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tests, increased survival of grafted NSCs, and differentiation of grafted NSCs into
neurons and astrocytes. These results are consistent with previous studies that
Akt1, protein which is known as a general mediator of cell survival signals, pro-
motes the survival of CNS neurons in vitro [23], [27]-[29], and provides favor-
able clinical outcome in animal models of ischemia [36].
In the present study we exposed F3 and F3.Akt1 hNSCs to hydrogen per-
oxide injury in vitro to study the neuroprotectiion provided by Akt1 under the
conditions of oxidative stress, and the results indicate that F3.Akt1 cells showed
a higher survival rate following H2O2 -induced injury as compared to the F3
controls (Figs. 2A-C). Following H2O2 treatment, phosphorylation of Akt1
and concomitant inactivation of caspase-3 were found in F3.AKt1 cells, while
in control F3 cells non-phosphorylation of Akt1 and an increase in active frag-
ment of caspase 3 were found (Fig. 2D). It is known that Akt1 phosphorylates
caspase-9 at Ser-196, thereby blocking cytochrome c-mediated caspas-9 activa-
tion and inactivation of caspase-3 leading to inhibition of proapoptotic signals
[37]. Recent studies have also shown that antioxidant enzymes such as glutathi-
one peroxidase-1 (Gpx1), Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and heme
oxygenase-1 (HO-1) are target substrates of activated Akt and results in modula-
tion of redox system and reduced toxic levels of reactive oxygen species (ROS) in
various cell types [38]-[40].
From as early as 8-day post-transplantation (PT) to 8-weeks PT, F3.Akt1 hN-
SCs induced an increased survival of transplanted NSCs in the host brain. Sur-
vival of transplanted F3.Akt1 hNSCs in ICH mouse brain was identified by hu-
man nuclear matrix antigen (hNuMA)- or LacZ/
β
-gal-positive reaction. The cell
count in ICH brain indicates that 40% increase in cell survival in F3.Akt1 group
over control F3 group and 100% increase at 8-weeks PT. It should be noted that
the majority of grafted F3.Akt1 cells differentiated into either neurons (
β
-tubulin
III-, NF-L- or NF-H-positive) or astrocytes (GFAP-positive) along the border of
lesion sites (Fig. 4). In addition, a majority of grafted F3.Akt1 hNSCs differenti-
ated into either neurons or astrocytes in response to signals provided in the local
microenvironment.
Previous studies have reported that the activation of PI3K/Akt signaling axis
promotes growth and survival of tumor cells, and genetic perturbation of this
pathway increases the survival of cancer cells [41]-[43]. For that reason, we were
very concerned about possible tumorigenesis in the animals transplanted with
F3.Akt1 hNSCs. Transplanted F3 or F3.Akt1 hNSCs in ICH lesion sites were im-
munoreaction-negative for cell proliferation marker Ki-67 indicating that hNSCs
do not proliferate actively in vivo environment. In addition, none of the animals
grafted with F3.Akt1 or F3 hNSCs showed tumor formation upon histological
examination even in animals with 6-months PT.
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