPS2, alternative name: MHHi001)41 and hCBiPSC242 (link) were cultured on Geltrex (Gibco #A1413302) coated cell culture plates in StemMACS full medium with supplements. The confluent hiPSCs were passaged every 5 days with Versene (Gibco #15040-066) in StemMACS (Miltenyi #130-104-368) full medium supplemented with 2 μM Thiazovivin (Selleckchem #S1459). HiPSC-derived CMs were differentiated and maintained as described previously43 (link),44 (link).
3
Directed Hematopoietic Differentiation of hPSCs
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Before haematopoietic differentiation, hPSCs were dissociated by Accutase (Sigma) and plated on growth factor‐reduced Matrigel (Corning)‐coated plates with thiazovivin (0.1 μM, Selleck). Firstly, at day 0, 40 ng/ml of BMP4 (Peprotech), 30 ng/ml of ACTIVINA (Sino Biological Inc.), 20 ng/ml of bFGF (Sino Biological Inc.), 6 μM CHIR99021 (Selleck) and 10 μM LY294002 (Selleck) were added to the basic medium (BM, mimics of the CustommTeSR1) of Dulbecco's‐modified Eagle's medium/F‐12 (GIBCO) supplemented with 1% insulin–transferrin–selenium (GIBCO), 70 μg/ml of vitamin C (Sigma). Second, 30 ng/ml of BMP, 1 μM A8301 (Selleck) and 2 μM IWR‐1‐endo (Selleck) were added to the BM on day 1. Then, on days 2–4 of differentiation, 40 ng/ml of vascular endothelial growth factor (Sino Biological Inc.) and 50 ng/ml of bFGF were added to the BM. Finally, 40 ng/ml of vascular endothelial growth factor, 50 ng/ml of bFGF, 10 μM SB431542 (Selleck), 10 ng/ml of stem cell factor (Peprotech), 50 ng/ml of thrombopoietin (Sino Biological Inc.), 10 ng/ml ofinterleukin 3 (Sino Biological Inc.) and 50 ng/ml of interleukin 6 (Sino Biological Inc.) were added in the BM at days 4–6 of differentiation and further haematopoietic commitment and maturation.
4
Optimized Culture of Pluripotent Stem Cells
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Cells were routinely maintained in E8 (made as above) on either Synthemax II-SC (625 ng/cm2) or 1:200 growth factor-reduced Matrigel (9 µg/cm2) and passaged every four days using 0.5 mM EDTA (as above). 2 µM thiazovivin (Selleck Chemicals) was added for the first 24 h after passage. Control hESC lines H7 (WA07) and H9 (WA09)47 (link) were supplied by WiCell Research Institute. All hESC and hiPSC lines were converted to E8/EDTA-based culture for at least 5 passages before beginning differentiation. For growth comparison experiment cells were grown in mTeSR1 (Stemcell Technologies). Cell lines were used between passages 20 and 83. All cultures (primary, pluripotent and differentiation) were maintained with 2 mL medium per 9.6 cm2 of surface area or equivalent. All pluripotent cultures were routinely tested for mycoplasma using a MycoAlert Kit (Lonza).
5
Optimized Culture of Pluripotent Stem Cells
6
Directed Differentiation of hESCs
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hESCs were dissociated using 0.5 mM EDTA (Life Technologies) in PBS without CaCl2 or MgCl2 (Corning, 21-040-CV) for 7 min at room temperature. Cells were plated at 3 × 105 cells per well of a 12-well plate in mTeSR1 medium (Stem Cell Technologies) supplemented with 2 µM thiazovivin (Selleck Chemicals) for the first 24 h after passage. Cells were fed daily for 3–5 d until they reached ≥90% confluence, at which time they were washed with PBS, and the medium was changed to basal differentiation medium (BDM) consisting of RPMI 1640 medium (Life Technologies, 11875-093) and B27 supplement minus insulin (Life Technologies, A1895601). For the first 24-h differentiation period, the BDM was supplemented with 300 ng/mL recombinant human Activin A and 2 µg/mL puromycin (Acros, 227420100). After 24 h, this medium was replaced with basic BDM supplemented with 6 µg/mL puromycin. BDM + 6 µg/mL puromycin was replaced every 48 h. At day 5, cells were collected and frozen as described above.
7
Routine Maintenance of Undifferentiated ESCs
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For routine maintenance, undifferentiated ESCs were cultured on growth factor-reduced Matrigel (Corning, USA)-precoated dishes in complete mTeSR™1 medium (STEMCELL Technologies, Canada) and passaged every ~ 4 days using 0.5 mM EDTA (Sigma, USA). Rho-associated protein kinase (ROCK) inhibitor thiazovivin (Selleck Chemicals, USA) was added during cell passaging to prevent dissociation-induced ESC apoptosis.
8
Human iPSC Culture and Maintenance
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Two wild-type human iPSC lines were utilized: WTC11 (XY, mono-allelic ACTN2-mEGFP, obtained from Coriell Institute #AICS-0075-085) and MSN02-4 (XX, generated from skin biopsy of a healthy 36-year-old at Icahn School of Medicine at Mount Sinai).68 ,69 (link) The iPSC lines were maintained in 6-well plates coated with a thin layer of Matrigel (6.6% v/v, Corning), diluted in DMEM (Gibco) and supplemented with penicillin/streptomycin (5%, ThermoFisher). iPSCs were maintained and fed daily with iPSC culture medium consisting of mTesR Plus (Stem Cell Technologies) supplemented with penicillin/streptomycin (1%, ThermoFisher). iPSCs were passaged every 3 to 4 days by first dissociating to single cells with Accutase (Innovative Cell Technologies, Inc) and then replating at a 1:10 ratio with iPSC culture medium, supplemented with Thiazovivin (2 μM, Selleckchem) for the first 24 h. iPSC cultures were incubated at 37°C in a normoxic environment (5% CO2). iPSCs were visually authenticated daily to ensure proper stem cell morphology without presence of spontaneous differentiation. Mycoplasma contamination was checked quarterly via PCR (ATCC).
9
CRISPR-Cas9 Genome Editing in H1 hESCs
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Cas9 protein and guide RNA were expressed through pX330 (Addgene). Guide RNAs (gRNAs) for the knockout and knock-in of HBO1 were designed on the website (CCTop, http://crispr.cos.uni-heidelberg.de ). The knockout donor plasmid contained a left homology arm, a LoxP-flanked PGK-puromycin cassette and a right homology arm. In addition, the left homology arm of the knock-in donor plasmid contained a 3 × FLAG tag. These donor plasmids were used for targeting.
After all the plasmids were constructed, then 4 μg of each pX330 plasmid and donor plasmid were electroporated into 1 × 106 H1 hESCs by Nucleofector 2b Device (Lonza). These hES cells were cultured in mTeSR1 medium with Thiazovivin (0.1 μM, Selleck) for 1 day, then screened by puromycin (1 μg/ml, Gibco).
To identify gene knockout or knock-in cell lines, Genomic DNA extracted by the TIANamp Genomic DNA Kit (Tiangen) was used in PCR experiments. For gene knockout, the KO-F/R primers were used to amplify a ∼2 kb product of the targeted integration. For gene knock-in, the KI-F/R primers were used to amplify a ∼2 kb product of the targeted integration. All gRNA sequences and primer sequences are listed inSupplementary Table S1 .
After all the plasmids were constructed, then 4 μg of each pX330 plasmid and donor plasmid were electroporated into 1 × 106 H1 hESCs by Nucleofector 2b Device (Lonza). These hES cells were cultured in mTeSR1 medium with Thiazovivin (0.1 μM, Selleck) for 1 day, then screened by puromycin (1 μg/ml, Gibco).
To identify gene knockout or knock-in cell lines, Genomic DNA extracted by the TIANamp Genomic DNA Kit (Tiangen) was used in PCR experiments. For gene knockout, the KO-F/R primers were used to amplify a ∼2 kb product of the targeted integration. For gene knock-in, the KI-F/R primers were used to amplify a ∼2 kb product of the targeted integration. All gRNA sequences and primer sequences are listed in
10
Culturing Healthy Human iPSCs
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The fully characterized hiPSC line is derived from dermal fibroblasts of a healthy male as previously published (Shinnawi et al., 2015 (link)). Informed consent was obtained after approval by the IRB committee of Amsterdam Medical Center. Colonies of hiPSCs were cultured in mTeSR-1 (StemCell Technologies) on plates coated with growth factor-reduced Matrigel (1:200 dilution, Corning). Cells were collected using 0.5 mM EDTA (Invitrogen), passaged every 4–6 days, and replated in mTeSR-1 supplemented with 2 μM thiazovivin (Selleck Chemicals). mTeSR-1 medium was replaced daily, except for the first day after passaging.
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