Following established protocols (Cai et al., 2008 ), control, LMNA T10I, LMNA R541C hiPSCs were grown in feeder-free differentiation conditions. For efficient hepatocyte differentiation, cells were incubated in definitive endoderm media with recommended supplements (Stem Cell Technologies) for 4 days in ambient O2 and 5% CO2, yielding homogeneous monolayer of definitive endoderm cells. At day 5, cells were incubated with recombinant human BMP-4 (20ng/mL; Peprotech) and recombinant human FGF basic (10ng/mL; R&D Systems) for 5 days in RPMI-B27 (with insulin) in 5% O2 and 5% CO2, yielding hepatic progenitor cells. At day 10, cells were incubated in RPMI-B-27 (with insulin) supplemented with recombinant human HGF (20ng/mL; PeproTech) for 5 days at 5% O2 and 5% CO2, yielding immature HLCs. Finally, at d15, cells were incubated with HCM Hepatocyte Culture Medium (Lonza) without EGF and supplemented with recombinant human oncostatin M (20ng/mL; R&D Systems) for 7 days in ambient O2 and 5% CO2, yielding mature HLCs, which were collected at day 23 for subsequent ChIP, immunofluorescence, and immunoblotting.
Hcm hepatocyte culture medium
Manufactured by Lonza
Sourced in United Kingdom
HCM Hepatocyte Culture Medium is a specialized cell culture medium designed for the maintenance and growth of primary human hepatocytes. It provides the necessary nutrients and components to support the in vitro culture of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (3 mentions)
Recombinant human hgf, by Thermo Fisher Scientific (2 mentions)
Rpmi b27, by Thermo Fisher Scientific (2 mentions)
Recombinant human oncostatin m, by R&D Systems (2 mentions)
Recombinant human bmp4, by Thermo Fisher Scientific (1 mentions)
Rpmi b27, by R&D Systems (1 mentions)
Recombinant human fgf basic, by R&D Systems (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Oncostatin m, by R&D Systems (1 mentions)
Activin a, by R&D Systems (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Cryopreserved human primary hepatocytes, by Lonza (1 mentions)
5 protocols using hcm hepatocyte culture medium
1
Directed Differentiation of hiPSCs into Mature Hepatocytes
2
Differentiation of hiPSCs to Hepatocytes
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hiPSC lines were passaged with Accutase (Innovative Cell Technologies, San Diego, CA) for 5 minutes at room temperature (RT) and plated for differentiation on Geltrex coated dishes following Stephen Duncan’s protocol with modifications.(5 ) For efficient DE differentiation, the DE kit (STEMCELL Technologies) was used for the first 4 days in ambient O2/5% CO2. Later, cells were cultured in RPMI‐B27 (with insulin) supplemented with BMP4 (20 ng/mL; PeproTech, Rocky Hill, NJ) and FGF2 (10 ng/mL; R&D Systems) for 5 days in 5% O2/5% CO2, followed by RPMI‐B‐27 (with insulin) supplemented with recombinant human HGF (20 ng/mL; PeproTech) for 5 days in 5% O2/5% CO2. The last stage required culturing cells in HCM Hepatocyte Culture Medium (Lonza Group AG, Basel, Switzerland) that was supplemented with recombinant human Oncostatin M (20 ng/mL; R&D Systems) for 5 days in ambient O2/5% CO2.(9 )
3
Hepatic Differentiation Protocol from Cells
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Hepatic differentiation was performed following the protocol described in [20 (link)]. Briefly, when cells reach 70% confluency, the medium was changed for RPMI1640 media containing B27 Supplements Minus Insulin (Invitrogen), 100 ng/mL Activin A (R&D Systems), 20 ng/mL fibroblast growth factor 2 (FGF2) (R&D Systems), and 10 ng/mL bone morphogenetic protein 4 (BMP4) (PeproTech) to induce endoderm. After 8 days of culture, dishes were moved to hypoxia (4% O2) in RPMI/B27 Supplement (Invitrogen) medium with 20 ng/mL BMP4 and 10 ng/mL FGF2 for 5 days. Next, the medium was changed to RPMI/B27 supplemented with 20 ng/mL hepatocyte growth factor (HGF, PeproTech) for an additional 5 days in hypoxia. The final stage of differentiation was in HCM hepatocyte culture medium (Lonza, but omitting the EGF) supplemented with 20 ng/mL Oncostatin M (R&D Systems) for 5 days in normoxia (21% O2). During that time, the medium was freshly prepared and changed daily.
4
Culturing Primary Human Hepatocytes
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Cryopreserved primary human hepatocytes (Lonza Biologics, United Kingdom) were cultured with HCM Thawing Medium, Hepatocyte Plating Medium and HCM Hepatocyte Culture Medium (Lonza Biologics, United Kingdom) according to supplier’s instructions. For some experiments cells were exposed to LPS from Klebsiella pneumoniae (Merck, United Kingdom) or tunicamycin (Merck, United Kingdom) according to the doses stated.
5
Differentiation of hiPSCs into Hepatocyte-Like Cells
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