No abstract

Porcine embryonic stem cells (ESC) have become an important model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, embryo quality and blastocyst formation have been major limitations for derivation of cloned embryonic stem-like cells. In this study, we tried to overcome these problems by treating with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. A porcine embryonic fibroblast (PEF) cell line was used as the source of donor cells injected into the enucleated oocytes. First, to confirm the effect of HDACi in cloned embryo quality, cloned embryos were treated with Scriptaid (histone deacetylase inhibitor). The Scriptaid-treated blastocysts (n = 26) showed significantly increased total cell number (29.50 ± 2.10; P < 0.05) than nontreated blastocysts (n = 21; 22.29 ± 1.50). Then, the cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free reconstructed 4-cell stage SCNT embryos were injected into empty zonae from hatched parthenogenic blastocysts. The blastocyst formation and total cell number of cloned blastocysts was significantly elevated for all the aggregates (76.3% and 83.18 ± 8.33 cells/blastocyst) compared with nonaggregated (31.0%, and 27.11 ± 1.67 cells/blastocyst; P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine embryonic stem-like cells derivation. Aggregated blastocyst showed higher primary colony formation percentage than nonaggregated cloned blastocysts (20.0 ± 12.3% v. 2.2 ± 1.35%, respectively; P < 0.05). In conclusion, the aggregation of pig SCNT embryos at the 4-cell stage could be a useful technique for improving the development rate and quality of cloned pig blastocyst and derivation efficiency of cloned embryonic stem-like cells.

In somatic cell nuclear transfer, serum starvation is a widely used method to synchronize donor cells at the quiescent stage (Go) of the cell cycle. However, it has been shown that serum starvation during culture of mammalian cells could induce cell death via apoptosis by removing growth factors and increasing intracellular stress. Therefore, apoptosis caused by serum starvation in somatic cells could induce damages to nuclear DNA contributing to a lower efficiency of nuclear transfer. This study was performed to characterize apoptosis during serum starvation of bovine embryonic fibroblasts (BEFs) and to determine the effects of BEFs treated with apoptosis inhibitors on the development of bovine embryos after nuclear transfer. BEFs, collected from a fetus with a 3–4-cm crown-rump length, were cultured for 7 days in starvation medium consisting of Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum to induce quiescence. Cells were also placed in starvation medium containing the apoptosis inhibitors, β2-macroglobulin (broad-range protease inhibitor: MAC; 1.4 pM) and glutathione (GSH: reactive oxygen species scavenger; 2.0 mM). Apoptosis of serum starved BEFs with or without apoptosis inhibitors were analyzed morphologically with light and electron microscope, and biochemically using a TUNEL assay. Somatic cell nuclear transfer was performed by our standard procedure as follows. Bovine oocytes were matured in vitro and enucleated after 22 h. Nuclear donor cells were collected randomly before injection. The reconstructed embryos were placed into the fusion chamber in a solution containing 0.28 M mannitol and aligned manually. A double pulse of 1.8 kV/cm for 15 μs was used to fuse the cells and activate the embryos simultaneously. The fused embryos were cultured for 4 min in 5 μ­M ionomycin and 4 h in 2 mM 6-DMAP. Then, embryos were moved to culture media and cultured in 5% CO2 and 39°C in 100% humidity. Development of NT embryos was recorded at 120 h post NT (morulae) and 168 h (blastocysts) with experiments being repeated three times. Serum starved BEFs showed typical morphology of apoptotic cells such as chromatin condensation and membrane blebbing. Also, when stained for DNA fragmentation by TUNEL assay, 22.6% ofBEFs showed apoptosis, in contrast to 0.1% in actively growing cells. However, when BEFs were cultured with MAC and GSH, the proportions of apoptotic BEFs were greatly reduced, 6.0% and 2.1%, respectively. After nuclear transfer with BEFs, embryos reconstructed with BEF treated with apoptosis inhibitors showed significant improvement in in vitro development compared to the controls (Table 1). In conclusion, while there are a number of factors affecting the nuclear transfer procedure, damage to the donor nuclei by serum starvation is likely to reduce the efficiency of the procedure; the addition of apoptosis inhibitors could reduce this unnecessary damage to donor nuclei and result in improvement in the development of nuclear transferred embryos. Further experiments are needed to assess the effect of apoptosis inhibitors on improvement of overall nuclear transfer efficiency. Table 1. Development of bovine embryos nuclear transferred with embryonic fibroblasts treated with or without apoptosis inhibitors

X-chromosome inactivation (XCI) is an epigenetically essential process for balancing dosage of X-linked genes between male and female eutherian. Importance of this complex and species-specific event has been highlighted recently in developmental and stem cell biology. However, the process has been confirmed only in restricted species, even though the species-specific studies are needed for comprehensive understanding of XCI in specific species. XCI is regulated by the various genes, many of which are coded on the X chromosome inactivation centre (XIC). Among the XIC-linked genes, especially non-coding RNA (ncRNA) like XIST, which is master gene for XCI, are known to regulate XIC. But the centre is not identified in various species. In this study, we identified XIC in pig and analysed the dosage differences of XIC-linked gene in porcine embryos. At first, the centre was searched in pig. The genomic length of the porcine XIC was similar to human XIC and the order and coding strand of the counterparts in pig XIC were same as the human XIC-linked genes. However, sequence comparison between human XIC-linked gene and its porcine counterpart showed that ncRNA around XIST were less conserved rather than protein-coding genes. This would be caused by rapid evolution of genomic region harboring ncRNA. The expression of XIC-linked genes was compared between male and female porcine embryonic fibroblast (PEF) to confirm that dosage compensation is completed in PEF. Most of the genes were not expressed sex-specifically, but two genes, XIST and an uncharacterized gene, LOC102165544, were expressed female preferentially in PEF. Interestingly, LOC102165544, which had low sequence homology with human JPX, was expressed about 2-fold higher in female PEF. This means that XIST and LOC102165544 are XCI-escaping genes. Among the XIC-linked genes, CHIC1, XIST, LOC102165544, and RLIM were stably expressed in embryonic stage, and XIST and LOC102165544 were up-regulated after morula formation. As XIST accumulation is a requisite for XCI initiation, expression levels of the 4 genes between male and female blastocysts were compared. Interestingly, expression levels of CHIC1 and RLIM were not different in male and female blastocysts. This means their dosage would be already compensated in porcine blastocyst. Additionally, to confirm loci of the 2 genes CHIC1 and RLIM harbor one of the inactive alleles in female blastocyst, the DNA methylation pattern was examined. One of the CHIC1 alleles was inactive but RLIM CpG site was hypo-methylated in female blastocyst. This would indicate that one of the RLIM alleles is transcriptionally inactivated by chromatin modification rather than by DNA methylation of the allele. Regulatory regions of XIST and LOC102165544 were demethylated in blastocyst and this showed XCI was not finished in porcine blastocyst. Conclusively, our results demonstrate the XCI already occurs in porcine blastocyst at least one gene but it is not completed.This work was supported by Next BioGreen21 program (PJ009493), Rural Development Administration, Republic of Korea.

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

Cellular reprogramming of committed cells into a pluripotent state can be accomplished by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. However, during reprogramming, it has been verified that failures of reactivating endogenous genes and epigenetic remodelling lead to partially reprogrammed cells exhibiting features similar to those of fully reprogrammed cells. In this study, partially reprogrammed induced pluripotent stem cells (pre-iPSC) were derived from porcine fetal fibroblasts via drug-inducible vector carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study aimed to investigate characteristics of pre-iPSC and reprogramming mechanisms. The pre-iPSC were stably maintained over an extended period having in vitro differentiation ability into 3 germ layers. The pluripotent state of pre-iPSC was regulated by modulation of culture condition. They showed naive- or primed-like pluripotent state in leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) supplemented culture conditions respectively. However, pre-iPSC could not be maintained without ectopic expression of transgenes. The cultured pre-iPSC expressed endogenous transcription factors (OCT4 and SOX2) except for NANOG known as gateway into complete reprogramming. In addition, endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occurred in female pre-iPSC. Given the results, we found that expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu for achieving faithful pluripotency in pig. Accordingly, investigating pre-iPSC could help us to improve and develop reprogramming methods via understanding reprogramming mechanisms in pig. This work was supported by the Next-generation BioGreen 21 Program (PJ0113002015), Rural Development Administration, Republic of Korea.

The lineage specification of the pre-implantation embryo is important to understand the developmental process, but it remains unclear because the expression of lineage-specific genes is distinct among species. Pigs have genetic and physiological traits similar to humans; however, there are differences in gene expression during the pre-implantation stage. To select a candidate gene that affects the formation of the inner cell mass (ICM) in porcine embryo, we conducted preliminary experiments. First, we measured the expression level of candidate genes for lineage specification in parthenogenetic-activated embryos. The expression of pluripotent genes peaked on Day 3 and thereafter decreased gradually. Next, we conducted immunocytochemistry. OCT4 was expressed in all cells in morula and Day 5 blastocyst, but some Day 7 blastocysts expressed OCT4 in both ICM and trophectoderm (TE), whereas others expressed OCT4 only in ICM. NANOG was not observed in the morula stage, whereas SOX2 was located in a restricted area. To examine the effect of SOX2 in ICM formation, we injected plasmid expressing Cas9 and guide (g)RNA using Lipofectamine for efficient transgene expression at the 2-cell stage to increase viability by inducing mosaicism. The expression of enhanced green fluorescent protein (EGFP) contained in the plasmid confirmed that the plasmid was operating normally. In SOX2-knockout (KO) early blastocysts, the numbers of total cells and SOX2- and NANOG-positive cells were greatly decreased, while OCT4 was expressed in most cells. As in early blastocysts, SOX2-KO late blastocysts had fewer cells expressing SOX2, NANOG, and SOX17 than control. To identify the transcriptional consequences of SOX2 reduction, we performed quantitative PCR analysis on non-injected and PX458-gRNA injected blastocysts. Injection of PX458-gRNA resulted in downregulation of NANOG, SOX17, and SMAD7, but not SOX2 and OCT4. Furthermore, proliferation-associated genes were downregulated in injected blastocysts. In conclusion, SOX2-targeted porcine embryos showed blastocoel formation, the inner cell mass formed poorly, and embryos have inefficient cells. Also, the depletion of SOX2 in porcine blastocysts downregulated pluripotent genes and proliferation genes. This work was supported by the BK21 Plus Program, the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2019R1C1C1004514), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-3-HD020).

Pigs are significant as a disease model in translational research. However, authentic porcine embryonic stem cells (ESC) have not yet been established showing limited capacities until now. In this study, a total of 7 primed ESC lines were derived from porcine embryos of various origins, including in vitro-fertilized (IVF), parthenogenetic activation (PA), and nuclear transfer (iPS-NT) from a donor cell with induced pluripotent stem cells (iPSC). We observed typical morphology, intensive alkaline phosphatase activity, and normal karyotype in all pESC lines. Also, the expression of pluripotency markers such as OCT4, Sox2, NANOG, SSEA4, TRA 1–60, and TRA 1–81 was shown in our pESC. We investigated expression of key markers of lineage commitment to confirm the differentiation potentials of the 7 cell lines to formation of EB and all 3 germ layers, such as AFP (endoderm), DESMIN (mesoderm), and CRABP2 (ectoderm) by RT-PCR and Cytokeratin 17 (endoderm), Desmin (mesoderm), and Vimentin (ectoderm) by immunofluorescence analysis. We also examined the XIST gene expression and nuclear H3K27me3 foci from all female cell lines for analysing epigenetic characteristics. Furthermore, we classified 2 colony types (normal and transformed colony) and 3 subpopulations of ES cells composed of transformed colonies with intrinsic morphological characteristics: petaloid rapidly self-renewing cells, small spindle-shaped cells, and large flattened cells. This result will help to approach the goal for establishing authentic naive pluripotent stem cells in pigs and it will make possible sophisticated genetic manipulation to create ideal animal models for preclinical research and studies of human diseases.This work was supported, in part, by a grant from the National Research Foundation of Korea Grant Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.