Background: Coating a culture plate with molecules that aid in cell adhesion is a technique widely used to produce animal cell cultures. Extracellular matrix (ECM) is known for its efficiency in promoting adhesion, survival, and proliferation of adherent cells. Gelatin, a cost-effective type of ECM, is widely used in animal cell cultures including feeder-free embryonic stem (ES) cells. However, the optimal concentration of gelatin is a point of debate among researchers, with no studies having established the optimal gelatin concentration. Methods: In this study, we coated plastic plates with gelatin in a concentrationdependent manner and assessed Young’s modulus using atomic force microscopy (AFM) to investigate the microstructure of the surface of each plastic plate. The adhesion, proliferation, and differentiation of the ESCs were compared and analyzed revealing differences in surface microstructure dependent on coating concentration. Results: According to AFM analysis, there was a clear difference in the microstructure of the surface according to the presence or absence of the gelatin coating, and it was confirmed that there was no difference at a concentration of 0.5% or more. ES cell also confirmed the difference in cell adhesion, proliferation, and differentiation according to the presence or absence of gelatin coating, and also it showed no difference over the concentration of 0.5%. Conclusions: The optimum gelatin-coating for the maintenance and differentiation of ES cells is 0.5%, and the gelatin concentration-mediated microenvironment and ES cell signaling are closely correlated.

Chicken embryonic stem (ES) cells have great potential and provide a powerful tool to investigate embryonic development and to manipulate genetic modification in a genome. However, very limited studies are available on the functional characterization and robust expansion of chicken ES cells compared to other species. Here, we have developed a method to generate chicken embryonic stem cell-like cells under pluripotent culture conditions. The chicken embryonic stem cell-like cells were cultivated long-term over several passages of culture without loss of pluripotency in vitro and had the specific expression of key stem cell markers. Furthermore, they showed severe changes in morphology and a significant reduction in pluripotent genes after siRNA-mediated NANOG knockdown. Collectively, these results demonstrate the efficient generation of chicken embryonic stem cell-like cells from EGK stage X blastoderm-derived singularized cells and will facilitate their potential use for various purposes, such as biobanking genetic materials and understanding stemness in the fields of animal biotechnology.

Parthenogenesis is maternally uniparental reproduction through the embryonic development of oocytes without fertilization. Artificial activation of mature oocytes could generate homozygous haploid embryos with the extrusion of the second polar body. However, the haploid embryos showed low embryo development in preimplantation embryos. In this study, we investigated whether the electronic fusion of the haploid embryos could enhance embryo development and ESC establishment in mice. Haploid embryos showed the developmental delay from 4-cell to the blastocyst stage. The haploid blastomeres of the 2-cell stage were fused electronically, resulting in that the fused embryos showed a significantly higher rate of blastocysts compared to non-fused haploid embryos (55% vs. 37%). Further, the embryonic stem cells (ESCs) derived from the fused embryos were confirmed to be diploid. The rate of ESC establishment in fused embryos was significantly higher compared to non-fused ones. Based on the results, we concluded that the electronic fusion of haploid embryos could be efficient to generate homozygous ESCs.

Runt related transcription factors (RUNX), a family of well-known transcription factors, play key regulatory roles in diverse biological processes, such as proliferation, differentiation, and DNA repair. Of RUNX family, RUNX3 is the least well characterized of the three family members. Nevertheless, the role of RUNX3 as a key regulator in essential biological pathways has been reported and inactivation of RUNX3 leads to a variety of disease, such as cancer, via regulation of Wnt signaling and K-ras mutations in many mammalian tissues. Recent studies using RUNX3-deficient cells and mice revealed an association with hematopoiesis and hypersensitivity to granulocytecolony stimulating factor. Nevertheless, protein dynamics associated with RUNX3 remain poorly understood. In the present study, we performed a large-scale protein study from Runx3 knockout (KO) mouse embryonic stem cells (mESC) using a stable isotope labeling by amino acids (SILAC)-based quantitative proteomics approach. The results showed that 67 proteins were significantly up and downregulated after Runx3 KO. Bioinformatic analyses that revealed that these proteins have diverse biological functions, such as substances transport and cellular structure. Thus, our results enhance our current understanding of the function of RUNX3 in mESCs and suggest potential roles for RUNX proteins in diverse diseases. Additionally, our results can be used as a database to help us understand the mechanism of action of RUNX3.

The establishment of porcine embryonic stem cells (ESCs) from porcine somatic cell nuclear transfer (SCNT) blastocysts is influenced by in vitro culture day of porcine reconstructed embryo and feeder cell type. Therefore, the objective of the present study was to determine the optimal in vitro culture period for reconstructed porcine SCNT embryos and mouse embryonic fibroblast (MEF) feeder cell type for enhancing colony formation efficiency from the inner cell mass (ICM) of porcine SCNT blastocysts and their outgrowth. As the results, porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days showed significantly increased efficiency in the formation of colonies, compared to those for 7 days. Moreover, MEF feeder cells derived from outbred ICR mice showed numerically the highest efficiency of colony formation in blastocysts produced through in vitro culture of porcine SCNT embryos for 8 days and porcine ESCs with typical ESC morphology were maintained more successfully over Passage 2 on outbred ICR mice-derived MEF feeder cells than on MEF feeder cells derived from inbred C57BL/6 and hybrid B6CBAF1 mice. Overall, the harmonization of porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days and MEF feeder cells derived from outbred ICR mice will greatly contribute to the successful establishment of ESCs derived from porcine SCNT blastocysts.

Somatic cell nuclear transfer derived embryonic stem cells (NT-ESCs) have significant advantages in various fields such as genetics, embryology, stem cell science, and regenerative medicine. However, the poor establishment of NT-ESCs hinders various research. Here, we applied fasudil, a Rho-associated kinase (ROCK) inhibitor, to develop somatic cell nuclear transfer (SCNT) embryos and establish NT-ESCs. In the study, MII oocytes were isolated from female B6D2F1 mice and performed SCNT with mouse embryonic fibroblasts (MEFs). The reconstructed NT-oocytes were activated artificially, and cultured to blastocysts in KSOM supplemented with 10 μM fasudil. Further, the blastocysts were seeded on inactivated MEFs in embryonic stem cell medium supplemented with 10 μM fasudil. A total of 26% of embryos formed into blastocysts in the fasudil treated group, while this ratio was 44% in the fasudil free control group. On the other hand, 30% of blastocysts were established NT-ESCs after exposure of fasudil, which was significantly higher than the control group (10%). The results suggest that fasudil reduced blastocyst development after SCNT due to inhibition of 2 cell cleavage while improved the establishment of NT-ESCs through the anti-apoptotic pathway.

Because of the physiological and immunological similarities between pigs and humans, porcine embryonic stem cells (ESCs) have been identified as important candidates in preliminary studies on human disease. A comparative understanding of pig ESCs with the human is required to achieve these goals. To gain insights into pig stem cells, the transcriptome of pig ES-like cells were compared with pig preimplantation embryos and human/mouse pluripotent stem cells by RNA-seq analysis. As a result, pig stem cells were more similar to late epiblasts of pig preimplantation embryos than early ICM as revealed by transcriptome analysis, suggesting that pig stem cells are in a developmentally primed state. Moreover, the physiological and biological functions of pig ESCs were more similar to those of human PSCs than to those of mouse PSCs, as determined by direct differentiation and GO/KEGG term analysis. Overall, our data indicate that pig ESCs are in a primed pluripotent state resembling human PSCs. Our findings will facilitate both the development of large animal models for human stem cell therapy and the generation of pluripotent stem cells from other domestic animals for agricultural use. This work was supported by the Korea Institute of Planning and Evaluation for Technology in food, agriculture, forestry, and fisheries (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-1-HD020), and partially supported by the grants from the Agenda Program of Rural Development Administration, Republic of Korea (No. PJ01362402)

Introduction Porcine embryonic stem cells (pESCs) derived from cloned embryos might be a useful animal model in biomedical research, however, establishment of cloned pESCs is difficult by its incomplete nuclear reprogramming. Here, we report the improved development competence of porcine cloned embryos by vitamin C (VC) supplement to establish the pESCs. Materials and Methods Slaughterhouse-derived oocytes were in vitro matured for 44h and parthenogenetic and cloned embryos were produced using matured oocytes. Both of embryos were cultured for 6 days in PZM-5 media and development rates were examined. Four different concentration of VC (0, 25, 50, 100, and 200 μg/ml) was supplemented in IVM and IVC media and preimplantation developments in the 5 groups were compared in both of embryos Results and Discussion In the cleavage rates of IVM group, significantly higher rate was shown in 50 mg/ml group than other groups (84.5 ± 0.6% vs. 69.8 ± 5.5, 75.7 ± 1.8, 80.4 ± 0.2, 72.4 ± 0.1%; P<0.05), respectively. Significantly higher rates of blastocyst development also were shown in 50 mg/ml group than other groups (27.0 ± 2.0% vs. 20.4 ± 1.4, 22.1 ± 1.3, 23.7 ± 1.2, 19.6 ± 1.3%; P<0.05), respectively. In the cleavage rate of IVC group, non-significantly different with each group (84.0 ± 1.3, 86.7 ± 1.0, 88.4 ± 1.4, 76.7 ± 3.0, 64.6 ± 4.4; P<0.05). In the blastocyst rate of IVC group, significantly higher rate was shown in 25mg/ml and 50 mg/ml group than other groups (22.3 ± 1.7, 23.8 ± 1.7% vs. 19.1 ± 1.3, 15.9 ± 1.0, 5.8 ± 1.5%; P<0.05) In conclusion, supplement of 50μg/ml of VC in IVM and IVC media enhanced the development of porcine parthenogenetic embryos and these results will be a helpful information in the development of porcine cloned embryos and derivation of its embryonic stem cells.

Matrix metalloproteinases (MMPs) have been known to affect to cell migration, proliferation, morphogenesis and apoptosis by degrading the extracellular matrix. In the previous studies, undifferentiated mouse embryonic stem cells (ESCs) were successfully proliferated inside the extracellular matrix (ECM) analog-conjugated three-dimensional (3D) poly ethylene glycol (PEG)-based hydrogel. However, there is no report about MMP secretion in ESCs, which makes it difficult to understand and explain how ESCs enlarge space and proliferate inside 3D PEG-based hydrogel constructed by crosslinkers containing MMP-specific cleavage peptide sequence. Therefore, we investigated what types of MMPs are released from undifferentiated ESCs and how extracellular signals derived from various niche conditions affect MMP expression of ESCs at the transcriptional level. Results showed that undifferentiated ESCs expressed specifically MMP2 and MMP3 mRNAs. Transcriptional up-regulation of MMP2 was caused by the 3D scaffold, and activation of integrin inside the 3D scaffold upregulated MMP2 mRNAs synergistically. Moreover, mouse embryonic fibroblasts (MEFs) on 2D matrix and 3D scaffold induced upregulation of MMP3 mRNAs, and activation of integrins through conjugation of extracellular matrix (ECM) analogs with 3D scaffold upregulated MMP3 mRNAs synergistically. These results suggest that successful proliferation of ESCs inside the 3D PEG-based hydrogel may be caused by increase of MMP2 and MMP3 expression resulting from 3D scaffold itself as well as activation of integrins inside the 3D PEG-based scaffold.

A lot of works have been dedicated to clarify the reasons why the establishment of embryonic stem cells (ESCs) from pig is more difficult than that from mouse and human. Several concomitant factors such as culture condition including feeder layer, sensitivity of cell to cell contact, definitive markers of pluripotency for evaluation of the validity and optimal timing of derivation have been suggested as the disturbing factors in the establishment of porcine ESCs. Traditionally, attempts to derive stem cells from porcine embryos have depend on protocols established for mouse ESCs using inner cell mass (ICM) for the isolation and culture. And more recently, protocols used for primate ESCs were also applied. However, there is no report for the establishment of porcine ESCs. Indeed, ungulate species including pigs have crucial developmental differences unlike rodents and primates. Here we will review recent studies about issues for establishment of porcine ESCs and discuss the promise and strategies focusing on the timing for derivation and pluripotent state of porcine ESCs.

Embryonic stem cell classically cultured on feeder layer with FBS contained ES medium. Feeder-free mouse ES cell culture systems are essential to avoid the possible contamination of nonES cells. First we determined the difference between ES cell and MEF by Oct4 population. We demonstrate to culture and to induce differentiation on feeder free condition using a commercially available mouse ES cell lines.

Although embryonic stem cells (ESCs) or ES-like cells are reported from many mammalian species other than the mouse, the culture system for murine ESCs may not be suitable to the other species. Previously many other research groups have modified either human or mouse ESC culture systems for bovine ESC culture. In this study, we compared three different culture mediums consisting of DMEM, -MEM or KnockOut-DMEM (KO), which are modified from human or mouse ESC culture system, for the generation of bovine ESCs. In this study, some pre-requisite events which are important for establishment and long-term propagation of ESCs such as inner cell mass (ICM) attachment on feeder cells, primary colony formation and sustainability after passaging. Once the ICM clumps attached on feeder cells, this was designated as passage 0. In regards to the rate of ICM attachment, -MEM was superior to the other systems. For primary colony formation, there was no difference between DMEM and -MEM whereas KO showed lower formation rate than the other groups. For passaging, the colonies were split into 2~4 pieces and passed every 5~6 days. From passage 1 to passage 3, DMEM system seemed to be appropriate for maintaining putative bovine ESCs. On the other hand, -MEM tended to be more suitable after passage 6. Although -MEM support to maintain a ES-like cell progenies to passage 15, all three culture systems which are modified from human or mouse ESC culture media failed to retain the propagation and long-term culture of putative bovine ESCs. Our findings imply that more optimized alternative culture system is required for establishing bovine ESC lines.

Various small molecules can be used to control major signaling pathways to enhance stemness and inhibit differentiation in murine embryonic stem cell (mESC) culture. Small molecules inhibiting the fibroblast growth factor (FGF)/ERK pathway can preserve pluripotent cells from stimulation of differentiation. In this study, we aimed to evaluate the effect of pluripotin (SC-1), an inhibitor of the FGF/ERK pathway, on the colony formation of outgrowing presumptive mESCs. After plating the zona pellucida-free blastocyst on the feeder layer, attached cell clumps was cultured with SC-1 until the endpoint of the experiment at passage 10. In this experiment, when the number of colonies was counted at passage 3, SC-1-treated group showed 3.4 fold more mESC colonies when compared with control group. However, after passage 4, there was no stimulating effect of SC-1 on the colony formation. In conclusion, SC-1 treatment can be used to promote mESC generation by increasing the number of early mESC colonies.

Porcine has been known to have a great impact on the studies of organ transplantation, biomaterial production and specific biomodel development such as transgenic animals. To achieve such therapeutic purposes, establishment of porcine embryonic stem cells (pESCs) will be needed. Especially, in vitro differentiation toward neural cells from pESCs can be a useful tool for the study of early neural development and neurodegenerative disorders. In addition, these cells can also be used in cell replacement therapies and drug development for neuroprotective and/or neurotoxic reagents. Although several studies reported the successful isolation of pES-like cells, it has been a big challenge to determine optimal conditions to generate pESCs without loss of pluripotency for a long time. The present study was performed for generation and characterization of putative pESCs, and differentiation into neurons and astrocytes. In this study, porcine blastocysts were produced by parthenogenetically activated oocytes. The putative pESCs were cultured in pESC growth media supplemented with a growth factor and cytokines (bFGF, LIF and SCF). Subculture of pESCs was conducted by mechanical dissociation using syringe needles after 4-5 days of incubation. As results, six putative pESC lines were maintained over thirty passages. The putative pESCs were compact, round, flat, and single layered, which were similar to human embryonic stem cell morphologically. Six pES-like cells were positive for alkaline phosphatase activity at every three passages. Furthermore, Oct-3/4, Sox-2, Nanog and SSEA-4 were shown to be expressed in those cells. Also, normal karyotypes of pESCs were observed by Giemsa-staining. Differentiation potential into the three germ layers of the putative pESCs was demonstrated by the formation of embryoid bodies (EB). Besides, the study of ESC is very important in aspect of its application to not only the cell-based replacement therapies but also cellular differentiation research. Our results also showed that RA and N2 supplements activated the neural differentiation in pESC5. Neurofilament-l60 were expressed in neural precursor cells. The expression of markers for specific neural lineages, such as Microtubule-associated protein-2 expressed in matured neuron, was also induced from embryonic neural progenitors. In summary, the pESCs were generated from the parthenogenetically activated blastocysts and the typical characteristics of the cells were maintained for the long term culture. Furthermore, it was successful to differentiate the pESCs into various neural lineages through in vitro neurogenesis system. Eventually, pESCs will be excellent biomedicine in incurable and/or zoonotic diseases by regenerating the damaged tissue.

Porcine blastocyst’s quality derived from in vitro is inferior to in vivo derived blastocysts. In this study, to improve in vitro derived blastocyst’s quality and then establish porcine ESCs (pESCs), we treated in vitro fertilized (IVF) embryos and parthenogenetic activated (PA) embryos with three chemicals: porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), resveratrol (RES) and β-mercaptoethanol (β-ME). The control group was produced using M199 media in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The treatment group is produced using M199 with 2 μM RES in IVM and PZM5 with 10 ng/mL pGM-CSF, 2 μM RES and 10 μM β-ME in IVC. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. In total, 1210 embryos in PA and 612 embryos in IVF evaluated. As results, we observed overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (p<0.05) in the treatment groups (54.5%) compared to the control group (43.4%) in PA and hatched blastocysts rates in day 6 and 7 were also increased significantly. Total cell numbers of blastocyst were significantly higher (p<0.05) in the treatment group (55.1) compared to the control group (45.6). In IVF, hatched blastocysts rates in day 7 were increased significantly, too. After seeding porcine blastocyst, the attachment rates were higher in the treatment group (36.2% in IVF and 32.2% in PA) than the control group (26.6% in IVF and 19.5% in PA). Also, colonization rates and cell line derivation rates were higher in treatment group than control group. Colonization rates of control group were 10.8% in IVF and 2.4% in PA, but treatment group were 17.75% in IVF, and 13.1% in PA. And we investigated the correlation between state of blastocysts and attachment rate. The highest attachment rate is in hatched blastocyst (78.35±15.74 %). So, the novel system increased quality of porcine blastocysts produced from in vitro, subsequently increased attachment rates. The cell line derivation rates were 4.2% (IVF) and 2.4% (PA) in control group. In treatment group, they were 10.0% (IVF) and 7.2% (PA). We established 3 cell lines from PA blastocysts (1 cell line in control group and 2 cell lines in treatment group). All cell line has alkaline phosphatase activity and express pluri-potent markers. In conclusion, the novel system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of porcine blastocysts produced from in vitro, subsequently increased derivation rates of porcine putative ESCs.

Pig embryonic stem cells (ESC) has been suggested to become important animal model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, the quality of cloned embryo and derivation rate of cloned blastocyst has been presented limits for derivation of cloned embryonic stem cell. In this study, we have tried to overcome these problems by aggregating porcine embryos. Zonafree reconstructed SCNT Embryos were cultured in micro-wells singularly (non-aggregated group) or as aggregates of three (aggregated groups) at the four cell stage. Embryo quality of the cloned embryos and attachment on feeder layer rate significantly increased in the aggregates. The aggregation of pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of pig cloned blastocyst and improvement in the percentage of attachment on the feeder layer of cloned embryos. * This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of Tuj1 increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous Igf2 may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.