In the present study, human NSCs over-expressing Akt1, F3.Akt1, showed strong resistant to the cell death stimuli than F3 cells in vitro and, F3.Akt1 cells transplanted into the brain of mouse ICH stroke model were found to increase survival of grafted cells, differentiate into the neuronal cells, and improve behavioral recovery in vivo.
The established F3.Akt1 cells showed normal growth pattern and morphology. They also expressed neural stem cell maker, nestin, and neuronal markers, NF-L, M and H but, F3.Akt1 cells did not showed a comparable level of GFAP, astrocytes marker, when compared to F3. In these results we confirm F3.Akt1 cells do not lose the properties of neural stem cells and still have the normal characteristics. And over expression of Akt1 gives cytoprotective effects to the hNSCs against cell death stimuli. In oxidative condition, F3.Akt1 cells showed increased survival rate by inducing phosphorylation of Akt1 and inhibition of caspase-3 cleavage. In this culture system, we could prove introducing Akt1 provides survival strength to the hNSCs.
Many of studies about neural transplantation, the aim of the cell transplantation is replacement or augmentation of losing cells. Selective neuronal loss such as in the case of Parkinson’s disease has already been considered a good candidate for neural replacement therapy. ICH is associated with considerable mechanical disruption of tissue in a large portion of the brain, and it was generally accepted that this disorder would be less likely to benefit from neural transplantation.
In the present study, an animal model of intracerebral hemorrhage (ICH) was used to
provide proof-of-principle that human NSCs over-expressing a survival factor Akt1 can be transplanted in the brain of animal models of neurological diseases, and produces beneficial effects of functional recovery and increased survival of grafted NSCs. Our results confirmed that the F3 hNSCs were transduced with Akt1 gene could survive well in the ICH animal brain following transplantation. From as early as 8 day post-transplantation to 8 weeks post-transplantation, F3.Akt1 cells provided functional recovery as determined by rotarod test and limb placement test and also induced an increased survival of transplanted NSCs in the host brain.
F3 hNSCs over-expressing Akt1 were able to survive much better than parental F3 NSCs, so that there were a 0.5-fold increase in cell survival of transplanted F3.Akt1 cells at 2 weeks post-transplantation and a 2-fold increase at 8 weeks, and a majority of grafted F3.Akt1 cells differentiated into neurons in the brain. These results indicate that F3.Akt1 NSCs at the ICH lesion sites are capable of preventing from the cell death and providing cytoprotective action in the ICH injury sites resulting in improved behavioral outcome.
But, for all the survival rate was increased in F3.Akt1 cells, there was no remarkable difference between F3 and F3.Akt1 groups in the behavioral test. In this study, we assessed in vivo test for 8 weeks so we suppose behavioral improvement may be different from the two hNSCs groups in more long-term period. We should do further study about this subject.
At 8 weeks post-transplanted, we found that the expression of the hNuMA+/GFAP cells of F3.Akt.1 was a small portion in ICH animals, which was not observed in transplantation of parental F3 cells. In the previous study using F3.VEGF cells in same animal models (Lee et al., 2007), they reported 55-65% of engrafted hNSCs were
differentiated into the astrocytes. A previous study reported Akt regulates the assembly and activity of basic helix-loop-helix (bHLH) transcription factor-coactivatior complexs to promote neuronal differentiation (Anne et al., 2003) and in fact, many transcription factors are known as the substrate of Akt1. We assumed that the activated phospho-Akt1 might translocate into the nucleus and it phosphorylates and activates transcriptional factors which then may regulate and determine the fate of engraft cells. However, despite we could not find significant differences in behavioral recovery between F3 and F3.Akt1 groups, we suggested that the extent of neuronal and glial differentiation may not affect the functional improvement.
As Akt is known to exert very strong control over cell survival and important influence on cell cycle control, it became a prime target in the search for cancer-related genes. In many studies have been reported that Akt gene is over expressed and constitutively active in many human cancers (Graff et al., 2000; .Brognard et al., 2001; Liu et al., 1999; Roy et al., 2002). We only focused on the survival effect of Akt1 in the present study so, we could not expect that what exactly happened in cellular level of the F3.Akt1 cell by the over expression of Akt1 and the result of the long-term survival in host brain.
We were very concerned about the tumorigenesis in the F3.Akt1 cells. Because F3, parental hNSCs were genetically engineered with oncogene v-myc and we introduced another gene, also known as oncogene, Akt1 into the F3 cells in this study. So, from the beginning we considered the potential of tumorigenesis of F3.Akt1 cells. But, 6 months post transplantation of F3.Akt1 cells ICH mice were not showed any kinds of signs of tumor. We killed the mice and stained the brain slides by the hematoxylin and eosin. Also a
previous study reported about the v-myc expression was undetectable after F3 cell transplantation 24 or 48h in F3 cells (Flax et al., 1998). Therefore it might be concluded that introducing oncogenes were not induced tumorigenesis in F3.Akt1 cells and even though those genes were used to immortalize and establish the cell line, it means just immortalization not beyond the word.
In the present study we used hydrogen peroxide and OGD system in vitro and ICH animal model in vivo, so we concerned about the role of Akt1 in the oxidative stress condition. The PI3K-Akt signal is well-known pathway for the cell survival and it exhibits anti-apoptotic effects against oxidative stress-induced damage in various cell types, including neural progenitor cells (Franke et al., 1997; Elyaman et al.,2002). This pathway mediates cell survival by phosphorylation and therefore inactivating several pro-apoptotic factors. For example, activated Akt phosphorylates and releases pro-apoptotic FHKR transcription factor proteins from DNA. The free FHKR then binds to 14-3-3 proteins, which forms a complex that is transported out of the nucleus, thereby functionally inactivating the transcription factor. Activated Akt also phosphorylates and inactivates pro-apoptotic GSK-3β protein. Furthermore, when Akt phosphorylation is inhibited, FHKR phosphorylation is blocked and cell death is markedly increased. That is, inactivation of these proteins then prevents oxidant-mediated apoptotic cell death. Recently, some studies were showed antioxidant enzymes such as, glutathione peroxidase-1 (Gpx1), Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and heme oxygenase-1 (HO-1) are became the new target substrate of activated Akt (Taylor et al., 2005; Rojo et al., 2004; Salinas et al., 2004).
Even we have not testified these antioxidant enzymes in vitro and in vivo system, we
suggest that activated Akt1 in hNSCs might influence on these proteins and then modulate the redox system and reduced toxic levels of reactive oxygen species (ROS) in the cell.
Further investigation is required to define the mechanism of F3.Akt1 cells’ survival effect and to widen understanding about the molecular signaling pathway.