Production of healthy cloned pigs with neural stem cells as nuclear donors

The objectives of the present study were to establish a porcine neural stem cell (NSC) line and to determine if these NSCs could be used to produce cloned pigs. NSCs were isolated from the brains of three embryonic day 30 fetal pigs and were induced to differentiate in vitro . NSCs and the differentiated cells were harvested for analysis of markers by immunostaining and reverse-transcription polymerase chain reaction (RT-PCR). The NSCs at passage 10 were used for nuclear transfer, and the cloned embryos at the two-cell stage were transferred into the oviducts of surrogate mothers. The results showed that three NSC lines (2 male and 1 female) were successfully established. All NSCs at passage 17 continued to express nestin and Sox2. NSCs could differentiate into neurons (TUBB3+), astrocytes (GFAP+), and oligodendrocytes (O4+). After NSC nuclear transfer, 2020 two-cell stage embryos formed. After embryo transfer, 6 of 10 surrogates were pregnant, and 40 piglets (18 males and 22 females) were born. Twenty-two of these piglets reached sexual maturity and were found to be fertile. The other piglets died within 45 days post-partum. In conclusion, 3 porcine NSC lines capable of self-renewal and differentiation were established, and the cloned embryos derived from these cells could develop to term. Thus, NSCs could be efficient alternative nuclear donors for pig cloning.