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Open Access Highly Accessed Research article

All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways

Jianfeng Lu12, Li Tan12, Ping Li12, Hui Gao3, Bo Fang12, Shoudong Ye12, Zhe Geng12, Ping Zheng3 and Houyan Song12*

Author Affiliations

1 Department of Molecular Genetics, Shanghai Medical School, Fudan University, Shanghai, PR China

2 Key Laboratory of Molecular Medicine, Ministry of Education, PR China

3 State Key Laboratory of Medical Neurobiology, PR China

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BMC Cell Biology 2009, 10:57  doi:10.1186/1471-2121-10-57

Published: 30 July 2009

Additional files

Additional file 1:

High does of RA (10-6 M) shows obvious noxious effect on cell survival. ESCs were cultured under monolayer differentiation conditions with low does of RA (N2B27 + 10-8 M RA), high does of RA (10-6 M) or without RA (N2B27 + 0 or N2B27 + ethanol). A, Intact cultures at different time points of monolayer differentiation, shown in phase contrast; B, Ratios of harvested cell number to plated cell number at day 10 (average of triplicates).

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Additional file 2:

RA increases the cell number of the monolayer culture at the early stage of neural induction. Ratios of harvested cell number to plated cell number at various time points were given (average of triplicates).

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Additional file 3:

RA shows less effect on neural differentiation when ESCs were plated at a high density (3 × 104 cells/cm2). Proportion of Sox1GFP+ cells at various time points was given (average of triplicates).

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Additional file 4:

Effects of inhibitors on cell viability or proliferation in RA-treated monolayer cultures. Monolayer differentiation of ESCs in RA-treated cultures (N2B27 + 10-8 M RA) exposed to FGF receptor tyrosine kinase inhibitor SU5402 (5 μM), MEK1/2 inhibitors PD184352 (4 μM) or PD0325901 (4 μM), or to equivalent amounts of DMSO diluents. Ratios of harvested cell number to plated cell number at 92 h were given (average of triplicates).

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Additional file 5:

GSK3 inhibitors block the neural specification of ESCs when RA is absent in the culture. A, B, LiCl inhibits the neural specification of ESCs when RA is absent in the culture. Monolayer differentiation of 46C ESCs in cultures without RA (N2B27) exposed to LiCl (10 mM) or to NaCl (10 mM) at day 4. A, Cultures of monolayer differentiation, shown in phase contrast or stained for Sox1GFP. B, Percentage of Sox1GFP+ cells scored by FACS. C, D, CHIR99021 inhibits the neural specification of ESCs when RA is absent in the culture. Monolayer differentiation of 46C ESCs in cultures without RA (N2B27) exposed to the GSK3 inhibitor CHIR99021 (3 μM) or to equivalent amounts of DMSO diluents. C, Cultures of monolayer differentiation, shown in phase contrast or stained for Sox1GFP. D, Percentage of Sox1GFP+ cells scored by FACS. Scale bars: A, C, 80 μm.

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Additional file 6:

SU 5402 inhibits the neural specification of ESCs when RA is absent in the culture. Monolayer differentiation of 46C ESCs in cultures without RA (N2B27 + 0) exposed to FGF receptor tyrosine kinase inhibitor SU5402 (5 μM) or to equivalent amounts of DMSO diluents at day 6. A, Cultures of monolayer differentiation, shown in phase contrast or stained for Sox1GFP. B, Typical FACS profile of Sox1GFP expression. C, Percentage of Sox1GFP+ cells scored by FACS. Scale bars: A, 80 μm.

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Additional file 7:

RT-PCR primer specifications.

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Additional file 8:

Q-PCR primer specifications.

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