NCCIT, HEK293 cells were obtained from ATCC. NCCIT cells were maintained in 10% FBS (Omega), 1× Glutamax-I supplement (100× stock, Invitrogen), 1xNon-essential amino acids (100x stock, invitrogen), and basal media RPMI 1640 (Hyclone). HEK293 cells were maintained in 10% FBS, 1x NEAA, 1x glutamax in DMEM-high glucose (Hyclone). hESCs (H9 line, Wi-Cell) were used at passage 60–67 and were expanded in feeder-free, serum-free medium, mTESR-1 from StemCell technologies. HESC HSF-1 (male) and HSF-8 (male) hESC were used at passage 20–28, cultured as described above and their characterization is described elsewhere31 . Cells were passaged 1:7 every 5–6 days by incubation with accutase (Invitrogen) and resultant small cell clusters (50–200 cells) were subsequently re-plated on tissue culture dishes coated overnight with growth-factor-reduced matrigel (BD Biosciences). ELF1 naïve hESC were obtained from Dr. Carol Ware and cultured as previously described14 (link), with 10ng/mL human recombinant LIF (R&D). Cell cultures were routinely tested and found negative for mycoplasma infection (MycoAlert, Lonza).
H9 line
Manufactured by WiCell
Sourced in United States
The H9 line is a well-established and widely-used human embryonic stem cell (hESC) line. It was derived from a preimplantation human embryo and has the ability to differentiate into a variety of cell types. The H9 line is a valuable tool for researchers studying human development, disease modeling, and regenerative medicine.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Glutamax 1 supplement, by Thermo Fisher Scientific (2 mentions)
Recombinant human lif, by R&D Systems (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Growth factor reduced matrigel, by BD (2 mentions)
Mycoalert, by Lonza (2 mentions)
Mtesr1, by STEMCELL (2 mentions)
Matrigel, by BD (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Chemically defined lipid concentrate, by Thermo Fisher Scientific (1 mentions)
Monothioglycerol, by Merck Group (1 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
Activin a, by R&D Systems (1 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (1 mentions)
Transferrin, by Roche (1 mentions)
Insulin, by Roche (1 mentions)
Bmp 2, by Thermo Fisher Scientific (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
8 protocols using h9 line
1
Maintenance and Culture of Cell Lines
2
Feeder- and Serum-free hESC and hiPSC Culture
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3
Maintenance and Culture of Cell Lines
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NCCIT, HEK293 cells were obtained from ATCC. NCCIT cells were maintained in 10% FBS (Omega), 1× Glutamax-I supplement (100× stock, Invitrogen), 1xNon-essential amino acids (100x stock, invitrogen), and basal media RPMI 1640 (Hyclone). HEK293 cells were maintained in 10% FBS, 1x NEAA, 1x glutamax in DMEM-high glucose (Hyclone). hESCs (H9 line, Wi-Cell) were used at passage 60–67 and were expanded in feeder-free, serum-free medium, mTESR-1 from StemCell technologies. HESC HSF-1 (male) and HSF-8 (male) hESC were used at passage 20–28, cultured as described above and their characterization is described elsewhere31 . Cells were passaged 1:7 every 5–6 days by incubation with accutase (Invitrogen) and resultant small cell clusters (50–200 cells) were subsequently re-plated on tissue culture dishes coated overnight with growth-factor-reduced matrigel (BD Biosciences). ELF1 naïve hESC were obtained from Dr. Carol Ware and cultured as previously described14 (link), with 10ng/mL human recombinant LIF (R&D). Cell cultures were routinely tested and found negative for mycoplasma infection (MycoAlert, Lonza).
4
Expansion of Human Embryonic Stem Cells
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The human embryonic stem cells (hESC), H9 line, was purchased from WiCell Research Institute (Madison, WI), and maintained and expanded on mouse embryonic fibroblasts (MEFs) as instructed by the provider.
5
Chemically Defined Culture of hPSCs
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Human embryonic stem cells (HESCs; H9 line, Wicell, Madison,WI) and the human induced pluripotent stem cell (hIPSC) line BHX (Cambridge Biomedical Research Centre hIPSC Core Facility), collectively termed HPSCs, were cultured under chemically defined conditions as described previously (Brons et al., 2007 (link)). Briefly, HPSCs were cultured in a chemically defined medium (CDM) containing bovine serum albumin fraction A (CDM-BSA) supplemented with Activin A (10 ng/ml, R&D systems) and FGF2 (12 ng/ml, R&D systems) on gelatine-coated plates. CDM-BSA comprised Iscove's modified Dulbecco's medium (Gibco) plus Ham's F12 NUT-MIX (Gibco) medium in a 1:1 ratio, supplemented with Glutamax-I, BSA (5 mg/ml; Europa Bioproducts), chemically defined lipid concentrate (Life Technologies), transferrin (15 µg/ml, Roche Diagnostics), insulin (7 µg/ml, Roche Diagnostics) and monothioglycerol (450 μM, Sigma).
6
Differentiation of Human Embryonic Stem Cells to Hepatocytes
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Human embryonic stem cell, H9 line, was purchased from WiCell Research Institute (Madison, WI, USA) under a Materials Transfer Agreement (No. 19-W0512), cultured and maintained on the embryonic fibroblast feeder layer (MEF) of CF-1 strain mice according to provider’s instructions. Differentiation of embryonic stem cells into hepatic progenitors underwent two critical stages under our culture condition, definitive endoderm (DE) was initially induced for 2 days with RPMI1640 medium supplemented with ActivinA (100 ng/mL) and Wnt3a (25 ng/mL), and then, fresh RPMI 1640 medium was replenished with 100 ng/mL Activin A, 0.5 mM sodium butyrate and 1 × B27 supplement for another 5 days. Afterward, the differentiation of hepatic progenitor cells was performed for 14 days under complete hepatocyte differentiation medium (HDM) containing IMDM medium supplemented with 20% FBS, 0.3 mM 1-thioglycerin, 0.5% DMSO, 100 nM dexamethasone, 0.126 U/mL insulin, HGF (20 ng/mL), FGF4 (20 ng/mL), BMP2 (10 ng/mL) and BMP4 (10 ng/mL) (all growth factors from Peprotech).
7
Maintenance of Pluripotent and Differentiated hESCs
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MAVS+/+ hESCs (derived from Line H9, WiCell Research Institute) and MAVS−/− hESCs grown on mouse embryonic fibroblast (MEF) feeder layers inactivated with mitomycin C were cultured with conventional DMEM/F12 (Gibco) culture medium [42 (link)]. hESCs were also maintained on precoated plates with Matrigel (BD Biosciences) using mTeSR medium. hESC-derived hMSCs were maintained with hMSC culture medium [43 (link)]. hNSCs were cultured according to a previous study [71 (link)].
8
Culturing Human Stem Cell Lines
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Wildtype (WT) human ESCs (hESCs, Line H9, WiCell Research Institute) and their genetic modified derivatives were maintained on feeder cells (mitomycin C-inactivated mouse embryonic fibroblast, MEF) in hESC culture medium containing DMEM/F12 (Thermo Fisher Scientific), 20% Knockout Serum Replacement (Thermo Fisher Scientific), 0.1 mM non-essential amino acids (NEAA, Thermo Fisher Scientific), 2 mM GlutaMAX (Thermo Fisher Scientific), 55 μM β-mercaptoethanol (Thermo Fisher Scientific), and 10 ng/ml bFGF (Joint Protein Central) or on Matrigel (BD Biosciences) in mTeSR medium (STEMCELL Technologies). Both Human MSCs (hMSCs) derived from hESCs and primary human MSCs were cultured in MEMα (Thermo Fisher Scientific) medium supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific (Cat:10099-141, Lot:1616964)), 0.1 mM non-essential amino acids (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific), and 1 ng/ml bFGF (Joint Protein Central, JPC).
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