Human ESCs were passaged with Accutase (Sigma) and plated at a density of 100,000 cells/cm2 in mTeSR with 10 μM Y27632 (Selleck) on Matrigel (BD), Laminin (BD), and collagen IV (BD) (3:1:1) mixed gel coated-plate (Corning). In the restriction of definitive endoderm (DEs) stage (S1), cells were cultured for 24 hrs in RPMI with B27 supplement (1:50, Gibco), 100 ng/ml Activin A (R&D) and 3 μM CHIR99021 (Selleck), and then treated with 100 ng/ml Activin A for 2 days. In the hepatic specification stage to get hepatic progenitor cells (S2), the culture medium was replaced with RPMI (Gibco) supplemented with B27 supplement (1:50), 20 ng/ml BMP4 and 10 ng/ml FGF2 for 5 days. And in the stage of hepatic maturation (S3), cells were cultured in Hepatocyte Culture Medium (HCM, Lonza) with 20 ng/ml HGF, 10 ng/ml OSM and 1 μM dexamethasone for 10–15 days. During stages 2 and 3, cells are fed every 48 hr. The final stage was also carried out with 10 μM SB203580 (Selleck), 50 μM Vitamin K2 (Sigma), 50 μM 2-APB (Tocris), or 0.5 μM Anisomycin (Selleck), and 0.1% dimethyl sulfoxide (DSMO) was used as control, as described in the text. Cells were photographed during differentiation using a Nikon phase contrast microscope (Nikon Microscopes).
Vitamin k2
Manufactured by Merck Group
Sourced in United States
Vitamin K2 is a fat-soluble vitamin that plays a crucial role in the human body. It is responsible for the regulation of calcium metabolism, contributing to healthy bone development and blood clotting processes. Vitamin K2 is an important component in various biochemical pathways within the body.
Automatically generated - may contain errors
Lab products found in correlation
Hepatocyte culture medium, by Lonza (2 mentions)
Glutamax supplement, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Laminin, by BD (1 mentions)
Activin a, by Selleck Chemicals (1 mentions)
Anisomycin, by Selleck Chemicals (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Chir99021, by Selleck Chemicals (1 mentions)
Collagen 4, by BD (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
Accutase, by Merck Group (1 mentions)
2 apb, by Bio-Techne (1 mentions)
Matrigel, by BD (1 mentions)
B 27 minus insulin, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium its, by Merck Group (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
Activin a, by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
7 protocols using vitamin k2
1
Differentiation of Human ESCs to Hepatocytes
2
Hepatocyte Differentiation Protocol
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When the cells attained a confluence of 40%, mTeSR™1 was replaced with RPMI 1640 (Gibco), containing 2% B27 minus insulin (Gibco), 100 ng/mL activin A (Peprotech), and 50 ng/mL Wnt 3a (R&D System) for 3 days. The medium was then replaced with IMDM (Procell) with 20% knockout serum replacement (Gibco), 1% GlutaMAX™ supplement (Gibco), 1% dimethyl sulfoxide (DMSO) (Sigma-Aldrich), 1% nonessential amino acids (NEAA) (Gibco), and 0.1 mM β-mercaptoethanol (β-ME) (Sigma-Aldrich) for 4 days. Next, the cultures were placed in IMDM containing 1% GlutaMAX™ supplement, 20 ng/mL oncostatin M (OSM) (Peprotech), 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech), 0.5 μM dexamethasone (Dex) (Sigma-Aldrich), and 1% insulin-transferrin-selenium (ITS) (Sigma-Aldrich) for 5 days. Finally, the differentiated cells were cultured in hepatocyte culture medium (HCM) (Lonza) supplemented with 10 ng/mL hepatocyte growth factor (HGF) (Peprotech), 0.5 μM Dex, 10 μM lithocholine acid (LCA) (Sigma-Aldrich), 10 μM vitamin K2 (Sigma-Aldrich), and 1% ITS for continuous cultivation.
3
Vitamin K2 Treatment in Alzheimer's Fly Model
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Vitamin K2 (Sigma, Saint Louis, Missouri, USA) was dissolved in anhydrous ethanol to obtain a 0.4 M stock solution. According to different concentrations, the flies were divided into five groups: (1)wild-type A307/w1118, no treatment with K2, (2)A307/Aβarc, no treatment with K2, (3)A307/Aβarc + 0.1 mM K2, treated with vitamin K2 at 0.1 mM, (4)A307/Aβarc + 0.5 mM K2, treated with vitamin K2 at 0.5 mM and (5)A307/Aβarc + 0.8 mM K2, treated with vitamin K2 at 0.8 mM. A total of 100 μL vitamin K2 solution was added into each vial containing 20 flies daily until 28 days. The flies were transferred into fresh food every 7 days.
4
Vitamin K2 Cytotoxicity and Apoptosis
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Vitamin K2 was purchased from Sigma (USA), with carbon 82.6–84.9%, EmM 17.4–18.9 and completely soluted in ethonal. Vitamin K2 was dissolved in 99.9% ethanol at a stock concentration of 50 mM; it was then diluted to working concentration with MEM or DMEM. Ethanol was added to cultures at 0.1% (V/V) as a solvent control. 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), N-acetyl cysteine (NAC) and Rhodamine 123 were purchased from Sigma Chemical Co. (St. Louis, MO). Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) kit was purchased from BD Biosciences (San Jose, CA). The caspase-3 specific inhibitor Z-DEVD-FMK, JNK inhibitor SP600125 and p38 inhibitor SB203580 were purchase from Calbiochem (San Diego, CA). The antibodies against caspase-3, p-JNK, p38 and p-p38 were purchase from Cell signaling Technology. The antibodies against PARP, cytochrome C, COX IV, Bax, Puma, Bcl-2 and JNK were purchase from Proteintech. The antibodies against Actin was purchase from Santa Cruz.
5
Recombinant Gas6 and Pros1 Purification
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cDNAs encoding either full-length mouse Gas6/Pros1 or Gas6/Pros1 lacking the Gla domain were sub-cloned into pCep4 vector (Invitrogen, Carlsbad, CA). PCR primers introduced a C-terminal His6 tag for purification and the constructs were transfected into HEK293 cells. Cells were grown to confluency in DMEM supplemented with 10% FBS, 0.25 mg/ml G418, 100 µg/ml hygromycin, and Pen/Strep and subsequently switched to serum-free Pro CDMa medium (Lonza, Walkersville, MD) supplemented with 10 µM vitamin K2 (Sigma, St. Louis, MO) and Pen/Strep for 72 hr. Conditioned medium was passed through a 0.22 µm filter, affinity purified through a nickel-NTA resin, and further purified on an anion exchange column (Mono Q 5/5; GE Healthcare, Pittsburgh, PA) in 20 mM Tris–HCl, pH 8.0 using a sodium chloride gradient.
6
Dual Drug Functionalized Titanium Implants
7
Screening of Bioactive Compounds
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L‐Ornithine, sodium benzoate, 5‐azacytidine, NaPB, resveratrol, Vitamin K2 and ammonium chloride were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Other collections of small compounds acting as dopamine D3 receptor inhibitor (43 compounds), targeting mammalian targets of rapamycin (mTOR) pathway (58 compounds), or tumour necrosis factor (TNF) pathway (76 compounds) were synthesized and provided by Dr. Wu Zhong's laboratory from the Beijing Institute of Pharmacology & Toxicology. Dulbecco's modified Eagle's medium (DMEM, with or without Phenol Red) was purchased from Gibco (Grand Island, NY, USA). Foetal bovine serum (FBS) was purchased from Nichirei Biosciences (Tokyo, Japan). Hochest 33342, MitoTracker® Green FM® and lipofectamine® 2000 were purchased from Invitrogen (Carlsbad, CA, USA).
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