Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, College of Animal Science and Technology, Guangxi University, Nanning, China.
Xenotransplantation. 2017 May;24(3). doi: 10.1111/xen.12300. Epub 2017 Apr 11.
Blastocyst complementation is an important technique for generating chimeric organs in organ-deficient pigs, which holds great promise for solving the problem of a shortage of organs for human transplantation procedures. Porcine chimeras have been generated using embryonic germ cells, embryonic stem cells, and induced pluripotent stem cells; however, there are no authentic pluripotent stem cells for pigs. In previous studies, blastomeres from 4- to 8-cell-stage parthenogenetic embryos were able to generate chimeric fetuses efficiently, but the resulting fetuses did not produce live-born young. Here, we used early-stage embryos from somatic cell nuclear transfer (SCNT) to generate chimeric piglets by the aggregation method. Then, the distribution of chimerism in various tissues and organs was observed through the expression of enhanced green fluorescent protein (EGFP).
Initially, we determined whether 4- to 8- or 8- to 16-cell-stage embryos were more suitable to generate chimeric piglets. Chimeras were produced by aggregating two EGFP-tagged Wuzhishan minipig (WZSP) SCNT embryos and two Bama minipig (BMP) SCNT embryos. The chimeric piglets were identified by coat color and microsatellite and swine leukocyte antigen analyses. Moreover, the distribution of chimerism in various tissues and organs of the piglets was evaluated by EGFP expression.
We found that more aggregated embryos were produced using 4- to 8-cell-stage embryos (157/657, 23.9%) than 8- to 16-cell-stage embryos (100/499, 20.0%). Thus, 4- to 8-cell-stage embryos were used for the generation of chimeras. The rate of blastocysts development after aggregating WZSP with BMP embryos was 50.6%. Transfer of 391 blastocysts developed from 4- to 8-cell-stage embryos to five recipients gave rise to 18 piglets, of which two (11.1%) were confirmed to be chimeric by their coat color and microsatellite examination of the skin. One of the chimeric piglets died at 35 days and was subsequently autopsied, whereas the other piglet was maintained for the following observations. The heart and kidneys of the dead piglet showed chimerism, whereas the spinal cord, stomach, pancreas, intestines, muscle, ovary, and brain had no chimerism.
To our knowledge, this is the first report of porcine chimeras generated by aggregating 4- to 8-cell-stage blastomeres from SCNT. We detected chimerism only in the skin, heart, and kidneys. Collectively, these results indicate that aggregation using 4- to 8-cell-stage SCNT embryos offers a practical approach for producing chimeric minipigs. Furthermore, it also provides a potential platform for generating interspecific chimeras between pigs and non-human primates for xenotransplantation.
囊胚互补是在器官缺失的猪中生成嵌合体器官的重要技术，这为解决人类移植手术中器官短缺的问题提供了很大的希望。已经使用胚胎生殖细胞、胚胎干细胞和诱导多能干细胞生成了猪嵌合体；然而，猪没有真正的多能干细胞。在以前的研究中，4-8 细胞期的孤雌胚胎的卵裂球能够有效地生成嵌合体胎儿，但所得到的胎儿不能产生活产幼仔。在这里，我们使用体细胞核移植（SCNT）的早期胚胎，通过聚集法生成嵌合仔猪。然后，通过增强型绿色荧光蛋白（EGFP）的表达观察嵌合体在各种组织和器官中的分布。
首先，我们确定 4-8 细胞期或 8-16 细胞期的胚胎更适合生成嵌合仔猪。通过聚集两个标记有 EGFP 的五指山小型猪（WZSP）SCNT 胚胎和两个巴马小型猪（BMP）SCNT 胚胎来生成嵌合体。通过毛色和微卫星及猪白细胞抗原分析鉴定嵌合体。此外，通过 EGFP 表达评估仔猪各组织和器官中嵌合体的分布。
我们发现，与 8-16 细胞期胚胎相比（100/499，20.0%），4-8 细胞期胚胎产生的聚集胚胎更多（157/657，23.9%）。因此，4-8 细胞期胚胎用于生成嵌合体。WZSP 与 BMP 胚胎聚集后囊胚发育率为 50.6%。将 4-8 细胞期胚胎发育的 391 个囊胚移植到 5 个受体中，产生了 18 头仔猪，其中 2 头（11.1%）通过皮肤的毛色和微卫星检查被确认为嵌合体。其中一只嵌合体仔猪在 35 天时死亡，并随后进行尸检，而另一只仔猪则被保留进行后续观察。死亡仔猪的心脏和肾脏有嵌合体，而脊髓、胃、胰腺、肠、肌肉、卵巢和大脑没有嵌合体。
据我们所知，这是首次报道通过聚集 SCNT 的 4-8 细胞期卵裂球生成猪嵌合体。我们仅在皮肤、心脏和肾脏中检测到嵌合体。总的来说，这些结果表明，使用 4-8 细胞期 SCNT 胚胎聚集提供了一种实用的方法来生成嵌合小型猪。此外，它还为在猪和非人类灵长类动物之间生成异种移植用的种间嵌合体提供了一个潜在的平台。
