Two sets of human iPS cells were provided by RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). Normal human iPS cells from nasal epithelial cells (Nips cells; control iPS cells)14 (link); iPS cells derived from the skin fibroblasts of a patient with PWS (iPWS cells) that had a paternal 15q11-q13 deletion and were created with the Sendai virus vector (DNAVEC Inc; Cyto Tune TMiPS vector) that individually carried OCT3/4, SOX2, KLF4, and c-MYC. Human iPS cells derived from a healthy individual (WT-iPS in Fig. 2 A) and a patient with PWS (HPS2846: M-iPWS) with abnormal methylation were created with the pCE-hOCT3/4, pCE-hSK, pCE-hUL, pCE-mp53DD, and pCXB-EBNA1 vectors from peripheral blood mononuclear cells15 (link). The iPS cells were maintained in feeder-free conditions and cultured in Cellartis DEF-CS medium (Takara Bio, Inc.) containing Cellartis DEF-CS 500 additives (DEF-CS GF-1 and GF-2) on-coated dishes with Cellartis DEF-CS COAT-1 (Takara Bio, Inc.). The iPS cells were passaged after reaching 70–80% confluence (every 3–5 days). To prevent cell death, iPS cells were cultured in medium containing the ROCK inhibitor Y27632 (Wako Pure Chemical Industries, Ltd) after cell passage.
Rock inhibitor y 27632
Manufactured by Fujifilm
Sourced in Japan, United States
ROCK inhibitor Y-27632 is a small molecule compound that selectively inhibits the activity of Rho-associated coiled-coil containing protein kinase (ROCK) enzymes. ROCK inhibition can influence various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Imatrix 511, by Nippi (4 mentions)
Stemfit ak02n, by Ajinomoto (2 mentions)
Accumax, by Innovative Cell Technologies (2 mentions)
Stemfit ak02n medium, by Ajinomoto (2 mentions)
Matrigel, by Corning (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
Glycerol 2 phosphate, by Merck Group (1 mentions)
L ascorbic acid 2 phosphate sesquimagnesium salt hydrate, by Merck Group (1 mentions)
Glutamax, by Fujifilm (1 mentions)
Non essential amino acids (neaa), by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Stemdiff, by STEMCELL (1 mentions)
Sb431542, by Fujifilm (1 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (1 mentions)
Matrigel coated plate, by BD (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
16 protocols using rock inhibitor y 27632
1
Generation of Human iPSCs from PWS Patients
2
Osteogenic Differentiation of iPSCs
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iPSCs were differentiated into osteogenic lineages using previously published protocols [24 (link)]. Briefly, 2 × 105 iPSCs were maintained in feeder-free conditions and differentiation was induced in gelatin-coated 24-well plates with 20% mTeSR1 medium (StemCell Technologies, Vancouver, BC, CA) and 80% osteogenic differentiation medium composed of KnockOut DMEM (Gibco) supplemented with 20% FBS (Gibco), 1% non-essential amino acids (Wako), 0.1 mM 2-mercaptoethanol (Sigma), 2 mM Gluta-MAX (Wako), 10 mM glycerol-2-phosphate (Sigma), 1 nM dexamethasone (Sigma), 50 µg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), and 1 µM all-trans-retinoic acid (Sigma), and 10 µM rock inhibitor Y-27632 (Wako). On day 2, the medium was replaced with 100% osteogenic differentiation medium without Y-27632 and changed on days 4 and 7.
3
Neural Stem Cell Induction Protocol
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Neural stem cell induction was conducted using the dual-SMAD inhibition protocol [35 (link)] with STEMdiff™ neural induction medium (NIM; StemCell Technologies, Inc., Vancouver, Canada). Human iPSCs were dissociated using the Gentle cell dissociation reagent (StemCell Technologies), and cell suspension were passed through a 40 μm cell strainer. Cells were resuspended in NIM supplemented with 10 μM SB431542 (FUJIFILM Wako Pure Chemical Corp.), 100 nM LD193189 (FUJIFILM Wako Pure Chemical Corp.), and 10 μM Rock inhibitor (Y27632) (FUJIFILM Wako Pure Chemical Corp.) and plated at a density of 2 × 105 cells/cm2 on Matrigel-coated plates (BD Biosciences, Bedford, MA, USA). NIM supplemented with 10 μM SB431542 and 100 nM LD193189 was replaced every 24 h after plating. On day 7 of neural induction, the differentiated cells were collected and used for flow cytometric analysis.
4
Culturing hiPSC Line 1383D2 on Laminin-511
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The hiPSC line, 1383D2, was obtained from the Center for iPS Cell Research and Application, Kyoto University (Kyoto, Japan). Cells were maintained on culture substrates coated with recombinant laminin-511 E8 fragments (iMatrix-511; Nippi, Inc., Tokyo, Japan) in a chemically defined and animal component-free medium (StemFit AK02N; Ajinomoto Co., Inc., Tokyo, Japan) following a previously published protocol [43 (link)]. The cells were cultured at a viable cell density of 7.5 × 103 cells/cm2 and subcultured every four days using TrypLE Select. For the first 24 h, 10 μM Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (Y-27632; Wako Pure Chemical Industries, Osaka, Japan) was used to enhance single hiPSC survival. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2, and the culture medium was replaced daily with fresh medium.
5
Culturing Induced Pluripotent Stem Cells
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We cultured the established iPSCs according to a previously described method 25 . Briefly, the culture plates were precoated with recombinant laminin‐511 E8 fragments (iMatrix‐511, Nippi) (0.5mg/cm2), and the iPSCs were cultured in Stem Fit medium (Ajinomoto) at 37°C with 5% CO2. The medium was changed every other day and was passaged every 7–10 days using 0.5× TrypLE Select (1× TrypLE Select diluted 1:1 with 0.5 mM EDTA/PBS(‐), Life technologies) and Rock inhibitor (Y‐27632, WAKO, Osaka, Japan).
6
Culturing Diverse Cell Lines for Mitotic Studies
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HeLa Kyoto, U2OS, and RPE‐1 cells were grown at 37°C in a 5% CO2 atmosphere in DMEM (Nacalai Tesque), supplemented with 10% FBS. The HeLa Kyoto cell line is a popular subline of HeLa cells, a human cervical carcinoma‐derived cell line, which is suitable for mitotic study due to its slow migration.24 In spheroid culture, cells were seeded to round‐bottomed ultra‐low attachment 96‐well plates (Corning) at 1000 cells/well in renal epithelial cell growth basal medium (REBM) without FBS, and supplemented with B‐27 Supplement (Thermo Fisher Scientific), N‐2 Supplement (Thermo Fisher Scientific), and 10 μM ROCK inhibitor Y‐27632 (FUJIFILM Wako).
7
CRISPR/Cas9-Mediated Genome Editing in iPSCs
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Transfection of the CRISPR/sgRNA plasmid into iPSCs was performed as described previously [26 (link)]. Electroporation was performed using a Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, iPSCs were harvested by treating with 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Thermo Fisher Scientific). Cells (1 × 106) were suspended in 100 μl electroporation buffer containing 5 μg of the donor plasmid and 5 μg of the CRISPR/sgRNA expression plasmid, and electroporated (condition: 1050 V, 20 ms, two pulses). After electroporation, the cells were subsequently plated on an iMatrix-511 (Nippi Inc., Tokyo, Japan)-coated 100-mm dish and cultured in StemFit® AK01 medium (Ajinomoto, Tokyo, Japan) supplemented with ROCK inhibitor (Y-27632; Wako Pure Chemical Industries Ltd.) for the first 24 h. Geneticin selection (300 μg/ml) was started 72 h after electroporation. Individual colonies were picked and expanded for 10 days after electroporation.
8
Maintenance of B2-3 Lung Reporter iPSCs
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The B2-3 lung reporter iPS cell line (Gotoh et al., 2014 (link)) was maintained at 37 °C and 5% CO2 in StemFit AK02N medium (Ajinomoto, Cat. No. RCAK02N) on tissue culture plates coated with 0.5 mg/mL silk iMatrix-511, Recombinant Human Laminin-511 E8 Fragment (Nippi, Cat. No. 892021) with a daily medium exchange (Gotoh et al., 2014 (link)). Cell passage was performed every 7 days during maintenance. Cells were first dissociated with Accumax (Innovative Cell Technologies, Cat. No. AM105-500) and 10 min incubation at 37 °C, then washed in StemFit AK02N medium supplemented with 10 µM ROCK inhibitor Y-27632 (Wako, Cat. No. 253-00513) and seeded onto iMatrix511-coated plates at a density of 1 × 103 cells/cm2 in StemFit AK02N medium with ROCK inhibitor for 48 h after seeding, and then cultured without ROCK inhibitor. All the cell lines were routinely tested as negative for mycoplasma contamination.
9
CTOS Viability Assay via ATP
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After washing the CTOSs with PBS, they were dissociated into single cells using 0.25% trypsin/EDTA and filtered through a 40 μm cell strainer (BD Falcon). Approximately 1x104 cells/100ul were seeded in poly-HEMA-coated 96-well plates and cultured in the FBS medium and 10 μM of ROCK inhibitor Y27632 (Wako, Osaka, Japan). Each drug was added and the CTOSs cultured for 7 days. ATP content was measured at day 7 by CellTiter-Glo Luminescent Cell Viability Assays (Promega, Madison, WI, USA) and adjusted to the content of the vehicle-treated control.
10
Maintenance of Human Induced Pluripotent Stem Cells
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All cell cultures were performed at 37°C with 5% CO2. 201B7 iPS cell line was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. After being cultured in SNL feeder/20% Knockout Serum Replacement (KSR) condition, hiPSCs were transferred and routinely maintained on Matrigel (hESC-qualified; Corning) with mTeSR1 (Stemcell Technologies) medium supplemented with 0.5× penicillin/streptomycin (Nacalai Tesque). Cells were passaged using the dissociation reagent containing 0.5 mM ethlendiaminetetraacetic acid disodium salt (EDTA•2Na) in Dulbecco's phosphate-buffered saline without calcium/magnesium (D-PBS(−); Nacalai Tesque). 2.5 μM of Rock inhibitor Y-27632 (Wako) was supplemented to the medium for one day after passage.
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