Introduction The objective of this study was to investigate the effects of staining of porcine cumulus-oocytes complexes (COCs) by brilliant cresyl blue (BCB) test prior to in vitro maturation may be used to select developmentally competent oocytes. Furthermore, milrinone can be used to promote developmental competence of porcine embryos produced during parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). Materials and Methods Slaughterhouse-derived porcine cumulus-oocyte-complexes (COCs) were exposed to BCB and treated oocytes divided into BCB+ (colored cytoplasm), BCB- (colorless cytoplasm) groups. After division into 2 groups, intracellular glutathione (GSH) and reactive oxygen species (ROS) of matured oocytes were compared. And, preimplantation development of PA and SCNT embryos were also compared between 2 groups. BCB- oocytes were exposed to milrinone with different concentrations (0, 50, 75, and 100μM) for 6 h prior to IVM for further development of embryos. Results and Discussion GSH was higher in BCB+ group than BCB- group whereas ROS was lower in BCB+ than BCBgroup. In parthenogenetic embryos, BCB+ oocytes group was significantly higher on maturation (87.5 vs 80.6, 71.3%), cleavage (88.6 vs 82.9, 76.3%), and blastocyst formation rates (34.3 vs 27.8, 25.3%) than control and BCB- oocytes groups, respectively. Moreover, ratio of ICM:TE cells were higher in BCB+ oocytes group (30.3% vs. 28.6, 26.4%, respectively) than other groups. In cloned embryos, the significant higher blastocyst formation rates were shown BCB+ groups (30.6% vs. 26.0, 20.1%) than BCB- groups. To improve the cytoplasmic maturation in BCB- oocytes, 4 different concentrations of milrinone (0, 50, 75, and 100μM) were supplemented in the IVM media for 6 h. BCB- oocytes supplemented with 75μM milrinone showed the significantly higher rates of blastocyst formation than other groups. Our results demonstrate that staining of porcine oocytes with BCB before IVM may be used for selection of good quality oocytes and milrinone supplementation can be used to improve embryo developmental competence of porcine embryos.

The nature of molecular mechanisms governing embryo development is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to examine the mode of programmed cell death during nuclear transfer embryos development in porcine. In particular, the relative employment of two major pathways in programmed cell death; e.g. apoptosis (type I) and autophagy (type II) was compared. Oocytes use in the study was matured in vitro in the presence of 10% FBS maturation medium. After nuclear transfer embryos were cultured for each programmed cell death control factor [Cysteamine(Cyst : 0.4mM), 3-methyladenine(3MA : 2.5mM) and Rapamycin(RP : 100nM)] in TCM-199 medium supplemented with 0.1% BSA. In this study results of among the blastocysts development in 3MA; PCNA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of IVF<Cyst < 3MA < RP. However Casp-3 and TNF-r RNA gene expression level decreased in the order of IVF < 3MA and RP< Cyst. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. And next experiments analysis of MMP expression patterns. Analysed this MMPs enzyme activation to evaluate the effectiveness of high quality brastocyst culture in porcine. In this results of the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each 3MA and RP treatment group, with the level of another treatment group being relatively higher. These results suggest that the autophagy activation culture medium is more effective for stable and innovative nuclear transfer embryos development.

Growth differentiation factor8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. Overall of the current studies, the GDF8 is detected in oviduct fluid and uterus which led us to suggest that the GDF8 may effect on preimplantation embryonic development and act paracrine role to correlate with successful late-blastocyst implantation in in vivo. The purpose of this study is the effects of GDF8 on porcine parthenogenesis (PA) embryo development during in vitro culture (IVC). We were investigated the effect of GDF8 supplement during PA embryo IVC by cleavage and blastocyst formation rate and patterning analysis. Data were analyzed by on way ANOVA, followed by Tukey’s range test. Respectively 0.2, 2 and 20 ng/mL of GDF8 were added during IVC followed experiment design as control, 0.2, 2, and 20 GDF8 supplement groups. After 48h of embryo culture time, no significant difference was observed on cleavage rate from the different concentration (0, 0.2, 2, and 20 ng/ml) of GDF8 supplement groups (65.7%, 66.0%, 66.3%, and 65.8%, respectively). After 120h of embryo culture time, the 0.2 and 2 group showed significantly (p<0.05) higher blastocyst formation rate than control (40.4% and 36.4% VS 40.4%, respectively). In embryo developmental pattern analysis, the 0.2 ng/ml GDF8 supplement groups showed significantly higher (p<0.05) 2-3 cell cleavage- and early blastocyst pattern compared with control (12.0% and 10.4% VS 6.6% and 6.2%, respectively). However there are no significantly different pattern was observed in other groups. In conclusion, the 0.2 ng/ml of GDF8 supplementation during porcine PA embryo IVC significantly changed embryonic developmental patterns. However there are further studies are required such as analysis of blastocyst total number, specific gene transcription pattern, and ICM/TE rate to make clarify and support the conclusion.

The use of pigs in neuroscience has increased over the past years because the pigs are closely related to humans in terms of anatomy and physiology. Especially, the blood-brain barrier (BBB) maintains the homeostatic microenvironment in the central nervous system (CNS) and they can provide a valuable tool for studying the neurobiology. However, only a few putative blood-brain barrier (BBB) models have been generated by co-culture of porcine primary cells. The fundamental problem is that they lose some of their phenotypes when maintained in vitro for long-term culture. To establish improved in vitro porcine BBB models, we differentiated novel brain microvascular endothelial cells (BMECs) from porcine induced pluripotent stem cells (iPSCs) using a modified human-based protocol. Briefly, the dissociated single cells from iPSCs were seeded in Geltrex. For differentiation, cells were maintained for 3 days of expansion and then switched to unconditioned medium (UM) lacking bFGF for 6-7 days. Then, we subcultured cells onto collagen/fibronectin coated plates and changed BMEC medium for 2-3 weeks. About two weeks later, we observed a cluster of round cells surrounded by spindle shaped adherent cells termed as colony-forming units (CFU) of putative BMECs. Over time, the cluster of cells disappears and remained adherent spindle-shaped cells showed properties of endothelial cells. Although further studies will be needed, this study would be a great comparative analysis of the porcine and human in vitro BBB model.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated a specific gene transcription levels in oocytes and cumulus cells (CC) after IVM by realtime PCR arry, and specific protein expression and activation levels in matured CCs by western blotting. Each concentration (0, 1, 10, and 100 ng/ml) of GDF8 was added in maturation medium (TCM199) during process of IVM. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science). Data are presented as the mean and Differences were considered significant at P < 0.05. After 44 h of IVM, oocytes are mechanically denuded from CCs with 0.1% of hyaluronidase, and then the separated oocytes and CCs were sampled following each group. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, the realtime PCR array was performed. In CCs the 1- and 10 ng/ml of GDF8 supplement group showed the transcription co-factors CBP and SP1, cell metabolic regulator MAPK1, and cumulus expansion related genes Has2, Cox-2, Ptx3 and Areg transcription levels were significantly distinguished with control when hierarchically clustered by Euclidean distance with average linkage method after IVM. In matured oocytes the 10- and 100 ng/ml of GDF8 supplement group showed the maternal factors JMJD3 and Zar1, transcriptional regulator FOXO1, Sirt1 and Sirt2, mitochondrial activity factor Sirt3, ACSL3 and ACADL, anti-apoptosis gene BCL-2, and oocyte secrete factor BMP15 mRNA transcription levels were significantly distinguished compared with control. To determine effect of GDF8 supplement during IVM, the GDF8 down steam canonical regulator SMAD2/3 protein phosphorylation levels analyzed in CCs by western blotting. The 10- and 100 ng/ml supplement groups showed significantly increase phosphorylated (P)-SMAD3 (1.56 and 1.34 times higher than control) protein levels (P < 0.05). In conclusion, supplement of GDF8 during IVM activates FOXO homolog transcription and induced cumulus cells expansion via activation of SMAD3 signaling in CCs. While process of IVM, the transcriptional landscape changes in CCs may consequently result maternal factors accumulation and mitochondrial activation in oocytes.

Little is known to date about neural development of pig and directed differentiation of porcine pluripotent stem cells (PSCs) to neuronal cells remains elusive. To determine whether soluble factors from glioblastoma multiforme (GBM) promoted the neural differentiation from porcine induced PSCs (iPSCs), cells were treated cultured media of GBM cells. First of all, we isolated and established primary GBM cell line (WHO grade IV). The cellular morphology of GBM cancer cell line are dendritic-like with positive expression in NESTIN, SOX2, VIMENTIN and GFAP using immunofluorescence analysis. G-banded karyotype from primary GBM cell line revealed severe numerical chromosomal aberrations. GBM-cultured medium (CM) treated iPSC-NPCs survive well in vitro when supplemented with a combination of growth factors, including EGF and bFGF. The GBM-CM treated differentiated cells showed an increased mRNA expression level of astrocyte marker, GFAP and the dopaminergic neuron marker, tyrosine hydroxylase (TH). However, there was no significant difference in mRNA expression level of oligodendrocyte marker, MBP. The protocol developed in the present study for large animal models might provide an exciting tool to bridge the present gaps in neuroscience studies between rodents and humans.

This study was conducted to investigate the effect of activation method on the endoplasmic reticulum (ER) stress induction, apoptosis and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by four activation methods; 1) electric stimulus (ES) (E), 2) ES+10 μM Ca-ionophore (A23187) treatment (EC), 3) ES+2 mM 6-dimethylaminopurine (6-DMAP) treatment (ED), or 4) ES+A23187 and 6-DMAP treatments (ECD). Parthenogenetic embryos were sampled to analyze x-box binding protein 1 (Xbp1) mRNA, ER stress-associated genes and apoptosis genes at 3 h after ES and the 1-cell and blastocyst stages. In the EC group, the band intensity of spliced Xbp1 (Xbp1s) mRNA was higher than those of the other groups at the 3 h and 1-cell stage, and higher than that of the E group at the blastocyst stage. Four ER stress-associated genes were expressed at the highest level in the EC group and weakly expressed in the ED group at 3 h after activation. However, most of the genes were highly expressed at the 1-cell and blastocyst stages with some variation in the EC and ECD groups. Expression of Bcl-2-associated X protein (Bax) and caspase-3 mRNA was significantly higher in the EC group than in the other groups at all development stages. The developmental rates to the blastocyst stage were higher in the ED and ECD groups than in the E and EC groups. These results suggest that the intracellular ER stress of parthenogenetic porcine embryos is affected by the activation method and subsequently lead to the apoptosis of embryos.

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 β- galactoside α -2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a (10 μM) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

The present study was aimed to determine the effect of green tea extract (GTE) and beta-mercaptoethanol (β-ME) supplementation in boar sperm freezing extender on sperm motility, viability and reactive oxygen species (ROS) level. Experimental groups were allocated into Lactose-egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/L GTE in LEY) and β-ME (50 μM β-ME in LEY). Spermatozoa extended with LEY were cooled to 5°C for 3 h and then kept at 5°C for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM (final sperm concentration: 1 × 108/mL). Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. Following thawing at 37°C for 25 sec, sperm viability and ROS level were measured using fluorescent double stain Fertility® and cytometry, respectively. Motility and viability of GTE supplemented-group were higher than those of control and β-ME without significance. ROS level in GTE group showed significantly lower than control (P < 0.05). In conclusion, GTE supplementation in boar sperm freezing extender can reduce ROS generation during freezing.

CD26, also known as Dipeptidyl peptidase IV (DPP-4), is a cell surface glycoprotein that belongs to the serine protease family and has wide spread organ distribution throughout the body. CD26 was previously characterized in immune cells but also has important metabolic functions which are not yet fully understood. Thus, we investigated the effect of CD26 in porcine parthenogenetic embryos. We attempted CD26 downregulation of porcine embryos by siRNA, and evaluated CD26 suppression of developmental competencies. Although the porcine embryos injected with CD26 siRNA were able to develop to the early stage, these embryos were decreased to form blastocysts. Our results indicated that CD26 is one of factors for the regulation of development of porcine embryos.

The coronavirus porcine epidemic diarrhea virus (PEDV) infects the cells lining the small intestine of a pig and, causes porcine epidemic diarrhea (PED). Owing to its highly infectious nature, PEDV has a substantial economic burden, which results in significant morbidity and mortality in piglets. In this study, the virucidal efficacy of a powder disinfectant containing a phosphate compound against PEDV was investigated. Virucidal efficacy was assessed as the infectivity of PEDV toward Vero cells after exposure of the virus to the disinfectant. PEDV was exposed to the disinfectant in the presence of either hard water (HW) or an organic matter suspension (OM). In the HW condition, PEDV was inactivated by 4-fold dilution of the disinfectant. In the presence of OM, the disinfectant showed virucidal activity with a 2-fold dilution. As the disinfectant possessed virucidal activity against PEDV, it should be an effective reagent for limiting the spread of animal viral diseases.

The aim of this study was to investigate change of plasminogen activators (PAs) and their inhibitors (PAIs) mRNA and protein expression level by heat stress in porcine endometrial cells. The endometrial epithelial cells were isolated from endometrial epithelium in porcine uterus and cultured in different temperature conditions (38.5 and 41.5℃) for 24 h. Expression of urokinase-type PA (uPA), tissue-type PA (tPA), PA inhibitor-1 (PAI-1) and -2 (PAI-2) mRNA in epithelial cells were analyzed using reverse transcription-PCR and protein levels were measured by immunofluorescence. In result, mRNA expression of uPA, tPA, PAI-1 and PAI-2 were decreased in 41.5℃ than 38.5℃ culture condition, however, significant differences were no detected. uPA, tPA and PAI-2 protein were mainly expressed in nucleus, whereas PAI-1 was distributed in cytoplasm and nucleus. uPA and tPA protein levels were increased by heat stress treatment and significant difference was only detected in tPA level (p<0.05). In contrast, two types of PAIs protein level were decreased in 41.5℃ cultured group compared with 38.5℃ group. In present study, tPA protein expression was upregulated by heat stress in porcine endometrial cells. This result suggest that change of tPA by heat stress may be related to blood flow into uterus and intrauterine microenvironments, and could directly and indirectly influence to reproductive performance in pigs.

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Endostatin is 20 KDa C-terminal fragment derived from type XVIII collagen and an endogenous inhibitor of tumor growth by inhibition of angiogenesis. In this study, we are developed knock-in vector consists of 5’ arm region (1.02 kb), human Endostatin cDNA, CMV-EGFP, and 3’ arm region (1.83 kb). To express Endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of Endostatin gene and inserted into exon 3 of the β -casein gene. If this knock-in vector is inserted into the porcine β-casein gene locus by homologous recombination, human Endostatin mRNA are expressed using the gene regulatory region of the β-casein. Also, the β-casein and Endostatin fusion protein is translated and Endostatin protein is separated by F2A self cleavage during translation. In conclusion, our knock-in vector may help to create transgenic pig expressing human Endostatin protein via the endogenous expression system of the porcine β-casein gene in the mammary gland.

CRISPRs(clustered regularly interspaced short palindromic repeats) / CRISPR - associated(CAS) system has been used genome editing technology. Genome stage modification using CRISPR/CAS9 system can be used to wide research for the gene functional study and therapeutics. However, improving of CRISPR/CAS9 system in efficiency is essential for application in various fields. Here, we treated various chemicals during the procine early embryo development to increase the mutation of target site by NHEJ(non-homologous end joining). Firstly, we confirmed the chemical toxicity after parthenogenetic activation and then check embryo puality using by counting of total cell number and TUNEL Assay in blastocyst satge. To check any improvement on mutation rate by NHEJ pathway. AZT(3′-Azido-3′-deoxythymidine, antiretroviral drug – 0.1 μM) was treated after injection of cas9 and sgRNA target to OCT4 exon 5 during the zygote stage, followed by PCR sequencing. As a result, AZT treated group shows a significantly increased in knock-out efficiency as a consequence of NHEJ. Nocodazole(anti-neoplastic agent – 200ng/ml), RO-3306 (specific inhibitor of CDK1 - 10 μM) and NU-7026(PKC signalling inhibitor - 50 μM) was treated after injection of cas9 and sgRNA with eGFP vector during the zygote stage(hpa8~hpa20) and checked a efficiency of knock-in by PCR sequencing. Interestingly, nocodazole treatment groups increased of insertion of eGFP sequence in blastocyst stage compared with non-treat group(control : 8.33%, nocodazole treatment : 16.67%). However, RO-3306 and NU-7026 made a no impact. In summary, CRISPR/CAS9 system with treatment of chemicals during porcine embryogenesis can be improving of site-specific mutation and enhancement of CRISPR genome editing.

Abnormal epigenetic reprogramming of donor nuclei is supposed to be one of the factors that causes low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone acetylase, and so development of SCNT embryos could be increased by treatment with TSA. In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from NT (nuclear transfer) by investigating the status of histone acetylation in TSA-treated and control NT embryos and the expression of developmental related genes. In this study, we found that incubating　NT embryos with 40nM TSA for 24h after activation could improved the blastocyst formation rate from 13.7% to 32.5%. Thechange in histone acetylation level as a reslut of TSA treatment were validated using immunofluorescence and confocal microscopy. Immunofluorescence results indicated that the level of aetylation at histone 3 lysine 18 (AcH3K18) was increased at early embryo development stage after TSA treatment. furthermore, we compared the expression patterns of several genes (developmental related genes; Oct4, Sox2, Nanog, Cdx2, the imprinting genes; igf2r). TSA treatment improved the expression of development related genes such as Oct4, Cdx2, Nanog as well as the imprinted genes like igf2r. In conclusion, our results demonstrated that TSA treatment improves the in vitro development of porcine NT embryos, increased the global histone acetylation (AcH3K18) and enhances the expression of some developmentally important genes (Oct4, Cdx2, Nanog) at blastocyst stages.

RNA Sendai virus (SeV) vector system has no risk of being integrated into the host genome. Sendai virus (SeV) vectors expressing pluripotent factors have been used to produce integration-free induced pluripotent stem cells (iPSCs) with high efficiency from various cell types in human and mouse. In this study, we generated iPSCs from pig ear fibroblast cells using the SeV vector expressing 4 human factors (POU5F1, SOX2, C-MYC, and KLF4). Colonies were emerged at Day 14 of transduction and expressed the classical pluripotency markers (POU5F1, NANOG, and SOX2) and surface marker (SSEA1). Furthermore, they showed a domed shape and could passage over 40 times under 2i (CHIR99021 and PD0325901)-LIF and MEF feeder culture condition having in vitro differentiation ability into 3 germ layers. Next, we examined the ability of six feeder free culture conditions to maintain piPSCs in a pluripotent state. piPSCs were plated on Matrigel coated dishes in different media: 1. CM: control media (LIF culture media); 2. CM-F: CM+100 ng Fetuin-A; 3. CM-N: CM+100 ng Nanog-TAT; 4. CM-2i: CM+3 uM CHIR99021+1 uM PD0325901; 5. CM-2iN: CM-2i+100 ng Nanog-TAT; 6. CM-2iN+100 ng Fetuin-A. However, piPSC could not maintain the typical self-renewal morphology on feeder free conditions regardless of culture media tested here. Further, expression of pluripotency-related genes (Oct4, Nanog and Klf4) of piPSCs cultured on feeder free conditions could not be compared with that of iPSCs cultured on MEF feeder plate. Our results suggest that integration free pluripotent stem cell from pigs could be generated by SeV vector system and maintained their pluripotency under 2i-LIF and MEF feeder culture condition, but further optimization of culture conditions may be required.

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.

노화와 재해 등으로 인한 인간의 치명적인 간 부전 문제를 해결하기 위한 대안 중의 한 축으로 교차분화 기법을 적용한 induced hepatocyte (iHep)연구가 진행되고 있지만, 분화효율은 극히 낮다. 본 연구에서는 돼지 섬유아세포에 교차분화기법을 사용하여 piHep을 만들고, 소분자 화합물(A83-01)의 첨가에 따른 교차분화의 효율개선을 시도 하 였다. piHep의 유도를 위해서 세가지 간 전사 인자를 [human hepatocyte nuclear factor 1 alpha (hHNF1A), human hepatocyte nuclear factor 4 alpha (hHNF4A), human forkhead box protein A3 (hFOXA3)]를 렌티바이러스를 이용하여 체세포에 도입하였다. 3가지 유전자가 도입된 세포는 기본 배양액으로 hepatocyte induction medium (HIM)을 사용하였고, 여기에 A83-01을 첨가군과 미 첨가군(대조군)으로 나누어 교차분화를 유도 하였다. 그 결과 대조군에서 4주 후에 불완전한 간세포의 형태가 낮은 빈도로 나타났지 만, A83-01 처리군은 도입 2주 후부터 epithelial-like cell의 형성을 통해 점점 다핵을 가 지는 성숙한 piHep(명확한 핵, 다각형의 세포질)의 특성이 확인되었다. 간세포로의 기능 적 분화를 검증하기 위해 albumin (ALB)과 transferrin (TF)의 mRNA의 발현을 real-time PCR을 통해 분석하였다. A83-01 처리군에서 ALB와 TF의 발현이 교차분화 4 주차까지 증가되었다. 섬유아세포 표지 인자인 vimentin의 발현은 A83-01의 처리와 상관 없이 모두 감소되었다. 대조군과 달리 A83-01처리군은 piHep의 유도를 위해 사용된 3가 지 간 전사인자들 중 HNF4Α와 FOXA3에서 교차분화 후 4주차까지 endogenic expression 을 보였다. 결론적으로 간세포 교차 분화의 효율 개선을 위해 사용된 A83-01은 돼지 섬 유아세포의 내재된 간 특이적 전사 인자들을 활성화시켜 간세포의 분화를 촉진함을 확인 할 수 있었다.

Due to their anatomical, physiological and genetic similarities, pig is attractive animal model in biomedical research. In the recent stem cell research era, porcine derived stem cells also gain attention due to its use for the preclinical application of human. Mesenchymal stem cells (MSCs) have been studied by many researchers over decade, and their prospect for clinical application is recognized. Although porcine derived MSCs (pMSCs) have confirmed to be differentiated into various types of cells, such as osteocyte, chondrocyte, neuronal cell, cardiomyocyte and pancreatic β cell, few report has been studied regarding hepatocyte differentiation in vitro. The present study was therefore aimed for bone marrow MSCs derived from pig femur to differentiate into hepatocyte. The cells were confirmed as MSCs by characterizing their morphology, lineage differentiation capacity and surface phenotype. They showed spindle like morphology and adipocytic, osteoblastic, and chondrocytic differentiation potentials and displayed positive expression of mesenchymal markers CD29, CD44 and CD90 while lacked the expression of hematopoietic marker CD45. Under appropriate differentiation conditions, MSCs displayed hepatocyte-like morphology depending on duration of differentiation. The differentiated MSCs into hepatocyte expressed hepatocyte-specific genes including hepatocyte nuclear factor 4 (HNF4), albumin (ALB), alpha fetoprotein (AFP), alpha-1-anti trypsin (A1AT). They also showed hepatocyte-like function, glycogen storage which is identified by PAS staining. Taken together, it concluded that the bone marrow MSCs have the potential to differentiate into hepatocyte. Further studies are needed on additional hepatocytic functional assays, such as low density lipoprotein (LDL) uptake and urea synthesis of differentiated MSC.

In all mammalian species, progesterone is essential in the preparation for and maintenance of pregnancy, if it occurs. Progesterone primes the endometrium for possible implantation and inhibits uterine contraction until birth. Aldo-keto reductases (AKRs) belong to a superfamily of NADPH-dependent reductases that act on a wide range of substrates, including simple carbohydrates, steroid hormones, and endogenous prostaglandins. 20-alpha hydroxysteroid dehydrogenase (20α-HSD; EC.1.1.1.149) enzyme belongs to the family of aldo-keto reductases. 20α-HSD predominantly converts progesterone into its biologically inactive form 20α-hydroxyprogesterone (20α -OHP), and plays a crucial role in the termination of pregnancy and initiation of parturition. In addition, the activity of 20α-HSD during the luteal phase is known to be inhibited by prolactin. We have been reporting on the molecular characterizations of placental and ovarian 20α-HSD in the bovine, pig, deer and monkey. In this study, we focused on the 20α-HSD expression in testis(6, 9, 12, 18 and 21 days after birth) of miniature pig. The protein expression and localization were detected by Western blotting and Immunohistochemical analysis. 20α-HSD protein was detected at molecular weight of 37-kDa by Western blot analysis. Also the RNA expression were detected by Reverse Transcription-PCR and quantification PCR. Additionally, We are going to analysis the function and role of 20α-HSD in the pig testis.