Use of human iPSCs was approved by the MUSC Stem Cell Research Oversight Committee. Human K3 iPSCs were generated previously and have been described elsewhere40 (link). The hepatocyte differentiation protocol has been published elsewhere21 ,25 ,40 (link) with minor modifications. Briefly, human iPSCs were seeded on Matrigel-coated tissue culture plates with a density of 7 × 105 cells/ml to form a monolayer. Cells were induced to form definitive endoderm in RPMI 1640 Medium (Invitrogen, Waltham, MA) supplemented with 2% B-27 Supplement minus insulin (Invitrogen), 10 ng/mL of BMP4 (Invitrogen), 20 ng/mL FGF2 (Invitrogen), and 100 ng/mL Activin A (Invitrogen) for two days, followed by 100 ng/mL Activin A for three days. Definitive endoderm was then converted to hepatocyte progenitor cells by addition of BMP4 (20 ng/mL) and FGF2 (10 ng/mL) for an additional 5 days. Immature hepatocytes were generated by the inclusion of Hepatocyte Growth Factor (20 ng/mL) (Invitrogen) for 5 days. Cells were finally induced to mature to hepatocyte-like cells by the addition of Oncostatin M (20 ng/mL) (Invitrogen) for 5 days.
Oncostatin m
Manufactured by Thermo Fisher Scientific
Sourced in United States
Oncostatin M is a recombinant protein that functions as a cytokine. It is primarily involved in the regulation of inflammatory and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (21 mentions)
Dexamethasone (dex), by Merck Group (13 mentions)
Il 22, by Thermo Fisher Scientific (10 mentions)
Il 1α, by Thermo Fisher Scientific (9 mentions)
Tnf α, by Thermo Fisher Scientific (9 mentions)
Nicotinamide, by Merck Group (8 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (8 mentions)
Il 17a, by Thermo Fisher Scientific (8 mentions)
Activin a, by Thermo Fisher Scientific (6 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Its premix, by BD (4 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Wnt3a, by R&D Systems (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
43 protocols using oncostatin m
1
Differentiation of Human iPSCs to Hepatocytes
2
Directed differentiation of hepatocytes
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HD was induced following our previously reported two-step protocol (Lee et al., 2004 (link)). In brief, 1.0 × 104/cm2 MSCs were seeded and cultured for 2 days in the maintenance medium. Two days post seeding, cells were treated with a step-1 differentiation medium, consisting of IMDM supplemented with 20 ng/mL hepatocyte growth factor and 10 ng/mL bFGF (R&D Systems), and 0.61 g/L nicotinamide (Sigma-Aldrich). Seven days later, cells were treated with a step-2 differentiation medium, consisting of IMDM supplemented with 20 ng/mL oncostatin M (Invitrogen), 1 μmol/L dexamethasone (Sigma-Aldrich), and 50 mg/mL ITS+ premix (BD Biosciences) for 3 weeks. The differentiation medium was changed once per week. To induce dHD in MSC-derived dHeps, we replaced the step-2 medium with maintenance medium (low-glucose DMEM supplemented with 10% FBS). A protocol with stepwise reduction of TGFβ1 was used to induce the lineage conversion of Heps. Specifically, Heps were cultured in an mMSC-maintenance medium with 10 ng/mL TGFβ1 for the first 7 days, then with 5 ng/mL TGFβ1 for another 7 days, and finally with 1 ng/mL TGFβ1 for the following 14 days. After 28 days, cells were cultured in maintenance medium without TGFβ1.
Other experimental procedures and statistical analysis are described inSupplemental Experimental Procedures .
Other experimental procedures and statistical analysis are described in
3
Efficient Hepatocyte Generation from iPSCs
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To generate hepatocytes from both control and patient iPSC clones, we employed an already described method that allow the efficient production of HLCs. (Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells [26]. Briefly, iPSC clones were plated on Matrigel and cultivated for 20–22 days changing the media composition as follows: 5 days in RPMI media supplemented with B27 and 100 ng/ml of Activin A (R&D), 5 days in RPMI media supplemented with 20 ng/ml BMP4 (R&D) and 10 ng/ml bFGF (Preprotech), 5 days in RPMI-B27 supplemented with 20 ng/ml hepatocyte growth factor (HGF, Invitrogen) and finally further 5–7 days in Hepatocyte Culture Medium (Lonza) supplemented with 20 ng/ml Oncostatin M (Invitrogen).
4
Differentiation of hiPSCs into Mature Hepatocytes
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Human‐inducible pluripotent stem cells (hiPSCs) were cultured with mTESR1 medium in a Matrigel‐coated culture dish. After the cell density reached 70%, the hİPSC medium was replaced with Roswell Park Memorial Institute / B27 medium containing 100 ng/mL Activin A (PeproTech), 50 ng/mL Wnt3a(R&D Systems), and 10 ng/mL HGF(PeproTech) for 3–5 days to induce endoderm cells. Subsequently, endodermal cells were differentiated to progenitor cells by changing RPMI medium with the knockout [KO]/DMEM containing 20% knockout serum replacement, 1 mm l ‐glutamine, 1% nonessential amino acid, 0.1 mm 2‐mercaptoethanol, and 1% dimethyl sulfoxide for further 4 days. In the final stage, cells were cultured with Iscove’s modified Dulbecco's (IMDM) medium containing 20 ng/mL oncostatin M (Invitrogen), 0.5 lM dexamethasone, and 50 mg/mL ITS premix(BD Biosciences) for 5 days in order to obtain mature hepatocytes [22 (link), 23 (link)]
5
3-Step Hepatocyte Differentiation from iPSCs
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Human iPS cells (ACS-1011) were obtained from ATCC (Manassas, VA) and maintained with the stem cell culture medium SFM XF/FF (ACS-3002). The three step protocol of hepatocyte-like cells induction from iPS cells was modified on the base of previous publications [19 (link)]. First, 70% confluent iPS cells were cultured with Roswell Park Memorial Institute (RPMI)/B27 medium containing 100ng/mL activin A or 10ng/ml TGF-β1, along with 50ng/mL Wnt3a, and 10ng/mL HGF (R&D Systems) for 3 days of endodermal induction. Then, RPMI/B27 was replaced by hepatic commitment medium (DMEM containing 20% knockout serum replacement, 1mM L-glutamine, 1% nonessential amino acids, 0.1mM 2-mercaptoethanol, and 1% dimethyl sulfoxide) for another 4 days. Finally, the cells were incubated in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20ng/mL oncostatin M (Invitrogen), 0.5μM dexamethasone, and 50 mg/mL ITS premix (BD Biosciences, San Jose, CA) for 5 days to induce hepatocyte maturation.
6
Inducing Hepatocyte-like Cells from Mouse Embryonic Fibroblasts
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Mouse embryonic fibroblasts (MEF) were prepared from 13.5-day post-coitum embryos. MEF were grown in DMEMc (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 2 mM Glutamax). In the reprogramming experiments two different media were used: hepatocyte conditioned medium (HCM) I and HCM II. HCM I is composed of IMDM:F12 (1:3), supplemented with 10 % FBS, 2 mM l -glutamine, penicillin/streptomycin, 10 ng/ml epidermal growth factor (EGF), 100 ng/ml fibroblast growth factor (FGF)2, 50 ng/ml vascular endothelial growth factor (VEGF), and 100 ng/ml transforming growth factor (TGF)β. HCM II is composed of IMDM:F12 (1:3), supplemented with 10 % FBS, 2 mM l -glutamine, penicillin/streptomycin, 10 ng/ml hepatocyte growth factor (HGF), and 10 ng/ml Oncostatin M. All media was purchased from Invitrogen (www.thermofisher.com ). Growth factors were purchased from R&D Systems (www.rndsystems.com ). iHepL cells exhibited enhanced attachment to the culture dishes and needed trypsinization for 30 min at 37 °C for passaging.
All cells were maintained at 37 °C with 5 % CO2 and were regularly examined with an Olympus CKX41 microscope. Images were taken on an Olympus FV1000 confocal mounted on an IX81 inverted microscope.
All cells were maintained at 37 °C with 5 % CO2 and were regularly examined with an Olympus CKX41 microscope. Images were taken on an Olympus FV1000 confocal mounted on an IX81 inverted microscope.
7
Hepatocyte Differentiation from Stem Cells
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siHBs were harvested using cell dissociation buffer (0.1 mg/mL EDTA, 0.5 mg/mL BSA) and seeded onto collagen I-coated plates in RPMI/B27, 10% Fetal Bovine Serum (FBS, Gibco, Waltham, MA, USA), 1% bovine serum albumin (BSA, Sigma, St. Louis, MO, USA), and 50 ng/mL HGF for four hours to enable cell attachment. The medium was then replaced by RPMI/ITS with 50 ng/mL HGF, 3 nM CHIR-99021 (Stemcell Technologies, Vancouver, WA, Canada) and 0.1 ng/mL TGF-β1 (Peprotech, Cranbury, NJ, USA), for 24 h. The medium was changed every day until the end of the differentiation with 20 ng/mL HGF, 0.05 nM dexamethasone (Sigma, St. Louis, MO, USA), 10 ng/mL oncostatin M (Peprotech, Cranbury, NJ, USA), 0.25 nM Compound E (Santa Cruz, Dallas, TX, USA), 2.5 nM SB431452 (Tocris, Bristol, UK) and 10 ng/mL vitamin K1 (Roche, Basel, Switzerland).
8
Expansion and Hepatic Maturation of hiPSC-Derived Liver Progenitor Cells
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To expand hiPSC-derived LPCs, we cultured the cells on mitomycin C-treated (Wako) HUVEC/MSC or hiPSC-derived NPC feeder cells (50,000 cells/cm2) in DMEM/F12 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (JRH Biosciences), penicillin-streptomycin-glutamine, insulin-transferrin-selenium, N2 supplement, MEM non-essential amino acids solution, L-glutamine (Life Technologies), ascorbic acid (1 mM), nicotinamide (10 mM), N-acetylcysteine (0.2 mM) (Sigma-Aldrich), dexamethasone (1 × 10−7 M), Y27632 (5 μM) (Wako), and A83-01 (2.5 μM) (Tocris) for 14 days. To induce hepatic maturation of hiPSC-derived LPCs, we cultured cells in HBM (Lonza) supplemented with HCM SingleQuots (excluding epidermal growth factor) and oncostatin M (20 ng/mL) (PeproTech) for 5 days.
9
Hepatic Differentiation of Human Mesenchymal Stem Cells
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To induce hepatic differentiation, the growth medium was replaced with differentiation medium described below when cells in passage four reached 80% confluency, as based on a previous protocol (3 (link)). Differentiation was induced by treating MSCs with liver-specific growth factors: Days 0–2, Iscove's modified Dulbecco's medium (IMDM, Gibco; Thermo Fisher Scientific, Inc.) with 20 ng/ml epidermal growth factor (PeproTech, Inc.) and 10 ng/ml basic fibroblast growth factor (bFGF; PeproTech, Inc.); days 3–9, IMDM supplemented with 20 ng/ml hepatocyte growth factor (PeproTech, Inc.), 10 ng/ml bFGF and 0.61 g/ml nicotinamide (Sigma-Aldrich; Merck KGaA); from day 9 onwards, IMDM containing 20 ng/ml oncostatin M (PeproTech, Inc.), 1 µmol/l dexamethasone (Sigma-Aldrich; Merck KGaA) and 50 mg/ml insulin/transferring/selenium (Sigma-Aldrich; Merck KGaA). The hepatic differentiation medium was replaced every 3 days. The progression of differentiation from HuMSCs to hepatocytes (at days 14 and 28) was analyzed.
10
Psoriasis-like Keratinocyte Stimulation
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HaCaT and HEKa cells (human keratinocyte cell lines) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2. Cells were stimulated with 10 ng/ml M5 (IL-1α, IL-17A, IL-22, oncostatin M, and TNF-α; PeproTech, USA) for 24 hours at 37°C to mimic a psoriasis-like environment in keratinocytes [17 (link)–20 (link)]. Cells were then either left untreated or pretreated with 1 μg/ml C23 (GRGFSRGGGDRGYGG synthesized from GenScript, Piscataway, NJ; dissolved in phosphate-buffered saline) for 30 minutes and then were stimulated with 1 μg/ml rhCIRP (APG886Hu01, Cloud-Clone Corp, Texas, USA) for 1 hour. After rhCIRP stimulation, cells were collected for qRT-PCR and western blot analysis.
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