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Background  The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycER TAM  transgene delivered by retroviral infection. Under the conditional 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 implantation, 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  transcript 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 sequencing 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-mycER TAM  transgene expression both  in vitro  following EGF, bFGF and 4-OHT withdrawal and  in vivo  following implantation in MCAo rat brain. The silencing of the c-mycER TAM  transgene  in vivo  provides an additional safety feature of CTX0E03 cells for potential clinical application.

c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain