• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.23-34
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• 2009

Objectives: Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. It is known that long-term in vitro culture of human bone marrow and adipose tissue derived-MSCs lead to a reduction of life span and a change of stem-like characters. The aim of our study was to examine whether stem cell properties of human umbilical cord-derived stem cells (HUC) could be affected by in vitro expansion. Methods: HUC were isolated from human umbilical cord and cultured for 10 passages in vitro. Morphology and population doubling time (PDT) were investigated, and changes of stem cell properties were examined using RT-PCR and immunocytochemistry during serial subcultures. Results: Morphology and PDT of HUC began to change slightly from the 7th passage (p7). Expression level of nestin and vimentin mRNAs increased along with the culture period from p4 until p10. In contrast, expression level of SCF mRNA decreased during the same culture period. Expression level of Oct-4 and HNF-4${\alpha}$ mRNAs was not significantly changed throughout the culture period until p10. Expression level of BMP-4, FGF-5, NCAM and HLA-ABC mRNAs appeared to increase as the culture continued, however, the difference was not significant. Immunocytochemical studies showed that HUC at p3, p6 and p9 positively were stained with antibodies against SSEA-3 and SSEA-4 proteins. Interestingly, staining intensity of HUC for ICAM-1 and HLA-ABC gradually increased throughout the culture period. Intensity against thy-1 and fibronectin antibodies increased at p9 while that against TRA-1-60 and VCAM-1 antibodies began to decrease at p6 until p9. Conclusions: These results suggest that HUC change some of their stem cell characteristics during in vitro culture. Development of culture system might be needed for the maintenance of characteristics.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.3
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• pp.439-445
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• 2017

Objective: Production of alpha-1,3-galactosyltransferase (${\alpha}GT$)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous ${\alpha}GT$ knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce ${\alpha}GT$-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods: Miniature pig fibroblasts were transfected with ${\alpha}GT$ gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous ${\alpha}GT$ gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-${\alpha}$-1,3-galactose, an epitope produced by ${\alpha}GT$. Using magnetic activated cell sorting, cells with monoallelic disruption of ${\alpha}GT$ were removed. Remaining cells with LOH carrying biallelic disruption of ${\alpha}GT$ were used for the second round NT to produce homozygous ${\alpha}GT$ gene-targeted piglets. Results: Monoallelic mutation of ${\alpha}GT$ gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous ${\alpha}GT$ gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous ${\alpha}GT$ knockout piglets. Conclusion: The present study demonstrates that the time required for the production of ${\alpha}GT$-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.