In utero transplantation of stem cells in cynomolgus monkeys

食蟹猴子宫内干细胞移植

基本信息

批准号：
12557083
负责人：
HANAZONO Yutaka
金额：
$ 4.8万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

To achieve human embryonic stem (ES) cell-based transplantation therapies, allogeneic transplantation models of non-human primates would be useful. We have prepared cynomolgus ES cells genetically marked with the green fluorescent protein (GFP). The cells were transplanted into the allogeneic fetus in utero, taking advantage of the fact that the fetus is immunologically too immature to induce immune responses to transplanted cells and that fetal tissue compartments are rapidly expanding and thus providing space for the engraftment. Cynomolgus ES cells were genetically modified to express the GFP gene using a simian immunodeficiency viral vector or electoroporation. These cells were transplanted in utero with ultrasound guidance into the cynomolgus fetus in the abdominal cavity (n=2) or liver (n=2) at the end of the first trimester. Three fetuses were delivered 1 month posttransplantation, and the other, 3 months posttransplantation. Fetal tissues were examined for transplanted cell progeny by quantitative PCR and in situ PCR of the GFP sequence. A fluorescent tumor, apparently derived from transplanted ES cells, was found in the thoracic cavity at 3 months posttransplantation in one fetus. However, transplanted cell progeny were also detected (〜1%) without teratomas in multiple fetal tissues. The cells were solitary and indistinguishable from surrounding host cells. Transplanted cynomolgus ES cells can be engrafted in allogeneic fetuses. The cells will, however, form a tumor if they "leak" into an improper space such as the thoracic cavity.

为了实现人类胚胎（ES）基于细胞的移植疗法，非人类素数的同种异体移植模型将是有用的。我们制备了用绿色荧光蛋白（GFP）遗传标记的cynomolgus ES细胞。将细胞移植到子宫中的同种异体胎儿中，利用胎儿在免疫学上过于不成熟，无法诱导免疫复杂的细胞来移植细胞，并且胎儿组织室正在迅速扩张，从而为植入提供了空间。 cynomolgus ES细胞进行遗传修饰，以使用猿猴免疫缺陷病毒载体或选举表达GFP基因。将这些细胞用超声引导在子宫内移植到腹腔（n = 2）或肝脏（肝脏（n = 2）结束时）中的cynomolgus胎儿。移植后1个月交付了三个胎儿，另一个胎儿在移植后3个月分娩。通过定量PCR和GFP序列的原位PCR检查了胎儿组织的移植细胞后代。在一个胎儿移植后3个月，在胸腔腔中发现了一种显然是从移植的ES细胞衍生的荧光肿瘤。然而，在多个胎儿组织中也没有检测到移植的细胞后代（〜1％）。细胞是固体的，与周围宿主细胞无法区分。可以在同种异体胎儿中刻有移植的cynomolgus ES细胞。但是，如果细胞“泄漏”到胸腔腔等不当空间中，它们将形成肿瘤。