Human pre‐implantation embryos were donated from patients who had completed their family after assisted reproduction treatment. Written informed consents were obtained from all the donors recruited and the study protocols were approved by the Institutional Review Boards of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (IRB number: UW 18‐017) and the Council of Reproductive Technology, Hong Kong (research license number: R5004). Cryopreserved day 2 human pre‐implantation embryos were thawed and cultured in G‐1 medium (Vitrolife) for 1 day before transferred to G‐2 medium (Vitrolife) and cultured till morula or early blastocyst stages when the zona pellucida of the embryos was removed using acid tyrode solution (Sigma Aldrich). The human embryos were then placed on the STO feeder cells in hEPSC medium supplemented with 10 ng mL−1 recombinant bFGF (R&D), 20 ng mL−1 recombinant activin A (PeproTech), and 5% FBS (Thermo Fisher Scientific). The embryos showed outgrowth after several days. The resultant cell colonies were dissected into small clusters using glass pipettes and transferred onto new STO feeder cells. After several passages, the colonies were digested with 0.05% Trypsin (Thermo Fisher Scientific) and passaged like other hEPSCs.
Recombinant activin a
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant activin A is a protein that plays a role in cell growth and differentiation. It is produced using recombinant DNA technology and can be used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
G1 medium, by Vitrolife (1 mentions)
G2 medium, by Vitrolife (1 mentions)
Recombinant bfgf, by R&D Systems (1 mentions)
Fibroblast growth factor 2 fgf2, by Thermo Fisher Scientific (1 mentions)
Recombinant human wnt3a, by R&D Systems (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
3 protocols using recombinant activin a
1
Derivation of human embryonic pluripotent stem cells
2
Differentiation of ESCs and PrE-ESCs into ETX Embryoids
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In method C (>5 replicates), following a 4- to 6-hour treatment of PrE-ESCs with Dox (1 μg/ml), Actin-GFP ESCs and PrE-ESCs were dissociated as described. Cell numbers were adapted aiming at assembling on day 0, 20 to 24 ESCs and 5 to 10 PrE-ESCs per well of a 384-well low adhesion plate (final volume of 50 μl/well). Using a multichannel, 50 μl of this cell suspension was pipetted into each well of a 384-well low attachment plate and incubated overnight at 37°C and 5% CO2. Dox (1 μg/ml) and LIF (20 ng/ml) were added to the ETX medium on the day of the aggregation. The following day, the wells were rinsed twice with ETX medium devoid of LIF and Dox. TSCs were dissociated as described, and a cell suspension containing 70 TSCs per 60 μl of ETX medium supplemented with recombinant activin A (0.1 ng/ml; PeproTech) was added to each well containing approximately 20 μl of ETX medium. The medium of the cell aggregates was refreshed on day 3 with IVC1 (20% FBS) and with IVC2 on days 4 and 5 (30% FBS). ETX embryoids of interest were manually picked with a pipette. To assess the efficiency, rows of a 384-well plate with each well containing one ETX embryoid were randomly chosen to evaluate development of the ETX embryoids.
3
Directed Differentiation of iPSCs to iHeps
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Stepwise-directed differentiation of the iPSCs into iHeps was achieved by a modification of the method reported previously (2) . Briefly, 1 to 5 × 10 4 UiPSCs at a passage number of 20-30 were plated onto six-well plates precoated with Matrigel (BD Biosciences). The next day, UiPSCs were seen as small colonies with ~60% confluence. The medium was changed to DMEM/F12 enriched with 100 ng/ml recombinant Activin A (PeproTech, Rocky Hill, NJ, USA), 100 ng/ml fibroblast growth factor-2 (FGF2; PeproTech) plus 50 ng/ml recombinant human Wnt3a (R&D Systems, Minneapolis, MN, USA) and was maintained for the next 3 days. Concentrations of KnockOut SR Xenofree medium (KSR; Life Technologies) was 0%, 0.2%, and 2.0% for the first, second, and final 24 h, respectively. For the next 8 days, cells were cultivated in DMEM/ F12 supplemented with 10% KSR, 1 mM NEAA, 1 mM L-glutamine, 1% dimethyl sulfoxide (DMSO; Sigma-Aldrich), and 100 ng/ml hepatocyte growth factor (HGF; PeproTech). Cells were then grown for 3 days in 10% KSR, 1 mmol/L NEAA, 1 mM L-glutamine, and 10 -7 mol/L dexamethasone (Sigma-Aldrich).
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