期刊
BMC NEUROSCIENCE
卷 10, 期 -, 页码 -出版社
BMC
DOI: 10.1186/1471-2202-10-86
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资金
- ReNeuron (RENE. L)
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 similar to 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.
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