Differentiation of human embryonic stem cells
A technology of cell differentiation and pluripotent stem cells, applied to embryonic cells, non-embryonic pluripotent stem cells, artificial cell constructs, etc., can solve problems such as simulating the developmental program of higher mammals
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[0349] Human embryonic stem cell culture
[0350] Human embryonic stem cell lines H1, H7, and H9 were obtained from WiCell Research Institute, Inc., (Madison, WI) and cultured according to instructions provided by the source institution. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeder layers in ES cell culture medium supplemented with 20% Knockout serum replacer, 100 nM of MEM non-essential amino acids, 0.5 mM β-mercaptoethanol, 2 mM L-glutamine and 4 ng / ml human basic fibroblast growth factor (bFGF) (all from Invitrogen / GIBCO) in DMEM / F12 (Invitrogen / GIBCO). MEF cells derived from E13 to 13.5 mouse embryos were purchased from Charles River. MEF cells were expanded in DMEM medium supplemented with 10% FBS (Hyclone), 2 mM glutamine and 100 mM of MEM non-essential amino acids. Subconfluent MEF cell cultures were treated with 10 mg / ml mitomycin C (Sigma (St.Louis, MO)) for 3 hours to prevent cell division, and then trypsinized and treated with 2 × 10 ...
example 2
[0352] Formation of definitive endoderm
[0353] The effect of activin A on the expression of definitive endoderm markers was detected. Activin A (100 ng / ml) was added to human embryonic stem cell populations cultured on mouse embryonic fibroblasts. Cells were continuously cultured in the presence of Activin A and harvested at the indicated times. By PCR ( figure 1 ), FACS (results are summarized in Table II) and immunohistochemistry ( figure 2 ) to detect the expression levels of definitive endoderm markers.
[0354] Activin A caused a time-dependent increase in the expression of CXCR4, GATA4, HNF-3β, Mixl1 and Sox-17 mRNA in the H9 cell line ( figure 1 , screen a). Significant upregulation of the anterior endoderm markers Cerberus, Otx-1 and Hex genes was also observed ( figure 1, screen b). After treatment with Activin A, an increase in CXCR4 protein was observed by FACS analysis. The expression of E-cadherin and N-cadherin was not altered after treatment with Ac...
example 3
[0356] Pancreatic endoderm formation
[0357] Growth factors known to induce differentiation of human embryonic stem cells into pancreatic endoderm are added to the cell culture. Specifically, activin A, bFGF and retinoic acid, known to induce pancreatic endoderm formation, were added to the cell cultures.
[0358] In the first series of experiments, activin A was added to populations of human embryonic stem cells in DMEM / F12 supplemented with 0% to 2% serum and activin A (100ng / ml) to induce fibroblasts in mouse embryos. Cells were cultured for up to seven days. exist image 3 Cells were harvested at the indicated time points and analyzed by PCR for the expression of the indicated genes ( image 3 , Figure 4 and Figure 5 ). exist image 3 , PCR analysis revealed that cells treated with activin expressed a broad spectrum of genes associated with endoderm development, including GATA4 ( image 3 , panel a), Sox-17 ( image 3 , panel b), HNF-3β ( image 3 , panel c)...
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