Background: Canine induced pluripotent stem cells (iPSCs) are an attractive source for veterinary regenerative medicine, disease modeling, and drug development. Here we used vitamin C (Vc) to improve the reprogramming efficiency of canine iPSCs, and its functions in the reprogramming process were elucidated. Methods: Retroviral transduction of Oct4, Sox2, Klf4, c-Myc (OSKM), and GFP was employed to induce reprogramming in canine fetal fibroblasts. Following transduction, the culture medium was subsequently replaced with ESC medium containing Vc to determine the effect on reprogramming activity. Results: The number of AP-positive iPSC colonies dramatically increased in culture conditions supplemented with Vc. Vc enhanced the efficacy of retrovirus transduction, which appears to be correlated with enhanced cell proliferation capacity. To confirm the characteristics of the Vc-treated iPSCs, the cells were cultured to passage 5, and pluripotency markers including Oct4, Sox2, Nanog, and Tra-1-60 were observed by immunocytochemistry. The expression of endogenous pluripotent genes (Oct4, Nanog, Rex1, and telomerase) were also verified by PCR. The complete silencing of exogenously transduced human OSKM factors was observed exclusively in canine iPSCs treated with Vc. Canine iPSCs treated with Vc are capable of forming embryoid bodies in vitro and have spontaneously differentiated into three germ layers. Conclusions: Our findings emphasize a straightforward method for enhancing the efficiency of canine iPSC generation and provide insight into the Vc effect on the reprogramming process.

Bovine somatic cell nuclear transfer (bSCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization (IVF). However, the efficiency of somatic cell cloning has remained low, and applications have been limited, irrespective of the nuclear donor species or cell types. One possible explanation is that the reprogramming factors of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we would like to introduce the aggregation method (agSCNT), a new experimental system that enables and increase oocyte volume and examined its subsequent development. Judgement by the blastocyst formation rate or total cell number was significantly higher in the agSCNT group than that in the SCNT group, and was very similar to that in the control IVF group. Moreover, the cleavage formation rate in the agSCNT group (61.5 ± 1.3) was higher than that in the SCNT group (39.7 ± 2.1), while still less than that in the IVF group (75.4 ± 1.3). We also analyzed the epigenetic modifications in bovine IVF, agSCNT, and untreated SCNT embryos. In conclusion, the present study demonstrated that agSCNT improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell numbers (TC).

It is still challenging to establish pESCs due to differences in the genetic backgrounds of mouse, human, and pig. So it is required to find pig specific pluripotency markers and cellular signaling. In this experiments, doxycycline-inducible vectors carrying OCT4, SOX2, NANOG, KLF4 and MYC known as reprogramming factors, were infected into pig stem cells for analyzing gene expression pattern. When cultured without doxycycline, pig stem cells were stably maintained in bFGF supplemented media. However, when treated with doxycycline, pig stem cells lost alkaline phosphatase activity and were differentiated within two weeks. And then, we investigated the expression of genes related to pluripotency in doxycycline-treated pig stem cells by using qRT-PCR. The qRT-PCR data revealed that expression of OCT4, CDH1 and FUT4 were significantly increased by OCT4 overexpression and OCT4 and FUT4 were also upregulated in SOX2-infected group. When infected with combination of two factors including OCT4 or SOX2, some groups could stably maintain at LIF supplemented media, having alkaline phosphatase activity. Given these data, although ectopic gene expression induced differentiation in pig stem cells, ectopic expression of OCT4 and SOX2 could upregulate pluripotent genes and overexpreession of two factors help pig stem cells adapt LIF-contained media. This study could improve understanding of pluripotent networks as well as aid in establishing bona fide pluripotent stem cells in pig.

Reprogramming is a process in which a developmentally determined cell fate is re-established to another fate by artificial modifications. Reprogramming to pluripotent state has been studied extensively in somatic cell nuclear transfer and induced pluripotent stem cells (iPSCs). Interestingly, recent studies showed that expression of a set of transcription factors also can induce transdifferentiation, also known as direct conversion or direct reprogramming. There are two major approaches in direct reprogramming: one is target cell-specific factor-mediated direct reprogramming and the other is epigenetic flexibility-inducing factor-mediated direct reprogramming. We are interested in generating induced neural cells via direct reprogramming by using pluripotency factors as epigenetic flexibility inducers and understanding the mechanistic basis of the process. We hope that these reprogramming technologies will provide a new paradigm of research in regenerative medicine as well as disease study and drug development.

Successful somatic cell nuclear transfer (SCNT) has been reported across a range of species using a range of recipient cells including enucleated metaphase II (MII) arrested oocytes, enucleated activated MII oocytes, and mitotic zygotes. However, the frequency of development to term varies significantly, not only between different cytoplast recipients but also within what is thought to be a homogenous population of cytoplasts. One of the major differences between cytoplasts is the activities of the cell cycle regulated protein kinases, maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK). Dependent upon their activity, exposure of the donor nucleus to these kinases can have both positive and negative effects on subsequent development. Co-ordination of cell cycle stage of the donor nucleus with the activities of MPF and MAPK in the cytoplast is essential to avoid DNA damage and maintain correct ploidy. However, recent information suggests that these kinases may also effect reprogramming of the somatic nucleus and preimplantation embryo development by other mechanisms. This article will summarise the differences between cytoplast recipients, their effects on development and discuss the potential role/s of MPF and or MAPK in nuclear reprogramming.

MicroRNAs (miRNAs) are approximately 22 nucleotides of small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The miRNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, metabolism, imprinting, and differentiation. Recently, a paper has shown that expression of the miRNA-302/367 cluster expressed abundantly in mouse and human embryonic stem cells (ESCs) can directly reprogram mouse and human somatic cells to induced pluripotent stem cells (iPSCs) efficiently in the absence of any of the four factors, Oct4, Sox2, c-Myc, and Klf4. To apply this efficient method to porcine, we analyzed porcine genomic sequence containing predicted porcine miRNA-302/367 cluster through ENSEMBL database, generated a non-replicative episomal vector system including miRNA-302/367 cluster originated from porcine embryonic fibro-blasts (PEF), and tried to make porcine iPSCs by transfection of the miRNA-302/367 cluster. Colonies expressing EGFP and forming compact shape were found, but they were not established as iPSC lines. Our data in this study show that pig miRNA-302/367 cluster could not satisfy requirement of PEF reprogramming conditions for pluripotency. To make pig iPSC lines by miRNA, further studies on the role of miRNAs in pluripotency and new trials of transfection with conventional reprogramming factors are needed.

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nu-clei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2’deoxycytidine (5-aza-dC), DNA methy-lation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molec-ular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptot-ic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-re-lated genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

Embryo reconstruction by somatic cell nuclear transfer (SCNT) has been used to demonstrate that mammalian somatic cells can dedifferentiate into a totipotent nucleus when introduced into an oocyte. This process of dedifferentiation of somatic cells after nuclear transplantation is defined as nuclear reprogramming, although this terminology gives little information on the molecular events that characterize this process. When planning on strategies for nuclear reprogramming by nuclear transfer (NT) one may suggest that converting the somatic nuclear configuration into an embryonic state is highly desirable, so this may promote a succession of events similar to those occurring during early embryo development. In the following part of this overview we will discuss the results of many studies that have investigated different aspects of nuclear remodelling after SCNT.

Induced pluripotent stem cells (iPSCs), generated by the overexpression of transcription factors Oct4, Sox2, Klf4 and c‐Myc in somatic cells, are pluripotent. iPSCs reprogrammed from differentiated cells get through a epigenetic modification during reprogramming and finally have the similar epigenetic state to embryonic stem cells (ESCs). In this study, these epigenetic changes were observed in reprogramming of uni‐parental parthenogenetic somatic cells. Furthermore, we have shown that parthenogenetic pattern of imprinted genes were changed during pluripotential reprogramming. Parthenogenetic neural stem cells (pNSCs) containing only maternal alleles regain the biparental imprinting patterns after reprogramming. However, we have yet to define whether the changed imprinted genes are maintained or reverted to the parthenogenetic state when the reprogrammed cells are differentiated into specialized cell types. To address this question, we compared genome‐wide expression profiles of biparental female neural stem cells (fNSCs), parthenogenetic neural stem cells (pNSCs), and NSCs differentiated from parthenogenetic maternal iPSC (miPS‐NSCs). Furthermore, this study establishes the correlation between the alteration of genome methylation and activation of imprinting genes in the parthenogenetic cells and reports for the first time that the silenced PWS‐related imprinted genes are activated in miPS‐NSCs. Our data demonstrated that pluripotential reprogramming of parthenogenetic somatic cells were able to reset the parthenogenetic imprinting patterns; reprogrammed miPSCs showed erasure of maternal methylation imprints and acquisition of methylation in paternally imprinted genes. Furthermore, the changed imprinting patterns were maintained when the reprogrammed cells are differentiated into specialized cell type. * This work was supported by the Next‐Generation BioGreen 21 program (Grant PJ008- 009) funded by the Rural Development Administration, Republic of Korea.

Oct4 and Nanog are transcription factors involved in pluripotency of stem cells. In general, Oct4 is up-regulated by Nanog. In previous results, however, Oct4 was differentially regulated by various doses of Nanog in P19 cells. High dose Nanog down-regulated the Oct4 expression. This negative feedback event was performed by DNMT and HDAC through the inhibitor assays (5-AZA-cytidine and trichostatin A). To identify the precise recruited sites for DNMT and HDAC, ChIP assay was performed in differential doses of Nanog. As a result, HDAC1, HDAC2, DNMT3A and Nanog were recruited to CR2, CR3, CR1, and CR4 upon high dose Nanog, respectively. Next, to investigate the differentiation potency of the cells upon high dose Nanog, RT-PCR with specific markers for three germ layers was performed. However, there was no increase for three germ layers in high dose Nanog treated cells except E-cadherin expression. E-cadherin is a specific marker for epithelial cells. Taken together, high dose Nanog induces Oct4 down-regulation and results in differentiating embryonic carcinoma cells to epithelial cell type. These results will be helpful for study on regulation of pluripotency-related genes in embryonic stem cells. * This study was supported by 2012 Post Doctoral Fellowship Program of National Institute of Animal Science, Rural Development Administration, Republic of Korea. This work received grant support from the Agenda Program (No.PJ007577), Rural Development Administration, Republic of Korea.

Limited success of somatic cell nuclear transfer(SCNT) is attributed to incomplete reprogramming of transferred donor cell. Several approachs, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors have been used to improve the efficiency of somatic cell nuclear transfer. Recently, it is reported that pre-treatment of somatic cells with undifferentiated cell extract, such as embryonic stem cell and mammalian oocytes is an attractive alternative ways to reprogramming control. The aim of this study was to evaluate the early development of porcine cloned embryos produced with porcine ear skin fibroblasts pre-treated with extract from porcine induced pluripotent stem cell (iPSC). For transport of porcine iPSC extract into cultured porcine ear skin fibroblasts, the ChariotTM reagent system was used. Treated cells were cultured for 3 days, and used for the analysis of histone H3K9 acetylation and SCNT The acetylation status of H3K9 was increased in cells treated with iPSC extract and cultured for 3 days compared with control. But, no significant difference was observed between the extract treated and control groups. After SCNT. no difference was observed in the rate of fusion (86.6% vs 86.2%) and embryo cleavage (86.6% vs 87.1%) between the extract treated and control groups. Also, no significant difference was noted in blastocyst rates (23.4% vs 28.4%) as well as cell numbers (43.8±10.8 vs 41.2±11.6) with extract treated group compared with control group. Overall apoptosis rate in blastocyst was not differences between the extract treated and control groups (4.6±3.5% vs 6.0± 5.8%). However, blastocyst rate with high apoptotic cells(>10% appototic cells) was significantly lower in extract treated group when compared with control group (7.1% vs 21.8%).. Our results demonstrated that pre-treatment of porcine ear skin fibroblasts using porcine iPSc extract had beneficial effect on the decreasing apoptosis in the blastocyst cultured in vitro, although there was no effect on the embryonic development.

Somatic cell nuclear transfer (SCNT) is an efficient technique which has been successfully applied to developmental biology, and resulted in the production of offspring from various species. It offers many opportunities in basic and medical research as well as endangered species preservation. On the other hand, embryonic stem (ES) cells are useful research tools for genetic engineering and developing disease models. In previous study, we established bovine IVF embryo derived ES cell line which can be grow indefinitely as undifferentiated cell state. In this study, we compared the effect of two different age cells (bovine ES cell; JNU-ibES-05 or adult ear fibroblast cell) on in vitro developmental potential of bovine SCNT embryo. To produce SCNT embryos, the ES cells or somatic cells were dissociated and transferred into enucleated MⅡ oocytes, and cleaved reconstructed embryos were cultured in CR1aa medium containing 10% FBS, 1 ug/ml epidermal growth factor (EGF) and 1 ug/ml insulin growth factor (IGF) for 8 days. In the result, blastocyst development rate was similar between ES cell treatment group and somatic cell treatment group, 27.7% (10/36) and 28.9% (11/ 38), respectively. However, there was particular difference in development speed from day 5 post SCNT, blastocyst expanding was 1 day faster in ES cell group than in somatic cell group. This difference was analyzed by semi-quantitative RT-PCR using pluripotency, growth and cell cycle gene markers. These results demonstrated that SCNT embryo using ES cell as a donor cell has better growth potential than somatic cell, and it will be a useful tool for a transgenic animal production.

The vast majority of embryo generated by Assisted Reproductive Technologies (ART) do not result in a live offspring and a multiple birth is the single biggest health risk associated with human fertility treatment, and the used of frozen embryos increased for medical or personal reasons. However, practical and ethical reasons might hamper study of human embryos. Therefore, animal models are necessary to elucidate the molecular and morphological changes during development. In the serial experiments, we employed mouse embryos and a Cdx-inducible ES cell system that transdifferentiates into TS cells. We found aberrant gene expression profiles including apoptosis associated (Bcl2), lineage formation related genes (Cdx-2, Tcfap2c, Oct4, and Nanog), and/or mitochondrial DNA replication related genes (mt-cox-1, mt-cox-2, Polg, Polg2, Tfam) in mouse embryos that showed developmentally retardation between morula to blastocyst transition or post implantation development after embryo transfer to surrogate mothers, compared to control embryos. To determine direct interaction between knockdown genes via siRNA approach and putative down-stream genes involved in blastocyst formation and further development, we carried out qPCR and Chip assay in either mouse embryos or the ES cells. qPCR and Chip assay results showed target gene directly bound to promoter regions of down-regulated genes in TS cells. In conclusion, we suggested that an increased understanding of epigenetic regulation of early embryonic development through animal models may ultimately lead to better methodologies for selecting more competent embryos and and/or protocols for augmenting embryos viability.