Cytological analysis of pregnancy-associated plasma protein-A expression in porcine neonatal testis

Ji-youn Kim,Keon Bong Oh,Sung June Byun,Sun-A Ock,Hwi-Cheul Lee,황성수,SangHyun Park,Wootae Ha,Jae-Seok Woo,Hyuk Song 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.3

The identification of biomarkers of a living tissues is essentially required to understand specific functions of the cells. In previous study, we reported IGFBP 3 as one of the putative biomarkers, by showing specific expression at porcine spermatogonial stem cells (SSCs) of early stage of porcine testis. In this study, we analyzed the expression of seven members of IGFBP family (IGFBPs) in SSCs and histological expression pattern of pregnancy-associated plasma protein-A (PAPP-A), which plays a role on the growth promoting enzyme by cleavage of IGFBPs in testis of 5 days old pig. RT-PCR analysis showed that IGFBP 1, 2, 3, 4, and 6 were expressed at high level specifically in porcine SSCs compared with whole testis. We performed immunohisotochemical staining of testis sections with PAPP-A and protein gene product 9.5 (PGP9.5) which are the known biomarkers for SSCs. We were not able to find co-expression of PAPP-A and PGP9.5; PAPP-A was expressed only in Sertoli cells and PGP9.5 expression was confirmed in spermatogonium. Additionally, we were able to confirm the GATA4 expression in Sertoli and Leydig cells as a regulator of Sertoli cell function was not detected PGP9.5 expressing cells, indicating indirect evidence of that cytolocalization of PAPP-A expression is limited in Sertoli cells. These results suggested that the PAPP-A expressed in Sertoli cells may play role on regulation of development and differentiation of testicular cells through the IGF axis in neonatal porcine testis.

Evaluation of phenotypic, functional and molecular characteristics of porcine mesenchymal stromal/stem cells depending on donor age, gender and tissue source

OCK, Sun-A,LEE, Yeon-Mi,PARK, Ji-Sung,SHIVAKUMAR, Sharath Belame,MOON, Seon-Woung,SUNG, Nak-Ju,LEE, Won-Jae,JANG, Si-Jung,PARK, Ju-Mi,LEE, Seung-Chan,LEE, Sung-Lim,RHO, Gyu-Jin JAPANESE SOCIETY OF VETERINARY SCIENCE 2016 The Journal of veterinary medical science Vol.78 No.6

<P>The biological properties of mesenchymal stem cells (MSCs) are influenced by donor age, gender and/or tissue sources. The present study investigated the cellular and molecular properties of porcine mesenchymal stromal/stem cells <B>(</B>MSCs) isolated from different tissues (adipose & dermal skin) and sex at different ages (1 week & 8 months after birth) with similar genetic and environmental backgrounds. MSCs were analyzed for alkaline phosphatase (AP) activity, CD90 and Oct3/4 expression, <I>in vitro</I> differentiation ability, senescence-associated <I>β</I>-galactosidase (SA-<I>β</I>-Gal) activity, telomeric properties, cell cycle status and expression of senescence (IL6, c-myc, TGFβ, p53 and p21)- and apoptosis (Bak and Bcl2)-related proteins. An age-dependent decline in AP activity and adipogenesis was observed in all MSCs, except for male A-MSCs. CD90 expression did not change, but SA<I>-β</I>-Gal activity increased with advancement in age, except in A-MSCs. Telomeric properties were similar in all MSCs, whereas expression levels of Oct3/4 protein declined with the advancement in age. p21 expression was increased with increase in donor age. Male derived cells have shown higher IL6 expression. The expression of p53 was slightly lower in MSCs of dermal tissue than in adipose tissue. Bak was expressed in all MSCs regardless of age, but up regulation of Bcl2 was observed in DS-MSCs derived at 1 week after birth. In conclusion, adipose tissue-derived MSCs from young female individuals were found to be more resistant to senescence under <I>in vitro</I> culture conditions.</P>

Small Molecules-Driven Pancreatic ß-Cells Differentiation of Bone Marrow Mesenchymal Stem Cells

Imran Ullah,Keon Bong Oh,Yurianna Shin,Youngim Kim,Sun A Ock 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

Diabetes mellitus, a hyperglycemic condition, in which the patients either fail to secrete insulin because of ß-cells destruction (type I) or shows insulin resistance (type II), affecting more than 300 million people around the globe. Generation of effective ß-cells for the treatment of diabetes is a key area in modern translational medicines. Here we tried to generate porcine induced pancreatic ß-cells (piPan ß-cells) from Gal- TKO+MCP porcine bone marrow mesenchymal stem cell (pBM-MSCs) by overexpressing set of transcription factors included EGFP along with step-wise induction of small molecules. pBM-MSCs were cultured in media with 5-azacytidine for 24 hours and then proceeded with transfection using episomal transfection system. Transfected cells were replaced with basal media containing growth factors cocktail, Activin A and 2 uM of A83-01 followed by retinoic acid treatment. Finally, cells were replaced by maintenance media until maturation. After 3 days of transfection, EGFP expression was observed showing successful transfection which disappeared completely after 21 days. pBM-MSCs attained definitive endoderm morphology after Activin A treatment and formed exact pancreatic clusters after 4 weeks and 8 weeks in maintenance media, respectively. After 4 and 8 weeks, piPan ß-cells were analyzed for the expression of pancreatic markers by immunofluorescence (Insulin, PAX6, Somatostatin) and RT-qPCR (GCG, INS, NKX6.1, PDX1 and NEUROD1), which showed significantly higher expression as compared with pBM-MSCs. Morphological changes and expression of pancreatic markers revealed successfully differentiation of PiPan ß-cells; however, more in depth studies for its functional characteristic and in vivo maintenance are still in progress.

In Vitro Culture of Rat Primary Hepatocytes Employing Monolayer and Spheroid Culture Environments

Imran Ullah,Yeongji Kim,Malgum Lim,Keon Bong Oh,Seongsoo Hwang,Yurianna Shin,Youngim Kim,Gi-Sun Im,Tai-Young Hur,Sun A Ock 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

Primary hepatocytes (PH) are considered as “gold standard” for drug screening because of its ability to express the entire set of drug metabolizing enzymes and transporters. Hepatocytes culturing and maintaining their hepatocyte fate in vitro is one of the major issue from last decade. The main problem in hepatocytes in vitro culturing is that, these cells rapidly loss their hepatic morphology and liver specific function in culture condition. In the present study, we isolated rat PH and cultured in monolayer (2D) as well as spheroid (3D) culture system. The 2D cultured PH hepatocyte showed an elongated hepatocyte morphology while, 3D cultured PH showed spheroid morphology with gradual decrease in diameter up to 7 days. After 7 days of in vitro culture, these cells were analyzed for the expression of hepatic markers (Alb, Tf, Afp) and apoptotic markers (Bax, Bcl2). Furthermore, PH in both culture systems was induced with two inducers i.e. 3-methylcholanthrene (3-MC, Cyp1a specific inducer) and dexamethasone (Cyp3a specific inducer) for 48 and 72 hours, respectively. The mRNA level of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PH, respectively. After 7 days in vitro culture, PH showed dramatic down regulation of hepatic markers in both culture systems. Furthermore, expression of apoptotic markers was higher in 2D cultured PH as compared to 3D. Cyp1a and Cyp3a mRNA level showed higher RNA content in 2D culture PH after 48 of induction. Therefore, we concluded that there was no significant difference found in two culture system and further studies are needed to find out the essential components for PH in vitro culture rather than culture system.

Growth Rate of Transgenic Pigs and Size of Pig Hearts for Xenotransplantation to Cynomolgus Monkey

Ock, Sun A,Oh, Keon Bong,Hwang, Seongsoo,Lee, Jungkyu,Kim, Youngim,Moon, Sun-Woung,Kwon, Dae-Jin,Yun, Ik Jin,Park, Eungwoo The Korean Society of Embryo Transfer 2014 한국동물생명공학회지 Vol.29 No.4

To compensate for the critical shortage of human organs for allotransplantation, xenotransplantation studies using genetically modified pigs are being performed in Korea. Two types of pigs that are used are ${\alpha}1,3$-galactosyltransferase gene knockout (GalT KO) pigs and GalT KO+hCD46 (human complement regulatory protein) pigs. The present study measured the gestation time, birth weight, daily growth rate, and heart weight of both kinds of transgenic minipigs. The gestation period for both types of pigs was 117~119 days. There was no difference in the body weight of GalT KO (-/+) and GalT KO (-/-) piglets, but GalT KO+hCD46 ($-^{hCD46+}/+$) pigs were significantly heavier at birth than were GalT KO+hCD46 ($-^{hCD46+}/-^{hCD46+}$) pigs. During the first 10 weeks of life, the daily weight gain of GalT KO+hCD46 ($-^{hCD46+}/-^{CD46+}$) piglets, which are considered the optimal type for xenotransplantation, was 0.19 kg. The weight of hearts from GalT KO piglets up to two months of age was affected more by body weight than by age. Transgenic pigs showed no differences in gestation period or reproductive ability compared with normal pigs. These results comprise basic data that may be used in xenotransplantation studies and transgenic animal production in Korea.

Comparative Characterization of Porcine Mesenchymal Stem Cells Derived from Bone Marrow Extract and Skin Tissues

Ock, Sun-A,Jeon, Byeong-Gyun,Rho, Gyu-Jin Mary Ann Liebert 2010 Tissue engineering. Part C, Methods Vol.16 No.6

<P>Mesenchymal stem cells (MSCs) offer a great promise for regenerative medicine. Present study compared the characterization of porcine MSCs (pMSCs) derived from bone marrow extract with adult ear and fetal skin-derived cells on morphology, cell growth, alkaline phosphatase activity, proliferation ability, expression of cluster of differentiation (CD) markers (CD29, 45, and 90), cell cycle, protein and mRNA levels of Oct-4, Sox-2, and Nanog, and lineage differentiation ability. Skin-derived cells exhibited alkaline phosphatase activity and differentiation ability like pMSCs. pMSCs possessed a longer doubling time than skin-derived cells, and there was no difference in the ratio of G0/G1 phase between pMSCs and skin-derived cells. Except for CD29 and 90, all cells were found negative for CD45. Protein and mRNA expression of Oct-4, Sox-2 and Nanog were observed with similar intensity in all cells. Taken together, pMSCs and skin-derived cells revealed similar characteristics, and suggested the possible supportive role of skin-derived cells with MSCs for the regeneration of damaged tissues in cell-based therapies.</P>

Growth Rate of Transgenic Pigs and Size of Pig Hearts for Xenotransplantation to Cynomolgus Monkey

Sun A Ock,Keon Bong Oh,Seongsoo Hwang,Jungkyu Lee,Youngim Kim,Sun-Woung Moon,Dae-Jin Kwon,Ik Jin Yun,Eungwoo Park 한국수정란이식학회 2014 한국동물생명공학회지 Vol.29 No.4

To compensate for the critical shortage of human organs for allotransplantation, xenotransplantation studies using genetically modified pigs are being performed in Korea. Two types of pigs that are used are α1,3-galactosyltransferase gene knockout (GalT KO) pigs and GalT KO+hCD46 (human complement regulatory protein) pigs. The present study measured the gestation time, birth weight, daily growth rate, and heart weight of both kinds of transgenic minipigs. The gestation period for both types of pigs was 117∼119 days. There was no difference in the body weight of GalT KO (—/+) and GalT KO (—/—) piglets, but GalT KO+hCD46 (—hCD46+/+) pigs were significantly heavier at birth than were GalT KO+hCD46 (—hCD46+/—hCD46+) pigs. During the first 10 weeks of life, the daily weight gain of GalT KO+hCD46 (—hCD46+/—CD46+) piglets, which are considered the optimal type for xenotransplantation, was 0.19 kg. The weight of hearts from GalT KO piglets up to two months of age was affected more by body weight than by age. Transgenic pigs showed no differences in gestation period or reproductive ability compared with normal pigs. These results comprise basic data that may be used in xenotransplantation studies and transgenic animal production in Korea.

Effect of Dimethyl Sulfoxide (DMSO) on Cryopreservation of Porcine Mesenchymal Stem Cells (pMSCs)

Ock, Sun-A,Rho, Gyu-Jin SAGE Publications 2011 Cell transplantation Vol.20 No.8

<P>Dimethyl sulfoxide (DMSO), a commonly used cryoprotectant in cryopreservation procedures, is detrimental to viability of cells. In this view point, a comparative study was carried out to evaluate the effect of DMSO on porcine mesenchymal stem cells (pMSCs). We compared the viability, colony forming unit-fibroblast (CFU-F) assay, expression of Bak and Bcl2 genes, Bcl2 protein antigen, and CD90 in pMSCs cryopreserved with 5%, 10%, and 20% DMSO. pMSCs isolated from bone marrow were characterized by alkaline phosphatase activity and the expression of transcription factors, such as Oct 3/4, Nanog, and Sox2. The cells were then cryopreserved by cooling at a rate of -1C/min in a programmable freezer and stored in liquid nitrogen. The results of survival of pMSCs cryopreserved at 5% DMSO were comparable to control group (fresh pMSCs). The survival and the number of colonies formed in cryopreserved pMSCs were inversely proportional to the concentration of DMSO. The number of colonies formed in pMSCs cryopreserved with all concentrations of DMSO was significantly (p < 0.05) lower than the control group. An increased tendency for Bak and Bcl2 gene expression was noticed in cryopreserved pMSCs at 3 h postthawing compared to control group. There was a close resemblance in higher level of expression of CD90 between control and cryopreserved pMSCs. Because there was no considerable difference in the results of pMSCs cryopreserved at 5% and 10% DMSO, this study strongly suggests the use of 5% DMSO in cryopreservation of pMSCs as an alternative to conventional 10% DMSO.</P>

Trans-differentiation Induction of Human-mesenchymal Stem Cells Derived from Different Tissue Origin and Evaluation of their Potential for Differentiation into Corneal Epithelial-like Cells

Sun-Woung Moon,Hyeon-Jeong Lee,Won-Jae Lee,Sun-A Ock,Sung-Lim Lee 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.2

The trans-differentiation potential of mesenchymal stem cells (MSCs) is employed, but there is little understanding of the cell source-dependent trans-differentiation potential of MSCs into corneal epithelial cells. In the present study, we induced trans-differentiation of MSCs derived from umbilical cord matrix (UCM-MSCs) and from dental tissue (D-MSCs), and we comparatively evaluated the in vitro trans-differentiation properties of both MSCs into corneal epithelial-like cells. Specific cell surface markers of MSC (CD44, CD73, CD90, and CD105) were detected in both UCM-MSCs and D-MSCs, but MHCII and CD119 were significantly lower (P < 0.05) in UCM-MSCs than in D-MSCs. In UCM-MSCs, not only expression levels of Oct3/4 and Nanog but also proliferation ability were significantly higher (P < 0.05) than in D-MSCs. In vitro differentiation abilities into adipocytes and osteocytes were confirmed for both MSCs. UCM-MSCs and D-MSCs were successfully trans-differentiated into corneal epithelial cells, and expression of lineage-specific markers (Cytokeratin-3, -8, and -12) were confirmed in both MSCs using immunofluorescence staining and qRT-PCR analysis. In particular, the differentiation capacity of UCM-MSCs into corneal epithelial cells was significantly higher (P < 0.05) than that of D-MSCs. In conclusion, UCM-MSCs have higher differentiation potential into corneal epithelial-like cells and have lower expression of CD119 and MHC class II than D-MSCs, which makes them a better source for the treatment of corneal opacity.

Characterization of porcine mesenchymal stem cellscompare with somatic cells homo ear skin fibroblasts and hetero fetal skin fibroblasts

Sun-A Ock,Jae- Hyeon Cho,Gyu-Jin Rho 한국동물생명공학회(구 한국동물번식학회) 2007 발생공학 국제심포지엄 및 학술대회 Vol.2007 No.1