Glycogen synthase kinase 3β inhibitor CHIR99021 induces human embryonic stem cells to differentiate into definitive endoderm cells

doi:10.3877/cma.j.issn.2095-3232.2014.03.013

Abstract

Abstract:

Objective

To investigate the feasibility of glycogen synthase kinase (GSK) 3β inhibitor CHIR99021 induces human embryonic stem cells to differentiate into definitive endoderm cells.

Methods

Human embryonic stem cells H1 cells were cultured in feeder-free medium (Essential 8). The expression of human embryonic stem cell pluripotency markers Oct4, Nanog were detected by immunofluorescence. Human embryonic stem cells were treated with CHIR99021 of different concentrations (0.33, 1.00, 3.00, 9.00, 27.00 μmol/L) and cultured for 3 d respectively. The untreated cells were taken as control. The expression levels of pluripotency related genes OCT4, SOX2 and definitive endoderm related genes GATA4, SOX17 were detected by fluorescent quantitative polymerase chain reaction (PCR). The experimental data were compared using one-way analysis of variance and LSD-t test.

Results

The expression of human embryonic stem cell pluripotency markers Oct4, Nanog could be detected by immunofluorescence. The human embryonic stem cells were observed in good growth when treated with CHIR99021 of different concentrations (0.33, 1.00, 3.00 μmol/L) . The human embryonic stem cells were observed in poor growth or dead when treated with the higher concentrations of CHIR99021. The expression of pluripotency related gene OCT4 decreased after treated with CHIR99021 of 1.00, 3.00 μmol/L concentration (LSD-t=-40.54, -59.12; P<0.05). The expression of SOX2 also decreased significantly (LSD-t=-20.46, -3.87; P<0.05). The expression of definitive endoderm related genes GATA4 increased after treated with CHIR99021 of 1.00, 3.00 μmol/L concentration (LSD-t=137.21, 65.29; P<0.05). The expression of SOX17 also increased significantly (LSD-t=50.93, 6.56; P<0.05).

Conclusions

Human embryonic stem cells can maintain a good pluripotent state in the feeder-free culturing system. CHIR99021 can effectively induce the human embryonic stem cells to differentiate into definitive endoderm cells.

Key words: Embryonic stem cells, Cell differentiation, Endoderm, Glycogen synthase kinase 3, CHIR99021

Cong Du, Dongbo Qiu, Nan Cai, Yabin Wang, Yuan Feng, Qi Zhang, Peng Xiang, Huimin Yi. Glycogen synthase kinase 3β inhibitor CHIR99021 induces human embryonic stem cells to differentiate into definitive endoderm cells[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2014, 03(03): 183-188.

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References 17

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