Differentiation of human embryonic stem cells
A pluripotent stem cell and cell technology, applied in embryonic cells, non-embryonic pluripotent stem cells, artificial cell constructs, etc., can solve the problem of not completely simulating the developmental procedures of higher mammals
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[0098] The role of insulin in the differentiation of human pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage Use: cluster planting
[0099] Previous studies have shown that high concentrations of FBS are detrimental to definitive endoderm (DE) formation from embryonic stem cells. See, eg, D'Amour et al., Nature Biotechnology, 2005, where induction of definitive endoderm from human embryonic stem cells was significantly increased when the FBS concentration was reduced from 10% FBS to 0.5-2% FBS. Similar observations were reported where the addition of 25ng / ml IGF or 200ng / ml insulin to 2% FBS for ES cells cultured in MEF-CM (mouse embryonic fibroblast conditioned medium) reduced the rate of activin-A treatment. The subsequent SOX17 expression was reduced by about 70%. See McLean et al., Stem Cells 25:29-38, 2007.
[0100] The observed inhibitory effect may be due to the presence of insulin or IGF in FBS, which triggers ...
example 2
[0111] The role of insulin in the differentiation of human pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage Use: single cell planting
[0112] Human embryonic stem cell line H1 (p40-p52) cells were used as single cells at 100,000 cells / cm 2 planted at a density of (1:30 dilution) (BD Biosciences; Cat. No. 356231) coated dishes supplemented with 16ng / ml FGF2 (Cat. No. Y0503, Sigma, MO) in MEF-CM (Mouse Embryo Fibroblast Conditioned Medium). 72 hours after seeding, cultures were differentiated into definitive endoderm (DE) as follows:
[0113] a. Supplemented with 2% fatty acid-free BSA (Cat. No. 68700, Proliant, Iowa), 0.0025 g / ml sodium bicarbonate (Cat. No. S3187, Sigma, Missouri), 1X GlutaMax TM (Cat. No. 35050-079, Invitrogen, California) and 100ng / ml Activin A (Andy Biotech, Minnesota) plus 20ng / ml WNT-3a (Cat. No. 1324-WN-002, The MCDB-131 (Cat. No. 10372-019 of Andy Biotechnology Company of Minnesota) (Catalo...
example 3
[0121] Role of IGF in the differentiation of human pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage: single cell planting
[0122] Human embryonic stem cell line H1 (p40-p52) cells were used as single cells at 100,000 cells / cm 2 planted at a density of (1:30 dilution) (BD Biosciences; Cat. No. 356231) coated dishes supplemented with 16ng / ml FGF2 (Cat. No. Y0503, Sigma, MO) in MEF-CM (Mouse Embryo Fibroblast Conditioned Medium). 72 hours after seeding, cultures were differentiated into definitive endoderm (DE) as follows:
[0123] a. Supplemented with 2% fatty acid-free BSA (Cat. No. 68700, Proliant, Iowa), 0.0025 g / ml sodium bicarbonate (Cat. No. S3187, Sigma, Missouri), 1X GlutaMax TM (Cat. No. 35050-079, Invitrogen, California) and 100 ng / ml GDF-8 (Cat. No. 120-00, Andy Biotech, Minnesota) plus 2.5 μM GSK3B inhibitor 14-propa- 2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1~2,6~.1~8,12~]twenty seven-1( 2...
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