Pregnenolone enhances the proliferation of mouse neural stem cells and promotes oligodendrogenesis, together with Sox10, and neurogenesis, along with Notch1 and Pax6

Background: Pregnenolone is a precursor of various steroid hormones involved in osteoblast proliferation, microtubules polymerization and cell survival protection. Previous reports focused on the effects of pregnenolone metabolites on stem cell proliferation and differentiation; however, the effects of pregnenolone itself has not been well explored. The present study aimed to investigate the role of pregnenolone on NSC proliferation and to determine the doses required for NSC differentiation as well as the various genes involved in its mechanism of action.

Methods: NSCs were isolated from the embryonic cortex of E14 mice, incubated for 5 days, and then treated with pregnenolone doses of 2, 5, 10, 15 and 20 μM for another 5 days. The number of neurospheres and neurosphere derived cells were then counted. Flow cytometry was used to evaluate the differentiation of NSCs into oligodendrocytes, astrocytes, and neurons. The expression level of Notch1, Pax6 and Sox10 genes were also measured by Real Time PCR after 5 days of treatment.

Results: Our data suggest that treatment with 10 μM pregnenolone is optimal for NSC proliferation. In fact, this concentration caused the highest increase in the number of neurospheres and neurosphere derived cells, compared to the control group. In addition, treatment with low doses of pregnenolone (5 and 10 μM) caused a significant increase in NSC differentiation towards immature (Olig2⁺) and mature (MBP⁺) oligodendrocyte cell populations, compared to controls. However, NSC differentiation into neurons (beta III tubulin ⁺ cells) increased in all treatment groups, with the highest and most significant increase obtained at 15 μM concentration. It is worth noting that pregnenolone at the highest concentration of 15 μM decreased the number of astrocytes (GFAP+). Furthermore, there was an increase of Sox10 expression with low pregnenolone doses, leading to oligodendrogenesis, whereas Notch1 and Pax6 gene expression increased in pregnenolone groups with more neurogenesis.

Conclusion: Pregnenolone regulates NSCs proliferation in vitro. Treatment with low doses of pregnenolone caused an increase in the differentiation of NSCs into mature oligodendrocytes while higher doses increased the differentiation of NSCs into neurons. Oligodendrogenesis was accompanied by Sox10 while neurogenesis occurred together with Notch1 and Pax6 expression.