Stevanato Lara, Corteling Randolph L, Stroemer Paul, Hope Andrew, Heward Julie, Miljan Erik A, Sinden John D
ReNeuron Limited, Surrey Research Park, 10 Nugent Road, Guildford, Surrey, GU2 7AF, UK.
BMC Neurosci. 2009 Jul 21;10:86. doi: 10.1186/1471-2202-10-86.
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.
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.
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.
人类神经干细胞系CTX0E03是为基于细胞的慢性中风残疾治疗而开发的。CTX0E03源自胎儿皮质脑组织，是一个克隆细胞系，包含通过逆转录病毒感染递送的c-mycERTAM转基因的单拷贝。在4-羟基他莫昔芬（4-OHT）的条件调节下，c-mycERTAM使CTX0E03细胞能够大规模稳定保存。在本研究中，我们研究了该转基因在体外生长停滞（去除表皮生长因子、碱性成纤维细胞生长因子和4-OHT）后以及脑内植入大脑中动脉闭塞（MCAo）大鼠脑内后的命运。在体外，从培养基中去除生长因子和4-OHT 4周后，c-mycERTAM转基因转录减少约75%。此外，免疫细胞化学和蛋白质印迹显示c-MycERTAM蛋白同时减少。为了检测转基因在体内的转录，在MCAo损伤后4周植入CTX0E03细胞（450,000个），并分别通过定量聚合酶链反应（qPCR）和定量逆转录聚合酶链反应（qRT-PCR）分析人类细胞存活情况和c-mycERTAM转录情况。
结果显示，在所有移植动物脑中均存在CTX0E03细胞，占注射细胞总数的6.3%至39.8%。植入前，CTX0E03细胞悬液中每个细胞含有215.7（标准误=13.2）个c-mycERTAM转录本拷贝。植入后，每个CTX0E03细胞的c-mycERTAM转录本拷贝数在1周时降至6.9（标准误=3.4），在4周时降至7.7（标准误=2.5）。体内样本的亚硫酸氢盐基因组DNA测序证实，c-mycERTAM沉默是通过转基因启动子序列的甲基化发生的。
总之，结果证实CTX0E03细胞在体外去除表皮生长因子、碱性成纤维细胞生长因子和4-OHT后以及在体内植入MCAo大鼠脑内后均下调了c-mycERTAM转基因表达。c-mycERTAM转基因在体内的沉默为CTX0E03细胞的潜在临床应用提供了额外的安全特性。
