Effects of Oestrogen on Neural Stem Cell Success in a Stroke Model

Stroke is a problem for the ageing population. The ageing population is increasing but there is no licensed therapy for chronic stages of the disease. Cell replacement has enormous potential to restore neurological function after stroke. Since evidence suggests that oestrogen may improve integration of neural stem cells in vivo. The study tested the hypothesis that transplantation of murine Maudsley hippocampal stem cell line clone 36 (MHP36) over-expressing oestrogen (Dax-1KD-MHP36), into mouse brain after a transient middle cerebral artery occlusion (MCAO), would promote functional recovery and improve integration and differentiation in vivo. Before the hypothesis could be addressed, the MHP36 cells were fully characterised for oestrogen, the in vivo murine model of stroke was established and since this is the first time the cells have been used in murine stroke model, the efficacy of these cells to restore functional recovery was established.

The expression of the enzyme that synthesises oestrogen (aromatase), oestrogen receptors (ERα, ERβ) and GPR30 protein in the MHP36 stem cells was investigated by immunofluorescence, Western blotting and reverse transcriptase PCR (RT-PCR). A lentiviral vector was used to silence dosage-sensitive sex reversal adrenal hypoplasia congenita region on X-chromosome gene 1 (Dax-1), to over-express oestrogen in MHP36 cells (Dax-1KD-MHP36). Adult C57BL/6 (male, 12-14 week old, weighing between 25 and 30 g) mice underwent transient MCAO and two days post-MCAO vehicle, oestradiol (E2), MHP36, Dax-1KD-MHP36 and MHP36 cells suspended in oestradiol (MHP36+E2) were transplanted into the brain ipsilateral to the injury. Sensorimotor function was assessed using cylinder and ladder rung tests at pre-MCAO and 2, 7, 14 and 28 days post-MCAO. Mice were perfusion fixed at 28 days post-MCAO for measurement of lesion (tissue loss) and histology.

Undifferentiated MHP36 cells were shown to express aromatase, ERβ and GPR30. ERα was absent in the MHP36 cells when compared to positive control (breast cancer) MCF-7 cells. Moreover, the genetically modified Dax-1KD-MHP36 cells were successfully characterised for over-expressing oestrogen with significantly reduced Dax-1, increased aromatase and oestrogen production when compared to MHP36 cells in vitro. Transplantation of Dax-1KD-MHP36 completely restored the sensorimotor function to pre-MCAO scores in both the ladder rung and cylinder test 28 days after transplantation post-MCAO. Function was not completely restored in any of the other treatment groups to pre-MCAO baseline scores and MHP36 and MHP36+E2 actually induced a significant bias towards the impaired limb in the cylinder test. In the ladder rung test, significant acceleration in recovery was observed by 14 days in the Dax-1KD-MHP36 group but not in any of the other groups. A significant increase in MAP-2 positive neurons and synaptophysin was observed in Dax-1KD-MHP36 when compared to vehicle. Lesion volume was significantly reduced only in Dax-1KD-MHP36 group when compared to vehicle.

In conclusion, the MHP36 cells express ERβ, GPR30 and aromatase, but do not express ERα. Aromatase and oestrogen production were significantly increased after genetic modification of the MHP36 cells. In vivo Dax-1KD-MHP36 cells completely restored functional recovery, which was not observed in any other group. This may be mediated via reduced tissue loss, improved synaptic plasticity and/or improved neuronal differentiation. Our approach therefore has great potential to improve the clinical application of cell replacement therapy, as well as gene therapy in patients suffering from stroke.