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

Stevanato L, Corteling RL, Stroemer P, Hope A, Heward J, Miljan EA, Sinden JD - BMC Neurosci (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: ReNeuron Limited, Surrey Research Park, 10 Nugent Road, Guildford, Surrey, GU2 7AF, UK. lara-stevanato@reneuron.com

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.

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In vitro characterisation of c-mycERTAM transcript and protein expression. CTX0E03 cells were cultured in growth medium (control) and in non-growth promoting medium (in the absence of growth factors and 4-OHT) for 1- and 4-weeks. Evaluation of c-mycERTAM gene transcript and protein levels were performed by qRT-PCR (A), ICC (B to E) and western blot (F). CTX0E03 cell ICC images shown in panels C to E are representative images of the control c-Myc (green), ERα (red) and overlay (Merge). Scale bars represent 50 μm. The western blots in panel F were quantified using densitometry normalised by α-tubulin (G).
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Figure 4: In vitro characterisation of c-mycERTAM transcript and protein expression. CTX0E03 cells were cultured in growth medium (control) and in non-growth promoting medium (in the absence of growth factors and 4-OHT) for 1- and 4-weeks. Evaluation of c-mycERTAM gene transcript and protein levels were performed by qRT-PCR (A), ICC (B to E) and western blot (F). CTX0E03 cell ICC images shown in panels C to E are representative images of the control c-Myc (green), ERα (red) and overlay (Merge). Scale bars represent 50 μm. The western blots in panel F were quantified using densitometry normalised by α-tubulin (G).

Mentions: Additional experiments were carried out in vitro to determine if a reduction in transgene transcription leads to a concomitant reduction in translated protein. Analyses were carried out on control CTX0E03 cells and those cultured for 1- and 4-weeks in the absence of growth factors and 4-OHT. In this non growth permissive condition, transgene transcription was found to decrease by 59.6% at 1-week and 73.4% at 4-weeks relative to control by qRT-PCR (Figure 4A). Analysis of c-MycERTAM protein was performed by colocalization of c-Myc and ERα antigen staining by immunocytochemistry (ICC; Figure 4B, C, D, E). The c-Myc and ERα antigens were always found to colocalize in the nucleus of a proportion of CTX0E03 cells, indicating that the antibodies were detecting the c-MycERTAM target (Figure 4E). Immunoreactive cells were counted and expressed as a proportion of total cells. These data showed control CTX0E03 cell cultures were approximately 52.0% positive for c-MycERTAM; whereas, following EGF, bFGF and 4-OHT withdrawal the number of CTX0E03 cells positive for c-MycERTAM were 42.5% at 1-week and 12.8% at 4-weeks (Figure 4B). Western blot analysis using the c-Myc and ERα antibodies demonstrated that total c-MycERTAM protein levels followed more closely the progressive drop observed by gene expression and were reduced by 53.8% at 1-week and 73.0% at 4-weeks following EGF, bFGF, and 4-OHT withdrawal (Figure 4F, G).


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

Stevanato L, Corteling RL, Stroemer P, Hope A, Heward J, Miljan EA, Sinden JD - BMC Neurosci (2009)

In vitro characterisation of c-mycERTAM transcript and protein expression. CTX0E03 cells were cultured in growth medium (control) and in non-growth promoting medium (in the absence of growth factors and 4-OHT) for 1- and 4-weeks. Evaluation of c-mycERTAM gene transcript and protein levels were performed by qRT-PCR (A), ICC (B to E) and western blot (F). CTX0E03 cell ICC images shown in panels C to E are representative images of the control c-Myc (green), ERα (red) and overlay (Merge). Scale bars represent 50 μm. The western blots in panel F were quantified using densitometry normalised by α-tubulin (G).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2725042&req=5

Figure 4: In vitro characterisation of c-mycERTAM transcript and protein expression. CTX0E03 cells were cultured in growth medium (control) and in non-growth promoting medium (in the absence of growth factors and 4-OHT) for 1- and 4-weeks. Evaluation of c-mycERTAM gene transcript and protein levels were performed by qRT-PCR (A), ICC (B to E) and western blot (F). CTX0E03 cell ICC images shown in panels C to E are representative images of the control c-Myc (green), ERα (red) and overlay (Merge). Scale bars represent 50 μm. The western blots in panel F were quantified using densitometry normalised by α-tubulin (G).
Mentions: Additional experiments were carried out in vitro to determine if a reduction in transgene transcription leads to a concomitant reduction in translated protein. Analyses were carried out on control CTX0E03 cells and those cultured for 1- and 4-weeks in the absence of growth factors and 4-OHT. In this non growth permissive condition, transgene transcription was found to decrease by 59.6% at 1-week and 73.4% at 4-weeks relative to control by qRT-PCR (Figure 4A). Analysis of c-MycERTAM protein was performed by colocalization of c-Myc and ERα antigen staining by immunocytochemistry (ICC; Figure 4B, C, D, E). The c-Myc and ERα antigens were always found to colocalize in the nucleus of a proportion of CTX0E03 cells, indicating that the antibodies were detecting the c-MycERTAM target (Figure 4E). Immunoreactive cells were counted and expressed as a proportion of total cells. These data showed control CTX0E03 cell cultures were approximately 52.0% positive for c-MycERTAM; whereas, following EGF, bFGF and 4-OHT withdrawal the number of CTX0E03 cells positive for c-MycERTAM were 42.5% at 1-week and 12.8% at 4-weeks (Figure 4B). Western blot analysis using the c-Myc and ERα antibodies demonstrated that total c-MycERTAM protein levels followed more closely the progressive drop observed by gene expression and were reduced by 53.8% at 1-week and 73.0% at 4-weeks following EGF, bFGF, and 4-OHT withdrawal (Figure 4F, G).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: ReNeuron Limited, Surrey Research Park, 10 Nugent Road, Guildford, Surrey, GU2 7AF, UK. lara-stevanato@reneuron.com

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.

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Related in: MedlinePlus