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ABSTRACT: The present study compared the efficiency of somatic cell nuclear transfer (SCNT) using porcine oocytes that were denuded of their cumulus cells at different maturation time. In pre-denuded group, the cumulus cells from cumulus-oocyte complexes (COCs) were removed at 29 hr post in vitro maturation (hpm) and followed by further culture for 12 hr. In control group, as a commonly followed procedure, cumulus cells were removed from COCs at 41 hpm. The majority of porcine oocytes at 29 hpm were observed in metaphase of the first meiotic division (MI). At 41 hpm, no significant (P>0.05) differences were observed in nuclear maturation and mitochondrial distribution of oocytes between pre-denuded and control groups. However, in pre-denuded group oocytes, metaphase II (MII) plate and spindle were located closely as 'adjacent' to the first polar body (PB1), resulting in an increased enucleation rates than in control group oocytes by blind enucleation method. Following SCNT and parthenogenesis (PA) using pre-denuded group and control group oocytes, no significant (P>0.05) differences were observed with respect to the development, total cell number, incidence of apoptosis and the expression profile of developmentally important genes (Pou5f1, Dnmt1, Dnmt3a, Igf2r, Bax, Bcl2 and Glut1) at the blastocyst stage. In conclusion, the removal of cumulus cells at 29 hpm in porcine oocytes increased the enucleation rates through proper positioning of PB1 without compromising the quality of SCNT embryos during preimplantation development. Hence, this could be a valuable strategy to improve the SCNT efficiency in a porcine model.
The Japanese journal of veterinary research 11/2012; 60(4):191-203. · 0.46 Impact Factor
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Jin Ho Lee,
Shin-Won Kim,
Uk-Kyu Kim,
Se Heang Oh,
Sang June-Kim,
Bong-Wook Park,
Jin-Hyun Kim,
Young-Sool Hah,
Deok Ryong Kim,
Gyu-Jin Rho,
Geun-Ho Maeng, Ryoung-Hoon Jeon,
Hee-Chun Lee,
Jong-Ryoul Kim,
Gyoo-Cheon Kim,
June-Ho Byun
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ABSTRACT: The purpose of this study was to generate tissue-engineered bone using human periosteal-derived osteoblasts (PO) and polydioxanone/pluronic F127 (PDO/pluronic F127) scaffold with preseeded human periosteal-derived CD146 positive endothelial-like cells (PE). PE were purified from the periosteal cell population by cell sorting. One of the important factors to consider in generating tissue-engineered bone using osteoprecursor and endothelial cells and a specific scaffold is whether the function of osteoprecursor and endothelial cells can be maintained in originally different culture medium conditions. After human PE were preseeded into PDO/pluronic F127 scaffold and cultured in endothelial cell basal medium-2 for 7 days, human PO were seeded into the PDO/pluronic F127 scaffold with PE, and then, this cell-scaffold construct was cultured in endothelial cell basal medium-2 with osteogenic induction factors, including ascorbic acid, dexamethasone, and β-glycerophosphate, for a further 7 days. Then, this 2-week cultured construct was grafted into the mandibular defect of miniature pig. Twelve weeks after implantation, the animal was sacrificed. Clinical examination revealed that newly formed bone was seen more clearly in the defect with human PO and PDO/pluronic F127 scaffold with preseeded human PE. The experimental results suggest that tissue-engineered bone formation using human PO and PDO/pluronic F127 scaffold with preseeded human PE can be used to restore skeletal integrity to various bony defects when used in clinics. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.
Journal of Biomedical Materials Research Part A 09/2012; · 2.63 Impact Factor
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ABSTRACT: This study compared the expression of genes involved in pluripotency, segregation of inner cell mass (ICM) and trophectoderm (TE), and primitive endoderm (PE) formation in porcine embryos produced by in vitro fertilization (IVF), parthenogenetic activation (PA), and nuclear transfer (NT) using either fetal fibroblasts (FF-NT) or mesenchymal stem cells (MSC-NT).
Blastocyst formation and total cell number were analyzed. The expression patterns of transcripts, including SRY-related HMG-box gene 2 (SOX2), reduced expression gene 1 (REX1/ZFP42), LIN28, caudal type homeobox 2 (CDX2), TEA domain family member 4 (TEAD4), integrin beta 1 (ITGB1) and GATA6 were assessed at the 4-8 cell and blastocyst stage embryos by real-time PCR.
Developmental rates to blastocyst stage and total cell number were higher in IVF and PA embryos than in NT embryos. But MSC-NT embryos had increased blastocyst formation and higher total cell number compared to FF-NT embryos. The relative expressions of transcripts were higher in blastocysts than in 4-8 cell stage embryos. The mRNA expression levels of SOX2 and REX1 were largely similar in embryos of different origins. However, the genes such as LIN28, CDX2, TEAD4, ITGB1 and GATA6 showed the differential expression pattern in PA and NT embryos compared to IVF embryos. Importantly, the transcript levels in MSC-NT embryos were relatively less variable to IVF than those in FF-NT embryos.
MSCs seem to be better donors for porcine NT as they improved the developmental competency, and influenced the expression pattern of genes quite similar with IVF embryos than that of FFs.
Journal of Assisted Reproduction and Genetics 05/2012; 29(8):723-33. · 1.84 Impact Factor
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Jin-Ho Lee,
Jin-Hyun Kim,
Se-Heang Oh,
Sang-June Kim,
Young-Sool Hah,
Bong-Wook Park,
Deok Ryong Kim,
Gyu-Jin Rho,
Geun-Ho Maeng, Ryoung-Hoon Jeon,
Hee-Chun Lee,
Jong-Ryoul Kim,
Gyoo-Cheon Kim,
Uk-Kyu Kim,
June-Ho Byun
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ABSTRACT: The aim of this study was to generate tissue-engineered bone formation using periosteal-derived cells seeded into a polydioxanone/pluronic F127 (PDO/Pluronic F127) scaffold with adipose tissue-derived CD146 positive endothelial-like cells. Considering the hematopoietic and mesenchymal phenotypes of adipose tissue-derived cells cultured in EBM-2 medium, CD146 positive adipose tissue-derived cells was sorted to purify more endothelial cells in characterization. These sorted cells were referred to as adipose tissue-derived CD146 positive endothelial-like cells. Periosteum is a good source of osteogenic cells for tissue-engineered bone formation. Periosteal-derived cells were found to have good osteogenic capacity in a PDO/Pluronic F127 scaffold, which could provide a suitable environment for the osteoblastic differentiation of these cells. Through the investigation of capillary-like tube formation on matrigel and the cellular proliferation of adipose tissue-derived CD146 positive endothelial-like cells cultured in different media conditions, we examined these cells could be cultured in EBM-2 with osteogenic induction factors. We also observed that the osteogenic activity of periosteal-derived cells could be good in EBM-2 with osteogenic induction factors, in the early period of culture. The experimental results obtained in the miniature pig model suggest that tissue-engineered bone formation using periosteal-derived cells and PDO/Pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells can be used to restore the bony defects of the maxillofacial region when used in clinics.
Biomaterials 08/2011; 32(22):5033-45. · 7.40 Impact Factor
