Piglets Cloned From Induced Pluripotent Stem Cells

Mouse embryonic stem (ES) cells have proven powerful in the generation of precisely genetically modified animals that can advance our knowledge of mammalian physiology and disease. In recent years, genetically modified pigs have also been produced by means of somatic cell nuclear transfer (NT). Pigs provide outstanding models for modeling human genetic diseases due to the striking similarities with human anatomy, physiology, and genetics. Yet, progress with porcine genetic engineering has been hampered by the lack of germline-competent pig ES cells. Somatic cells exhibit limited proliferation and have an extremely low frequency of homologous recombination (less than10) compared to ES cells. Hence, only a few knockout pig models have been reported thus far.Remarkably, induced pluripotent stem cells (iPSCs) have been generated by the reprogramming of somatic cells from multiple mammalian species using defined cocktails of transcription factors. Mouse iPSCs have passed the crucial test of germ line contribution and generation of all-iPSCs mice through tetraploid embryo complementation. This indicates that bona fide iPSCs are, if not identical, very similar to ES cells. Likewise, cloned mice have been obtained from mouse iPSCs by the NT method. These studies indicate that iPSCs can substitute ES cells for the purpose of genomic manipulation.Pig iPSCs (piPSCs) have been reported by several groups. These piPSC lines could be differentiated into the three germ layers in vitro and in vivo. Only one group reported the piPSCs they generated could pass the crucial test of germ line chimera production, but the germ line chimera was detected only by PCR analysis that was not convinced. More research should be done to obtain germline-competence piPSCs. Nevertheless, piPSCs are capable of long-term proliferation, which potentially allows lengthier and therefore potentially more sophisticated in vitro manipulation. More importantly, they are similar to ES cells in many aspects, suggesting that they may have as well high efficiency of homologous recombination. Hence, if iPSCs were suitable for cloning pigs by NT, gene-targeted pigs could potentially be produced much more efficiently than using fibroblasts as nuclear donors. In the present study, we explored the feasibility of generating cloned pigs from piPSCs.We used piPSC lines generated by different groups using various strategies. The first2piPSC lines, iPF4-2and iPM6-11, were induced from porcine ear fibroblasts (PEFs) and porcine bone marrow cells (pBMCs), respectively, of a10-week-old Danish Landrace pig using lentiviral vector over-expressing human transcription factors induced with doxycycline (DOX). The third piPSC line, hsC13, was induced from porcine fetal fibroblasts (PFFs) by retroviral over-expression of human transcription factors. The last3piPSC lines (JN2, KSR-4, and5%O2-1) were produced by retroviral over-expression of mouse or porcine transcription factors into pBMCs or PFFs.In the first round of NT experiments, we used undifferentiated piPSCs as donor cells and transferred the nuclei into enucleated metaphase Ⅱ (MⅡ) oocytes by the electrical fusion method. A total of11923cloned embryos reconstructed with the6piPSC lines were introduced into71surrogate mothers. Among these mothers,25were pregnant as detected by ultrasonography on day24-26following embryo transfer, but none of them developed to term. These results were disappointing considering that mouse iPSCs have been successfully used as donor cells for creating living cloned mice. We then hypothesized that inappropriate silencing of the exogenous transcription genes in the piPSCs may underlie the discrepancy. To test this hypothesis, we performed a second round of experiments, this time using piPSCs that were allowed to spontaneously differentiate for4-6days as donor cells. For this we focused on iPF4-2, iPM6-11, KSR-4and5%O2-1. After spontaneous differentiation, piPSCs became enlarged and flattened, and developed an epithelium-like morphology. For iPF4-2and iPM6-11, all the exogenous were reduced significantly after differentiation. On the other hand, the expression level of all4exogenous genes in KSR-4differentiated cells did not decrease significantly as in iPF4-2and iPM6-11. Some of the exogenous genes in5%O2-1differentiated cells even expressed at a higher level compared to the undifferentiated one. Except for Sox2, the expression level of extrogenous genes was higher in KSR-4and5%O2-1differentiated cells than in iPF4-2and iPM6-11differentiated ones.The differentiated piPSCs were transferred into enucleated MⅡ oocytes, and it was encouraging to observe that the resulting NT embryos had a significantly increased rate of blastocyst development compared to undifferentiated cells. Reconstructed embryos derived from differentiated iPF4-2and KSR-4cells were transferred into surrogate mothers. A total of545cloned embryos from KSR-4differentiated cells were transferred into3surrogates,2of whom became pregnant but lost their pregnancy between days35to50. A total of1135iPF4-2differentiated cell-NT embryos were transferred into7recipient surrogate mothers,3of whom became pregnant. To detect the early development of the cloned fetuses, one pregnant surrogate was sacrificed after36days of gestation, and2fetuses were retrieved by caesarean section. The2cloned fetuses were morphologically normal and the corresponding fetal fibroblasts grew normally and were positive for EGFP, a maker in iPF4-2. After113days of gestation,1of the pregnant surrogates using iPF4-2cells naturally delivered a piglet and a mummy. But the piglet died within the first hour after birth. The other pregnant surrogate using iPF4-2cells gave cesarean birth to a live and healthy cloned pig after114days of gestation. This piglet survived for32days. Both piglets had a white coat resembling the coat color of the pig (Danish Landrace) from which piPSCs, iPF4-2were derived. The fibroblasts isolated from the2piglets were both positive for EGFP. To further confirm that the fetuses and piglets were generated from iPF4-2piPSCs, we analyzed Oct4, Sox2, and EGFP transgene integration, as well as microsatellite DNA. Tissues from the2fetuses and born piglets contained the exogenous transgenes, as demonstrated by PCR. Analysis of microsatellite DNA revealed as well that the genomes of the fetuses and piglets were the same as iPF4-2cells, but different from the surrogate. These findings proved that the piglets were cloned from the piPSCs.In order to investigate the cause of death and the organ development status, pathological section was performed. All the investigated organs of the piglets died at birth showed normal morphology, and the cause of death was unclear. Some mniotic fluid got into pulmonary alveoli that maybe cause the death. Pathological section analysis indicated the cause of piglets died at32day was Myeloencephalitis. Organs of the two piPSC cloned piglets showed normal morphology. The death was not caused by inherent organ developmental defects of piPSC cloned piglets that meant normal development piglets could be cloned from piPSCs.In summary, here we have described the generation of live piPSC-NT piglets from a piPSC line established with a DOX-induced system. This system allows controllable silencing of the exogenous genes and may explain why the NT failed using dedifferentiated piPSCs. The generation of live piglets from piPSCs by NT potentially represents an important step toward a more efficient production of genetically engineered pigs using piPSCs. The latter may facilitate the more generalized use of pigs for human disease modeling and potentially also for agriculture.