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

ABSTRACT: 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-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM 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-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM 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-mycERTAM 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-mycERTAM transcript per cell. After implantation the c-mycERTAM 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-mycERTAM silencing occurred through methylation of the transgene promoter sequence. CONCLUSION: In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM 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-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.

SUBMITTER: Stevanato L

PROVIDER: S-EPMC2725042 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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

Stevanato Lara L Corteling Randolph L RL Stroemer Paul P Hope Andrew A Heward Julie J Miljan Erik A EA Sinden John D JD

BMC neuroscience 20090721

<h4>Background</h4>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-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growt ...[more]

PMID: 19622162

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Project description:Neural stem cell (NSC) therapy is a promising therapeutic strategy for stroke. Researchers have frequently carried out genetic modification or gene editing of stem cells to improve survival or therapeutic function. However, NSC transplantation carries the risk of immune rejection, and genetic modification or gene-editing might further increase this risk. For instance, recent studies have reported on manipulating the stem cell genome and transplantation via the insertion of an exogenous gene derived from magnetotactic bacteria. However, whether transgene-modified stem cells are capable of inducing immunological reactions has not been explored. Although NSCs rarely express the major histocompatibility complex (MHC), they can still cause some immunological issues. To investigate whether transgene-modified NSCs aggravate immunological responses, we detected the changes in peripheral immune organs and intracerebral astrocytes, glial cells, and MHC-I and MHC-II molecules after the injection of GFP-labeled or mms6-GFP-labeled NSCs in a rat model. Xenogeneic human embryonic kidney (HEK-293T) cells were grafted as a positive control group. Our results indicated that xenogeneic cell transplantation resulted in a strong peripheral splenic response, increased astrocytes, enhanced microglial responses, and upregulation of MHC-I and MHC-II expression on the third day of transplantation. But they decreased obviously except Iba-1 positive cells and MHC-II expression. When injection of both mms6-GFP-labeled NSCs and GFP-labeled NSCs also induced similar responses as HEK-293T cells on the third days, but MHC-I and MHC-II expression decreased 3 weeks after transplantation. In addition, mms6 transgene-modified NSCs did not produce peripheral splenic response responses as well as astrocytes, microglial cells, MHC-I and MHC-II positive cells responses when compared with non-modified NSCs. The present study provides preliminary evidence that transgenic modification does not aggravate immunological responses in NSC transplantation.

Dataset Information

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

Publications

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

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