On day 0 (start of differentiation) H9 human pluripotent stem cells were treated with 1mg/ml Collagenase B (Roche) for 1 h, or until cells dissociated from plates, to generate embryoid bodies. Cells were collected and centrifuged at 300 rcf for 3 min and resuspended as small clusters of 50–100 cells by gentle pipetting in differentiation media containing RPMI (Gibco), 2 mM/L L-glutamine (Invitrogen), 4×104 monothioglycerol (MTG, Sigma-Aldrich), 50 μg/ml ascorbic acid (Sigma-Aldrich). Differentiation media was supplemented with 2ng/ml BMP4 and 3 μmol Thiazovivin (Millipore). Embryoid bodies were cultured in 6-cm dishes (USA Scientific) at 37°C in 5% CO2, 5% O2, and 90% N2. On day 1, the media was changed to differentiation media supplemented with 30 ng/ml BMP4 (R&D Systems) and 30 ng/ml Activin A (R&D Systems), 5ng/ml bFGF (R&D Systems), and 1 μM Thiazovivin (Milipore). On day 3, embryoid bodies were harvested and washed once with DMEM (Gibco). Media was changed to differentiation media supplemented with 5 ng/ml VEGF (R&D Systems) and 5 μmol/L XAV (Stemgent). On day 5, media was changed to differentiation media supplemented with 5 ng/ml VEGF (R&D Systems). After day 8, media was changed every 3–4 days to differentiation media without supplements until approximately day 30.
Thiazovivin
Manufactured by Merck Group
Sourced in United States, Germany
Thiazovivin is a small molecule compound that functions as a selective inhibitor of the enzyme Rho-associated protein kinase (ROCK). It is commonly used in cell culture and laboratory research settings to modulate cellular processes and signaling pathways that are influenced by ROCK activity.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Matrigel, by Corning (6 mentions)
Chir99021, by Merck Group (3 mentions)
Essential 8 medium, by Thermo Fisher Scientific (3 mentions)
Matrigel, by BD (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Accutase, by Merck Group (2 mentions)
A83 01, by Bio-Techne (2 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (2 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Glutamax 1, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (2 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Accutase, by BD (2 mentions)
Mtesr1 medium, by STEMCELL (2 mentions)
27 protocols using thiazovivin
1
Directed Differentiation of hPSCs into Endothelial Cells
2
Precise Gene Editing in iPSCs
3
Maintenance and Differentiation of Human Embryonic Stem Cells
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H9 human embryonic stem cells were grown on matrigel-coated dishes (Corning). They were cultured in hESC medium that was incubated with mouse embryonic fibroblasts (MEFs) for 24 h. hESC medium consisted of DMEM/F12 supplemented with 2 mM L-glutamine (Invitrogen), 20% knockout serum replacement (Gibco), penicillin/streptomycin (Invitrogen), nonessential amino acids (Invitrogen), and 0.1 mM β-mercaptoethanol (Invitrogen). Medium was supplemented with basic fibroblast growth factor (Sigma-Aldrich, 8 ng/μL) and changed daily to secure pluripotency of hESCs. For maintenance of the culture, cells were grown in colonies and passaged manually by cutting the colonies with a needle. For transfection and clonal expansion hESCs were grown as single cells and passaged using accutase (Sigma-Aldrich) at ratios 1:4–1:10. For single cell culturing, culture medium was supplemented with ROCK inhibitor Thiazovivin (2 μM, Sigma-Aldrich) 1 h before dissociation and during plating. For H3K9me3 ChIP-seq experiments, hESCs were cultured on MEFs as described in Jacobs et al. (2014) (link).
4
Stem Cell Differentiation Regulator Compounds
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Recombinant peptide and small molecules are as follows: A83-01 (Tocris 2939), Y-27632 (Enzo Life Science ALX-270-333-M005), Recombinant Mouse TGF-β 1 (Novoprotein CA59, R&D Systems 7666-MB-005), GW788388 (Sigma-SML0116-5 mg), LY-364947 (Sigma-L6293-5MG), SD-208 (Sigma-S7071-5MG), Thiazovivin (Sigma-SML1045-5 mg) and SR-3677 (Sigma-SML0774-5 mg).
5
Expansion of Healthy Human iPSCs
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Commercially obtained ATCC hiPSC cells, originating from a healthy man (ATCC, CS-1026), were cultured daily with Essential 8TM medium (Gibco, A1517001, Waltham, MA, USA), in GeltrexTM LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix-coated wells (Gibco, A1413302). The cells were passaged once the confluency reached 80–100%. They were dissociated with TrypLE Express Enzyme (Gibco, 12605010) for 5 min at 37 °C, after which Essential 8TM medium supplemented with 1 µM thiazovivin (Sigma, 420220, St. Louis, MO, USA) was added. The cell suspension was then transferred to a Falcon tube and centrifuged for 3 min at 300 RCF. Finally, the cells were seeded at 15.000 cells/cm2 in Essential 8TM medium with 1 µM thiazovivin overnight, after which the medium was refreshed with plain Essential 8TM medium.
6
Human ESC Line Maintenance and Differentiation
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Human ESC line used in these studies was approved by the Ethics Commission of the TUM Faculty of Medicine (# 447/17S). Authorization to use the hESC-TN was granted by the Central Ethics Committee for Stem Cell Research of the Robert Koch Institute to AM (AZ 3.04.02/0131). Generation of the hESC-TN line was described in Zawada et al. (2023) (link). hESCs were maintained on Matrigel-coated plates (Corning, 354277) in Essential 8 medium (Thermo Fisher Scientific, A1517001) containing 0.5% penicillin/streptomycin (Thermo Fisher Scientific, 15140-122) under standard culture conditions (37°C, 5% CO2); medium was refreshed every day. Cells were passaged every 4 days with 0.5 mM EDTA (Invitrogen, AM92606) in PBS without Ca2+ and Mg2+ (PBS−/−; Thermo Fisher Scientific, 10010023). To promote better cell survival, 2 µM ROCK inhibitor Thiazovivin (Sigma-Aldrich®, SML1045) was added for 24 h after passaging. Cells were differentiated according to differentiation protocols described below.
7
iPSC Cultivation and Dopaminergic Differentiation
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iPSCs were cultivated in the growth medium containing KnockOut DMEM, 15% KnockOut Serum Replacement, GlutaMAX-I, 0.1 mM NEAA, 1% penicillin-streptomycin (all from Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM 2-mce (Sigma-Aldrich, Darmstadt, Germany), and 10 ng/mL bFGF (SCI Store, Moscow, Russia) onto a gelatin-coated plate with mouse embryonic fibroblasts (MEF) treated with Mitomycin C from Streptomyces caespitosus (Sigma-Aldrich, Darmstadt, Germany). For DA differentiation, iPSCs were passaged onto Matrigel-GFR (Corning, New York, NY, USA) in Essential 8 Medium (Thermo Fisher Scientific, Waltham, MA, USA). iPSCs were passaged 2 times a week at a ratio of 1:8–1:10 with the addition of 2 μg/mL Thiazovivin (Sigma-Aldrich, Darmstadt, Germany). Dissociation of iPSC colonies was carried out using TrypLE (Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured in a CO2 incubator (37 °C, 5% CO2).
8
Directed Cardiac Differentiation of hESCs
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A hESC line, BG01 was obtained from the WiCell Research Institute (Madison, WI, USA). BG01 hESCs were maintained on Matrigel-coated plates in E8 medium (Thermo Fisher Scientific, Waltham, MA, USA). For cardiac differentiation, BG01 hESCs were dissociated into single cells with Accutase (Sigma-Aldrich, Saint Louis, MO, USA) and then seeded onto a Matrigel-coated at 32,000 cells/cm2 in E8 supplemented with 2 μM Thiazovivin (Sigma-Aldrich), a Rho kinase inhibitor from day −3 to day 0. At day 0, cells were treated with 6 µM CHIR99021 (Sigma-Aldrich) in the basal medium RPMI 1640 (Thermo Fisher Scientific), L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich), and bovine serum albumin (BSA, Sigma-Aldrich) medium for 1 day. At 2 days, cells were treated with 2 µM IWP2 (Tocris Bioscience, Ellisville, MO, USA) in RPMI 1640 + B27 minus insulin (RPMI/B27-Insulin) medium for 48 h. Cells were treated with 10 ng/mL FGF2 (Peprotech, Rocky Hill, NJ, USA), 5, 10, 25, 50, and 100 ng/mL FGF4 (Sigma-Aldrich), 10 ng/mL FGF10 (Peprotech), 200 µg/mL AA, or 10 ng/mL FGF4 + 200 µg/mL AA (FGF4+AA) in RPMI/B27-Insulin medium from day 5 to day 15.
9
Optimized Culture Conditions for Tissue Engineering
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Type I collagen gel (cellMatrix type 1-A) was purchased from Nitta Gelatin, Inc. (Osaka, Japan). Ham's F-12 nutrient mix, advanced DMEM/F12, N-2, B-27, gentamicin/amphotericin B, HEPES, and GlutaMAX-I were obtained from Gibco (Waltham, MA, USA). Matrigel was acquired from BD Biosciences (Franklin Lakes, NJ, USA). mEGF and mNoggin were purchased from PeproTech (Rocky Hill, NJ, USA). CHIR99021, [Leu15]-Gastrin 1, SB202190, nicotinamide, N-acetylcysteine, and thiazovivin were obtained from Sigma-Aldrich (St. Louis, MO, USA). A83-01 was acquired from Tocris Bioscience (Bristol, UK). Y-27632 and Gentle Cell Dissociation Reagent were purchased from STEMCELL Technologies (Vancouver, Canada). Penicillin and streptomycin were acquired from Welgene Inc. (Gyeongsan-si, Republic of Korea). FGF10 was purchased from ATGen (Seongnam, Republic of Korea).
10
ROCK1 Inhibitor-Based Cell Viability Assay
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ROCK1 inhibitor-Thiazovivin and Y27632 (Supplementary Fig. 7 ), MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] and dimethyl sulfoxide (DMSO) were procured from Sigma (Steinheim, Germany).
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