文章基本信息

标题：c-MycER TAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain
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作者：Lara Stevanato ; Randolph L Corteling ; Paul Stroemer 等
期刊名称：BMC Neuroscience
印刷版ISSN：1471-2202
电子版ISSN：1471-2202
出版年度：2009
卷号：10
期号：1
页码：1
DOI：10.1186/1471-2202-10-86
语种：English
出版社：BioMed Central
摘要：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.