Differentiation toward cardiomyocyte lineage was induced using small molecules to modulate the WNT signaling pathway and was optimized according to a small molecule-based monolayer method as previously described (21 (link)). Briefly, hESC cultured in mTeSR1 medium were digested into single cells by Accutase (Sigma-Aldrich) and plated onto Matrigel-coated culture dishes at a density of 4 x 105 cells/12 well in mTeSR1 medium with ROCKi inhibitor (Y-27632; Selleck) for 24 h. After 1 d, Y-27632 was withdrawn from the medium, and Cells were then induced with mTeSR1 medium for 48 h. On day 0, cells were treated with cardiac differentiation medium for 24 h, consisting of RPMI/1640 and CHIR99021 (4 μM; Selleck). On day 1, the medium was exchanged by fresh RPMI/1640, and cells were induced for another 48 h. Subsequently, on day 3, the medium was changed to RPMI/1640 medium containing IWP2 (5 μM; Selleck) for 2 d. After 2 d (day 5), the medium was exchanged for RPMI/1640 medium for another 2 d. For maintenance of the obtained cardiomyocytes, at day 7, the medium was changed to RPMI/1640 containing insulin (10 μg/ml) and Vitamin C (200 μg/ml), and half the medium was exchanged every day until day 15. Contracting cells were seen from day 9 to day 15.
Y 27632
Manufactured by Selleck Chemicals
Sourced in United States, Germany, China, United Kingdom, Canada
Y-27632 is a cell-permeable, selective and potent inhibitor of Rho-associated protein kinase (ROCK). It functions by inhibiting ROCK activity, which plays a crucial role in regulating cellular processes such as cytoskeleton organization, cell migration, and cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (35 mentions)
Matrigel, by Corning (28 mentions)
Chir99021, by Selleck Chemicals (25 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (25 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (24 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (16 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (16 mentions)
A83 01, by Bio-Techne (15 mentions)
Accutase, by STEMCELL (15 mentions)
Hepes, by Thermo Fisher Scientific (13 mentions)
Rock inhibitor, by Selleck Chemicals (12 mentions)
Matrigel, by BD (12 mentions)
Dmem f12, by Thermo Fisher Scientific (11 mentions)
Accutase, by Merck Group (11 mentions)
Accutase, by Thermo Fisher Scientific (11 mentions)
N acetylcysteine, by Merck Group (10 mentions)
Mtesr1, by STEMCELL (10 mentions)
Fgf10, by Thermo Fisher Scientific (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Nicotinamide, by Merck Group (9 mentions)
319 protocols using y 27632
1
Cardiomyocyte Differentiation from hESCs
2
Cardiomyocyte Differentiation from hESCs
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Differentiation toward cardiomyocyte lineage was induced using small molecules to modulate the WNT signaling pathway and was optimized according to a small molecule-based monolayer method as previously described (21 (link)). Briefly, hESC cultured in mTeSR1 medium were digested into single cells by Accutase (Sigma-Aldrich) and plated onto Matrigel-coated culture dishes at a density of 4 x 105 cells/12 well in mTeSR1 medium with ROCKi inhibitor (Y-27632; Selleck) for 24 h. After 1 d, Y-27632 was withdrawn from the medium, and Cells were then induced with mTeSR1 medium for 48 h. On day 0, cells were treated with cardiac differentiation medium for 24 h, consisting of RPMI/1640 and CHIR99021 (4 μM; Selleck). On day 1, the medium was exchanged by fresh RPMI/1640, and cells were induced for another 48 h. Subsequently, on day 3, the medium was changed to RPMI/1640 medium containing IWP2 (5 μM; Selleck) for 2 d. After 2 d (day 5), the medium was exchanged for RPMI/1640 medium for another 2 d. For maintenance of the obtained cardiomyocytes, at day 7, the medium was changed to RPMI/1640 containing insulin (10 μg/ml) and Vitamin C (200 μg/ml), and half the medium was exchanged every day until day 15. Contracting cells were seen from day 9 to day 15.
3
In vitro 3D Blastoid Culture
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In vitro 3D culture of blastoids was performed as previously reported for human blastocysts (Xiang et al., 2020 (link)). Briefly, individual human blastoids were collected by mouth pipette, transferred into a 96-well plate (corning #3474), and cultured in mIVC1 [IVC1 supplemented with 0.22% (v/v) sodium lactate (L7900, Sigma-Aldrich), 1 mmol/L sodium pyruvate (P4562, Sigma-Aldrich), and 10 μmol/L Y-27632 (S1049, Selleck)] for 2 days. Then, half of the medium was changed by mIVC2 [IVC2 supplemented with 0.22% (v/v) sodium lactate (L7900, Sigma-Aldrich), 1 mmol/L sodium pyruvate (P4562, Sigma-Aldrich), and 10 μmol/L Y-27632 (S1049, Selleck)] for 24 h. The blastoids were transferred into mICV2 containing 10% Matrigel on the next day. Half of the medium was changed every day thereafter. mIVC1 and mIVC2 were pre-equilibrated in the incubator for at least 6 h before use. The blastoid culturing conditions were as follows: 37.2°C, 6% CO2, and 5% O2.
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In vitro 3D culture of blastoids was performed as previously reported for human blastocysts (Xiang et al., 2020 (link)). Briefly, individual human blastoids were collected by mouth pipette, transferred into a 96-well plate (corning #3474), and cultured in mIVC1 [IVC1 supplemented with 0.22% (v/v) sodium lactate (L7900, Sigma-Aldrich), 1 mmol/L sodium pyruvate (P4562, Sigma-Aldrich), and 10 μmol/L Y-27632 (S1049, Selleck)] for 2 days. Then, half of the medium was changed by mIVC2 [IVC2 supplemented with 0.22% (v/v) sodium lactate (L7900, Sigma-Aldrich), 1 mmol/L sodium pyruvate (P4562, Sigma-Aldrich), and 10 μmol/L Y-27632 (S1049, Selleck)] for 24 h. The blastoids were transferred into mICV2 containing 10% Matrigel on the next day. Half of the medium was changed every day thereafter. mIVC1 and mIVC2 were pre-equilibrated in the incubator for at least 6 h before use. The blastoid culturing conditions were as follows: 37.2°C, 6% CO2, and 5% O2.
4
Culturing Patient-Derived iPSCs
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Patient-derived iPSCs were cultured in StemMACS™ iPS-Brew XF medium (Miltenyi Biotec, #130-107-086 and #130-107-087) until cells reached 90 % of confluency. Once cells reached confluency, they were passaged using 0.5 mM EDTA (Invitrogen, #15575-038) and seeded again on Matrigel-coated plates (Corning, #356231). StemMACS™ iPS-Brew XF plus 10 μM of ROCK inhibitor Y-27632 (Selleck Chemicals #S1049) were used for culturing cells, with Y-27632 being removed after 24 h. Cells were maintained in a 37 °C incubator, with 5 % CO2.
5
Corneal Endothelial Cell Transplantation for Dysfunction
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CEC-like cells were transplanted according to our previous method11 (link)
. Briefly, the corneal endothelium was mechanically scraped with a lacrimal passage irrigator (Shandong Weigao) from the Descemet’s membrane to create monkey corneal endothelial dysfunction models. 50 µl aqueous humor was first extracted from the models’ anterior chamber. Then, 4.0 × 105 CEC-like cells suspended in 50 µl culture medium supplemented with 1.6 µg of Y-27632 (Selleck) were injected into the anterior chamber of four monkeys. And 50 µl culture medium supplemented with 1.6 µg of Y-27632 was injected into the anterior chamber of one monkey. Four monkeys that had cells injected were the experimental group and the other one monkey that had no cells injected was the control group. Peribulbar and subconjunctival injection of triamcinolone and dexamethasone were given after surgery. The eyes of all monkeys were kept in a face-down position for 6 hours under general anesthesia. 0.3% Tobramycin and 0.1% Dexamethasone were given topically 3–4 times a day. The corneas were examined by a slit-lamp microscope (Topcon), Visante OCT (Carl Zeiss), non-contact specular microscopy (Topcon), tenonometer (Suzhou liuliu), gonioscope (Volk), B-ultrasonography (Suoer), and fundus camera (Carl Zeiss) at certain times. The contralateral normal eyes of the monkeys were observed as the normal group.
. Briefly, the corneal endothelium was mechanically scraped with a lacrimal passage irrigator (Shandong Weigao) from the Descemet’s membrane to create monkey corneal endothelial dysfunction models. 50 µl aqueous humor was first extracted from the models’ anterior chamber. Then, 4.0 × 105 CEC-like cells suspended in 50 µl culture medium supplemented with 1.6 µg of Y-27632 (Selleck) were injected into the anterior chamber of four monkeys. And 50 µl culture medium supplemented with 1.6 µg of Y-27632 was injected into the anterior chamber of one monkey. Four monkeys that had cells injected were the experimental group and the other one monkey that had no cells injected was the control group. Peribulbar and subconjunctival injection of triamcinolone and dexamethasone were given after surgery. The eyes of all monkeys were kept in a face-down position for 6 hours under general anesthesia. 0.3% Tobramycin and 0.1% Dexamethasone were given topically 3–4 times a day. The corneas were examined by a slit-lamp microscope (Topcon), Visante OCT (Carl Zeiss), non-contact specular microscopy (Topcon), tenonometer (Suzhou liuliu), gonioscope (Volk), B-ultrasonography (Suoer), and fundus camera (Carl Zeiss) at certain times. The contralateral normal eyes of the monkeys were observed as the normal group.
6
Cell Proliferation Assay with Pharmacological Modulators
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Cell proliferation rates were determined with a cell counting kit-8 (CCK8) assay (Dojindo, Tokyo, Japan) following the manufacturer's instructions. FLS at a density of 2.5 × 104 mL−1 were seeded into 96-well plates for 12 hours, followed by individual treatment with 10 μM cyclopamine (Selleckchem, Houston, TX, USA), 1 μM purmorphamine (Sigma-Aldrich, St. Louis, MO, USA), 20 μM Y27632 (Selleckchem, Houston, TX, USA), or a combination of 20 μM Y27632 and 1 μM purmorphamine. Specifically, cyclopamine or purmorphamine dissolved at 10 mM in dimethyl sulfoxide (DMSO) was diluted to the final concentration using DMEM containing 10% FBS. Cells in the control group were treated with vehicle only.
7
Hepatic Organoid Differentiation in Spinner Flasks
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Disposable 125‐mL spinner flasks (Corning, New York, NY) were inoculated with 2.5 million cells in 25 mL expansion medium (EM), including 10% vol/vol Matrigel. Due to single cell seeding, 10 mM Y‐27632 (Rho kinase‐inhibitor; Selleckchem, Munich, Germany) was added to the medium during the first week of culture. Rotation speed was set to 85 rpm. Every 2‐3 days, new medium was added to the spinner flasks.
For hepatic organoid differentiation in spinner flasks, organoids were primed for 2 days with the addition of bone morphogenetic protein 7 (BMP‐7; 25 ng/mL) to the EM. Subsequently, to separate organoids from the EM, the organoid suspension was filtered through a 70‐µm cell strainer. The organoids were transferred to a new spinner flask and differentiated for 12 days in human organoid differentiation medium (DM) supplemented with 10% Matrigel. The procedure is schematically outlined in Supporting Fig.S4 .
For hepatic organoid differentiation in spinner flasks, organoids were primed for 2 days with the addition of bone morphogenetic protein 7 (BMP‐7; 25 ng/mL) to the EM. Subsequently, to separate organoids from the EM, the organoid suspension was filtered through a 70‐µm cell strainer. The organoids were transferred to a new spinner flask and differentiated for 12 days in human organoid differentiation medium (DM) supplemented with 10% Matrigel. The procedure is schematically outlined in Supporting Fig.
8
PDAC Organoid Viability Assay
9
Establishment of Colorectal Tumor Organoids
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CRC specimens were provided by the Peking University Third Hospital. Fresh tumor samples were stored in the antibiotic-containing DMEM medium with 10% fetal bovine serum (C04001-500, VivaCell) after surgically resected and transported to the laboratory at 4°C for immediate processing. The establishment and culture of colorectal tumor organoids was performed as previously described (Wang et al., 2022 (link)). Briefly, after being washed gently at least five times with pre-chilled 1X DPBS, tumor tissues were cut into small pieces using surgical scissors and digested with 2.5 mg/mL type II and type IV collagenase (17101015 and 17104019, Invitrogen) to obtain single-cell suspensions. After digestion, the suspension was passed through a 40 μm cell strainer to remove undigested parts, and then centrifuged at 800 ×g for 5 min. The pellet was resuspended with Matrigel (356231, Corning) and dispensed into a 24-well cell culture dish. After 30 min of solidification of Matrigel, conditioned medium was then added. Conditioned medium was prepared according to the previously reported protocol (Miyoshi and Stappenbeck, 2013 (link)). At early passages, 10 μmol/L ROCK inhibitor Y-27632 (S6390, Selleck) was supplemented to the medium.
10
Differentiating Human hESCs into Neurons
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Human neurons were derived by differentiating human hESCs using a small-molecule cocktail as described before, with minor adjustments [42 ]. N2B27 medium (N2B27 basal medium; 50% DMEM/F12 and 50% Neurobasal medium supplemented with 0.5 × N2 and 0.5 × B27, 1 mM GlutaMAX, and 1 × Penicillin–Streptomycin) was used throughout the differentiation protocol. Before induction, hESCs were passaged in mTeSR™ Plus and replated to form a uniform monolayer of cells. When the cells had reached around 95% confluence, medium was replaced with dual SMAD inhibition medium: N2B27 supplemented with 2 µM dorsomorphin (Selleckchem S7306) and 10 µM SB431542 (Sigma S4317). On day 10, the cells were replated in 1:2 ratio using 200 U/mL Collagenase IV (Gibco) onto Matrigel-coated dishes in N2B27 supplemented with 10 µM Y-27632 (Selleckchem S1049). From day 11 to day 20, N2B27 was supplemented with 100 ng/mL of FGF8 (PeproTech AF-100–25). On day 20, cells were detached using 0.5 mM EDTA and replated in N2B27 at 1:8 ratio. The next day following the split, N2B27 was supplemented with 20 µM DAPT (Selleckchem S2215) and the medium was replaced every 2 days.
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