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, 43 (5), 523-532

9-cis-Retinoic Acid and 1,25-dihydroxy Vitamin D3 Improve the Differentiation of Neural Stem Cells Into Oligodendrocytes Through the Inhibition of the Notch and Wnt Signaling Pathways

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9-cis-Retinoic Acid and 1,25-dihydroxy Vitamin D3 Improve the Differentiation of Neural Stem Cells Into Oligodendrocytes Through the Inhibition of the Notch and Wnt Signaling Pathways

Saeedeh Saeb PhD et al. Iran J Med Sci.

Abstract

Background: Differentiating oligodendrocyte precursor cells (OPCs) into oligodendrocytes could be improved by inhibiting signaling pathways such as Wnt and Notch. 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) can ameliorate oligodendrogenesis. We investigated whether they could increase oligodendrogenesis by inhibiting the Wnt and Notch signaling pathways.

Methods: Cortical neural stem cells were isolated from 14-day-old rat embryos and cultured using the neurosphere assay. The cells were treated in 4 different conditions for 1 week: the negative control group received only the basic fibroblast growth factor, the positive control group received only T3 without growth factors, the RA group was treated with 9-cis-RA, and the Vit D3 group was treated with 1,25(OH)2D3. The effects of 9-cis-RA and 1,25(OH)2D3 on the level of the myelin basic protein (MBP) and the gene expression of the SOX10, MBP gene, HES5, and LRP6 were studied using flow cytometry and real-time PCR. The data were analyzed using one-way ANOVA with GraphPad Prism. A P value less than 0.05 was considered significant.

Results: The mRNA expressions of the SOX10, MBP, and MBP gene were significantly increased in the treated groups compared with the negative control group; the increase was similar in the 9-cis-RA group and the positive control group. Furthermore, 9-cis-RA significantly decreased the expression of the HES5 gene, a Notch signaling pathway transcription factor, and 1,25(OH)2D3 significantly reduced the expression of the LRP6 gene, a Wnt signaling pathway co-receptor.

Conclusion: It seems that 9-cis-RA and 1,25(OH)2D3 are good candidates to improve the differentiation of OPCs into oligodendrocytes.

Keywords: 1,25-dihydroxy vitamin D3; 9-cis-retinoic acid; Notch signaling pathway; Oligodendrocyte precursor cells; Receptors, Wnt; Cell differentiation.

Figures

Figure1
Figure1
Differentiation and morphological maturation of the oligodendrocyte precursor cells in different culture conditions after 7 days. A) Negative control; B) Positive control; C) Vitamin D3 group; D) Retinoic acid group. Scale bar: 100 µm.
Figure2
Figure2
Effects of 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the mRNA expression of the Sry-related HMg-Box gene 10 (SOX10) and myelin basic protein (MBP) genes in the different groups. A) Real time-polymerase chain reaction (RT-PCR) data: NC, Negative control; PC, Positive control; Vit D3, Group treated with 1,25(OH)2D3; RA, Group treated with retinoic acid. B and C) RT-PCR analyses for the SOX10 and MBP genes, respectively (**P<0.01 and P<0.001 ***indicate significant differences between the groups.) Data are given as mean±SEM, n=3.
Figure3
Figure3
Representative flow cytometry histograms, showing the expression of the myelin basic protein (MBP) in the different culture conditions after 7 days. A) Negative control; B) Positive control; C) Group treated with retinoic acid (RA); D) Vitamin D3 group
Figure4
Figure4
Effects of 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the mRNA expression of the HES family BHLH transcription factor 5 (HES5) and low-density lipoprotein receptor-related protein 6 (LRP6) genes as well as the myelin basic protein (MBP) in the different groups. A) Percentage of the MBP positive cells in the different treated groups; B) Real-time polymerase chain reaction (RT-PCR) data: NC, Negative control; PC, Positive control; Vit D3, Group treated with 1,25(OH)2D3; RA, Group treated with retinoic acid (RA); C and D) RT-PCR analyses for the HES5 and LRP6 genes, respectively (**P<0.01 and P<0.001*** indicate significant differences between the groups). Data are given as mean±SEM, n=3.

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