In utero transplantation of stem cells in cynomolgus monkeys

为了实现基于人类胚胎干细胞的移植治疗，非人类灵长类动物的异体移植模型将是有用的。制备了具有绿色荧光蛋白（GFP）基因标记的食蟹ES细胞。这些细胞在子宫内被移植到异体胎儿中，利用了胎儿在免疫上还不成熟，无法诱导对移植细胞的免疫反应，以及胎儿组织区室正在迅速扩大，从而为移植提供了空间的事实。利用猴免疫缺陷病毒载体或电穿孔对食蟹蟹ES细胞进行基因修饰，使其表达GFP基因。这些细胞在超声引导下在子宫内移植到食蟹胎儿的腹腔（n=2）或肝脏（n=2），孕早期结束。三个胎儿在移植后1个月出生，另一个在移植后3个月出生。采用定量PCR和GFP序列原位PCR检测胎儿组织中移植细胞的子代。移植后3个月，在一名胎儿的胸腔内发现了一个荧光肿瘤，显然来源于移植的胚胎干细胞。然而，移植细胞后代在多个胎儿组织中也检测到（~ 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。移植的食蟹蟹胚胎干细胞可以移植到异体胎儿中。然而，如果这些细胞“渗漏”到不合适的地方，比如胸腔，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.