Abstract
Retinoic acid (RA) plays multiple roles in the nervous system, including induction of neural differentiation, axon outgrowth and neural patterning. Previously, RA for neural differentiation of embryonic stem (ES) cells always relies on embryoid bodies (EBs) formation. Here we report an in vitro adherent monoculture system to induce mouse ES cells into neural cells accompanied with RA. RA (1 μM) treatment, during initial 2 days of differentiation, can enhance the expression of neural markers, such as Nestin, Tuj1 and MAP2, and result in an earlier neural differentiation of ES cells. Furthermore, RA promotes a significant increase in neurite elongation of ES-derived neurons. Our study also implies that RA induced to express Wnt antagonist Dickkopf-1 (Dkk-1) for neural differentiation. However, the mechanisms of RA triggering neural induction remain to be determined. Our simple and efficient strategy is proposed to provide a basis for studying RA signaling pathways in neural differentiation in vitro.
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Abbreviations
- Dkk-1:
-
Dickkopf-1
- EB:
-
Embryoid body
- ES:
-
Embryonic stem
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- LIF:
-
Leukemia inhibitory factor
- MAP2:
-
Microtubule-associated protein 2
- MEF:
-
Embryonic fibroblast
- NeuN:
-
Neuronal nuclei
- Oct4:
-
Octamer-4
- Pax6:
-
Paired box gene 6
- RA:
-
Retinoic acid
- Sfrp2:
-
Secreted frizzled-related protein-2
- Sox1:
-
Sex determining region Y-box 1
- Sox2:
-
Sex determining region Y-box 2
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Acknowledgments
We would like to thank Dr. Duanqing Pei for kindly providing us with mouse ES cells R1. This study was supported by the National Basic Research Program of China (973 program, 2007CB947804), NSFC-Guangdong Joint Fund (U0972001/L02), National Natural Science Foundation of China (30700213/C090204) and Natural Science Foundation of Guangdong Province, China (07007215).
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11033_2011_800_MOESM2_ESM.tif
Figure S1. Images of immunostaining for neural marker Sox1, Pax6, Nestin, Tuj1 and NeuN evaluate neural conversion from mouse ES cells in N2B27 with different concentration of RA after 6 days of differentiation. RA at a concentration of 10−6 is best for cell survival and neural conversion. Scale bar = 100 μm. (TIFF 1482 kb)
11033_2011_800_MOESM3_ESM.tif
Figure S2. The expression of pluripotency gene Oct4 and neural genes Sox1, Pax6, Nestin, Tuj1 and MAP2 changed in RA-N2B27 compare with N2B27 analyzed by quantitative real-time PCR. (TIFF 73Â kb)
11033_2011_800_MOESM4_ESM.tif
Figure S3. Densitometry analysis shows expression level of pluripotency gene Oct4 and neural genes Sox1, Tuj1 and NeuN in both RA-N2B27 and N2B27 culture system. (TIFF 92Â kb)
11033_2011_800_MOESM5_ESM.tif
Figure S4. The expression of Wnt antagonist Dkk-1 in RA-N2B27 compare with N2B27 analyzed by quantitative real-time PCR. (TIFF 63Â kb)
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Xu, J., Wang, H., Liang, T. et al. Retinoic acid promotes neural conversion of mouse embryonic stem cells in adherent monoculture. Mol Biol Rep 39, 789–795 (2012). https://doi.org/10.1007/s11033-011-0800-8
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DOI: https://doi.org/10.1007/s11033-011-0800-8