Production of Transgenic Porcine Embryos Reconstructed with Induced Pluripotent Stem-Like Cells Derived from Porcine Endogenous Factors Using piggyBac System.

The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days. The iPS-like cells demonstrated pluripotent characteristics such as self-renewal, high proliferation, expression of pluripotent markers, and aggregation ability. The embryo development through somatic cell nuclear transfer (SCNT), cleavage rate, and blastocyst formation rate did not show any significant differences. However, the total cell number of blastocysts was significantly increased with the established cell line. In conclusion, the iPS-like cell line, generated from porcine transcriptional factors using the PB transposon system, demonstrated pluripotency with the capacity for unlimited self-renewal, and could be used as donor cells to produce cloned embryos by SCNT. These cells will be suitable for gene modification and would contribute to the stability or safety of pig models in biomedical research.

Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology.

Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.

Replacement of glutamine with the dipeptide derivative alanyl-glutamine enhances in vitro maturation of porcine oocytes and development of embryos.

The presence of glutamine (Gln) in in vitro maturation (IVM) and in vitro culture (IVC) medium is a more potent factor for improving porcine oocyte and embryo development than other amino acids. However Gln is inherently unstable and spontaneously breaks down into ammonia, and therefore interferes with proper development. To avoid this adverse effect, Gln was replaced in the present study with its stable dipeptide derivative alanyl-glutamine (Ala-Gln) and the effects of this replacement on porcine IVM and IVC were evaluated. Replacement of Gln with Ala-Gln during IVM did not improve nuclear maturation, however numbers of early cleaved embryos were significantly increased after activation. Blastocyst formation rates were also significantly improved by using Ala-Gln during IVM. Replacement of Gln with Ala-Gln during IVC significantly increased total cell numbers in blastocysts. Blastocyst formation rate was also significantly higher when Ala-Gln was used in both IVM and IVC. In conclusion, the use of Ala-Gln rather than Gln gives better results for development in both porcine IVM and IVC.

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels.

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 µg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 µg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 µg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 µg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.

Oxamflatin improves developmental competence of porcine somatic cell nuclear transfer embryos.

Abstract Aberrant epigenetic nuclear reprogramming of somatic nuclei is a major cause of low success in cloning. It has been demonstrated that treatment of histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos by alteration of epigenetic status. The aim of the present study was to investigate the effect of oxamflatin, a novel HDACi, on the developmental competence of porcine SCNT embryos. Treatment with 1 µM oxamflatin for 9 h after activation of SCNT embryos increased both in vitro and in vivo developmental competence. Treatment of SCNT embryos with 1 µM oxamflatin significantly increased blastocyst rate and total cell number in blastocysts (33.3±6.0 and 73.1±1.6, respectively) than that of controls (10.3±3.7 and 54.1±3.5, respectively) or scriptaid (16.4±4.6 and 64.4±2.1, respectively). Moreover, oxamflatin showed significant higher overall cloning efficiency from 0.9% to 3.2%, whereas scriptaid demonstrated 0% to 1.8%. In conclusion, these results indicate that oxamflatin treatment improves the developmental competence of porcine SCNT embryos.

Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture.

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus-oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10(-6)M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4×10(-6)M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine-paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.

Developmental competence of porcine oocytes after in vitro maturation and in vitro culture under different oxygen concentrations.

In this study, we investigated the effect of two oxygen concentrations (5 and 20%) during in vitro maturation (IVM) and during in vitro culture (IVC) on porcine embryo development and analysed differences in gene expression between cumulus-oocyte complexes matured under 5 or 20% oxygen and the resulting blastocysts cultured under 5% or 20% oxygen following parthenogenetic activation. There was no significant difference in oocyte maturation rate. However, the numbers of resulting blastocysts were significantly increased in the 5% IVC group compared with the 20% IVC group. Moreover, the M20C5 treatment group (23.01%) supported greater blastocyst development compared with the M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. However, total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each treatment altered the expression of genes in different patterns. GLUT1, G6PD and LDHA were up-regulated in cumulus cells that had been matured in low oxygen, suggesting a higher glucose uptake and an increase in anaerobic glycolysis, whereas cyclin B1 (CCNB) and MnSOD (Mn-superoxide dismutase) were upregulated in cumulus cells that had been matured in high oxygen, which suggests a higher activity of mitosis-promoting factor and antioxidant response. In spite of these differential effects on cumulus cells, oocytes could mature normally regardless of different oxygen concentrations. Therefore, it can be concluded that high oxygen concentration during in vitro maturation and low oxygen during in vitro culture may alter the expression of multiple genes related to oocyte competence and significantly improves embryo development (p < 0.05) but not blastocyst quality.

Effect of oocyte-secreted factors on porcine in vitro maturation, cumulus expansion and developmental competence of parthenotes.

The oocyte is known from recent studies in the mouse, cow, sheep and human to be a central regulator of follicular cell function. However, in the pig, little information is known about the regulation of cumulus expansion by oocyte-secreted factors and oocyte quality. We investigated the possible effects of oocyte-secreted factors during in vitro maturation on cumulus expansion and on porcine oocytes as judged by subsequent embryonic development after parthenogenetic activation. Cumulus-oocyte complexes (COC) from antral follicles of pig ovaries collected from a local abattoir were divided into control and treatment groups and were cultured in tissue culture medium 199 supplemented with follicle-stimulating hormone. Treatment groups consisted of increasing numbers of denuded oocytes (DO) co-cultured with COC (at ratios of COC to DO of 1:1, 1:2, 1:3, 1:4 and 1:5). After incubation for 44 h, cumulus expansion and maturation rates were assessed and oocytes were activated parthenogenetically. Cumulus expansion in the 1 COC:4 DO and 1 COC:5 DO groups was low and altered because full dispersion of the outer layer did not occur. Cell viability was not affected, as measured by the automated cell counter, but scanning electron microscopy revealed only a scanty extracellular matrix. Blastocyst rate was significantly higher in the 1 COC:4 DO (34.4%) and in the 1 COC:5 DO (34.9%) groups (p < 0.05) when compared with other groups. Maturation rate, cleavage rate and total cell number showed no significant difference between control and treatment groups. Amplification by reverse transcription polymerase chain reaction (RT-PCR) showed up-regulation of growth differentiation factor 9 (GDF9) in the cumulus cells in the 1 COC:4 DO group at 44 h. We conclude that denuded porcine oocytes could improve the maturation of COC as evidenced by increased blastocyst development in the 1 COC:4 DO, even though cumulus expansion was poor. This improvement could be a result of the GDF9 up-regulation.

Blastocysts derived from adult fibroblasts of a rhesus monkey ( Macaca mulatta) using interspecies somatic cell nuclear transfer.

In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus-oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5-5.5% CO2 under various conditions (37-39 °C and 5-20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.

The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell-oocyte complexes and effects on parthenogenetic embryo production.

The addition of 9-cis retinoic acid to the oocyte maturation culture medium has a beneficial effect on in vitro fertilized embryos. However, the mechanism of this activity is not known. Therefore, this study was done to elucidate the effect of 9-cis retinoic acid on parthenogenetic embryo production and its signaling pathway and molecular function during in vitro maturation of porcine cumulus cell-oocyte complexes (COCs). Concentrations of 0, 5, 50, and 500 nM 9-cis retinoic acid were added to the in vitro maturation medium, and the embryos were assessed after parthenogenetic activation. Cumulus cells and oocytes from the in vitro matured COCs were separated and subjected to RT-PCR and real-time RT-PCR for detecting retinoic acid receptors and measuring expression of prostaglandin-endoperoxide synthase1 and 2. The addition of 5 nM 9-cis retinoic acid to the maturation medium was beneficial for parthenogenetic embryo production. The effect of 9-cis retinoic acid was exerted directly through the oocytes via the retinoic acid receptor alpha and retinoid X receptor gamma signaling pathways and indirectly through the cumulus cells by the retinoic acid receptor beta and gamma and retinoid X receptor alpha and beta signaling pathways. The addition of 5 nM 9-cis retinoic acid-stimulated cumulus cells reaches full expansion by suppressing their excessive expression of prostaglandin-endoperoxide synthase 2. This study shows that 9-cis retinoic acid can exert its beneficial effect on parthenogenetic embryo production in pigs by multidimensional pathways affecting oocyte maturation.

Short-term treatment with 6-DMAP and demecolcine improves developmental competence of electrically or Thi/DTT-activated porcine parthenogenetic embryos.

Treatment with 6-dimethylaminopurine (6-DMAP) or demecolcine (DE) for several (at least 2) hours after artificial activation is known to improve in vitro development of porcine embryos. However, several reports have also shown that treatments with these chemicals induce apoptosis. The aim of this study was to find out whether short-term treatment with 6-DMAP and DE combined with electrical or thimerosal/dithiothreitol (Thi/DTT) activation had a beneficial effect on development of parthenogenetically activated porcine oocytes. We additionally treated embryos with 6-DMAP (2 mM) and/or DE (0.4 microg/ml) for a short time (40 min) after an electrical pulse (EP) or Thi/DTT. As a result, short-term treatment with 6-DMAP and DE successfully induced development of electrically or Thi/DTT-activated porcine parthenogenetic embryos with no significant difference in cleavage rate, blastocyst formation rate and total cell number compared with long-term treatment. To find optimal activation protocol, cleavage rate, blastocyst formation rate and total cell number were compared between EP and Thi/DTT treatments. Thi/DTT + 6-DMAP + DE showed significantly higher blastocyst formation rate (36.1 ± 3.5%) and total cell number (46.9 ± 1.0) than other groups (EP + 6-DMAP + DE, EP + Thi/DTT + 6-DMAP + DE: 23.3 ± 3.0%, 42.2 ± 1.1 and 17.2 ± 2.7%, 36.7 ± 1.5, respectively). In conclusion, this study demonstrates that short-term treatment with 6-DMAP and DE is as effective as the standard long-term treatment and Thi/DTT + 6-DMAP + DE exerts a synergistic effect.

Effect of recipient breed on delivery rate of cloned miniature pig.

The miniature pig is regarded as a better organ donor breed for xenotransplantation than other pig breeds because the size of their organs is similar to that of humans. To improve efficiency of cloned miniature pig production, we analysed the effect of breed difference between donor cells and embryo recipients on pregnancy rate and delivery rate. Cloned porcine embryos derived from domestic or miniature pig donor cells were transferred to domestic or miniature recipient pigs. Delivery rate was significantly higher when embryos reconstructed with miniature pig donor cells were transferred to miniature pig recipients as compared with that of embryos transferred to domestic pig recipients. However, pregnancy rates were similar between the two groups. The breed of donor cells, but not of embryo recipients, seems likely to affect litter size. From a 13 610 gene cDNA microarray, 1551 (11.7%) genes showed significantly different levels of expression between the fetuses of the two breeds. Vascular endothelial growth factor and c-kit ligand genes related to implantation and maintenance of pregnancy were significantly down-regulated in miniature pigs. In conclusion, the differential gene expression in fetuses interferes with proper fetal/maternal interactions, and results in late-stage pregnancy loss. Our results indicate that the miniature pig is the preferred embryo recipient breed than domestic pig for producing cloned miniature piglets.

Effects of human neural stem cell transplantation in canine spinal cord hemisection.

OBJECTIVES: Previous works have reported that the transplantation of neural stem cells (NSCs) may improve functional recovery after spinal cord injury (SCI), but these results have been mainly obtained in rat models. In the present work, the authors sought to determine whether the transplantation of human NSCs improves functional outcome in a canine SCI model and whether transplanted NSCs survive and differentiate. METHODS: Human NSCs (HB1. F3 clone) were used in this work. Lateral hemisection at the L2 level was performed in dogs and either (1) Matrigel (200 microl) alone as a growth-promoting matrix or (2) Matrigel seeded with human NSCs (10(7) cells/200 microl) were transplanted into hemisected gaps. Using a canine hind limb locomotor scale, functional outcomes were assessed over 12 weeks. Immunofluorescence staining was performed to examine cell survival, differentiation and axonal regeneration. RESULTS: Compared with dogs treated with Matrigel alone, dogs treated with Matrigel + human NSCs showed significantly better functional recovery (10.3 +/- 0.7 versus 15.6 +/- 0.7, respectively, at 12 weeks; p<0.05). Human nuclei-positive cells were found mainly near hemisected areas in dogs treated with Matrigel + NSCs. In addition, colocalization of human nuclei and neuronal nuclei or myelin basic protein was clearly observed. Moreover, the Matrigel + NSC group showed more ascending sensory axon regeneration. CONCLUSIONS: The transplantation of human NSCs has beneficial effects on functional recovery after SCI, and these NSCs were found to differentiate into mature neurons and/or oligodendrocytes. These results provide baseline data for future clinical applications.

Epiblast isolation by a new four stage method (peeling) from whole bovine cloned blastocysts.

We have established a new 4 stage epiblast isolation method from whole bovine cloned blastocysts without using immunosurgery. The new "peeling" method consists of dissolution of the zona pellucida (first stage), elimination of mural trophoblast (second stage), isolation of primitive endoderm and epiblast from polar trophoblast (third stage), and isolation of epiblast from primitive endoderm (fourth stage). The bovine cloned blastocyst consists of 4 different types of cells showing abundant alkaline phosphatase activity. The epiblast origin of isolated cells was confirmed by in vitro differentiation of isolated cells to tubulin beta3-positive neurons and by embryoid body formation. The bovine cloned blastocyst origin of isolated epiblasts was confirmed by microsatellite analysis and mitochondrial DNA sequencing analysis. This new method might accelerate establishment of somatic cell nuclear transfer derived embryonic stem cell lines from bovine and other mammals.

Effects of melatonin on in vitro maturation of porcine oocyte and expression of melatonin receptor RNA in cumulus and granulosa cells.

Melatonin is a multifunctional molecule that mediates several circadian and seasonal processes in animal reproduction. Melatonin and its metabolites are antioxidants and free radical scavengers. We investigated the effects of melatonin on porcine oocyte maturation and embryo development. We then investigated the local expression of the melatonin receptor 1 (MT1) gene in cumulus cells, granulosa cells, and the oocytes with the reverse transcription-polymerase chain reaction (RT-PCR) method. We further evaluated the antioxidant effects [reactive oxygen species (ROS) levels in cumulus-oocytes complexes] of melatonin supplementation during in vitro maturation (IVM). Compared with control, melatonin supplementation (10 ng/mL) during IVM resulted in a greater proportion of oocytes extruding the polar body (75.6% versus 84.6%). Significantly greater proportion of parthenogenetically activated oocytes developed to blastocysts when the in vitro medium was supplemented with melatonin; however, cleavage frequency and blastocyst cell number were not affected by the treatment. RT-PCR analysis revealed the expression of MT1 gene in cumulus and granulosa cells but not in oocytes. Melatonin-treated oocytes had significantly lower levels of ROS than did control (untreated) oocytes. We conclude that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine IVM. Some of the observed effects may be mediated by receptor binding and while others may have been receptor independent, e.g., direct free radical scavenging.

Patient-specific embryonic stem cells derived from human SCNT blastocysts.

Patient-specific, immune-matched human embryonic stem cells (hESCs) are anticipated to be of great biomedical importance for studies of disease and development and to advance clinical deliberations regarding stem cell transplantation. Eleven hESC lines were established by somatic cell nuclear transfer (SCNT) of skin cells from patients with disease or injury into donated oocytes. These lines, nuclear transfer (NT)-hESCs, grown on human feeders from the same NT donor or from genetically unrelated individuals, were established at high rates, regardless of NT donor sex or age. NT-hESCs were pluripotent, chromosomally normal, and matched the NT patient's DNA. The major histocompatibility complex identity of each NT-hESC when compared to the patient's own showed immunological compatibility, which is important for eventual transplantation. With the generation of these NT-hESCs, evaluations of genetic and epigenetic stability can be made. Additional work remains to be done regarding the development of reliable directed differentiation and the elimination of remaining animal components. Before clinical use of these cells can occur, preclinical evidence is required to prove that transplantation of differentiated NT-hESCs can be safe, effective, and tolerated.