c-MycERTAM Transgene Silencing in a Genetically Modified Human Neural Stem Cell Line Implanted into MCAo Rodent Brain - Genetic Engineering: Recent Developments in Applications - page 78

regulation by 4-hydroxytamoxifen (4-OHT), c-mycER TAM enabled large-scale
stable banking of the CTX0E03 cells. In this study, we investigated the fate of
this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal)
in vitro and following intracerebral implantation into a mid-cerebral artery
occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors
and 4-OHT from the culture medium, c-mycER TAM transgene transcription is
reduced by ~75%. Furthermore, immunocytochemistry and western blotting
demonstrated a concurrent decrease in the c-MycER TAM protein. To examine
the transcription of the transgene in vivo, CTX0E03 cells (450,000) were
implanted 4-weeks post MCAo lesion and analysed for human cell survival
and c-mycER TAM transcription by qPCR and qRT-PCR, respectively.
Results
The results show that CTX0E03 cells were present in all grafted animal brains
ranging from 6.3% to 39.8% of the total cells injected. Prior to implanta-
tion, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of
the c-mycER TAM transcript per cell. After implantation the c-mycER TAM tran-
script copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at
1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequenc-
ing of the in vivo samples confirmed c-mycER TAM silencing occurred through
methylation of the transgene promoter sequence.
Conclusion
In conclusion the results confirm that CTX0E03 cells downregulated c-myc-
ER TAM transgene expression both in vitro following EGF, bFGF and 4-OHT
withdrawal and in vivo following implantation in MCAo rat brain. The si-
lencing of the c-mycER TAM transgene in vivo provides an additional safety fea-
ture of CTX0E03 cells for potential clinical application.
background
Stem cell therapy is a facet of regenerative medicine that aims to ameliorate the
damage caused to the brain by the grafting of healthy “reparative” cells. Pioneer-
ing studies implanting mouse neural stem cells (NSCs) into the brains of stroke
animals have demonstrated significant recovery in motor and cognitive tests [1-5].
These findings provide a rational approach to the development of a cell based ther-
apy for ischemic stroke. A substantial and consistent supply of allogeneic NSCs is
required in order to treat a large patient population. Unfortunately, human NSCs
are somatic stem cells and susceptible to genetic and phenotypic changes and loss
of biological activity following extensive tissue culture expansion [6-8].