In utero transplantation of stem cells in cynomolgus monkeys

为了实现基于人胚胎干（ES）细胞的移植治疗，非人灵长类动物的异基因移植模型将是有用的。我们已经制备了用绿色荧光蛋白（GFP）遗传标记的食蟹猴ES细胞。利用胎儿在免疫学上太不成熟而不能诱导对移植细胞的免疫应答以及胎儿组织区室迅速扩增从而为植入提供空间的事实，将细胞移植到子宫内的同种异体胎儿中。使用猿猴免疫缺陷病毒载体或电穿孔对食蟹猴ES细胞进行遗传修饰以表达GFP基因。在妊娠早期结束时，在超声引导下将这些细胞在子宫内移植到腹腔（n=2）或肝脏（n=2）中的食蟹猴胎儿中。移植后1个月分娩3例，移植后3个月分娩1例。通过GFP序列的定量PCR和原位PCR检查胎儿组织中移植的细胞后代。移植后3个月，在一个胎儿的胸腔中发现了一个明显来自移植ES细胞的荧光肿瘤。然而，在多个胎儿组织中也检测到移植的细胞后代（约1%）没有畸胎瘤。这些细胞是孤立的，与周围的宿主细胞难以区分。移植的食蟹猴ES细胞可以移植到同种异体胎儿中。然而，如果这些细胞“泄漏”到胸腔等不适当的空间，就会形成肿瘤。

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.