• Clinical and Experimental Reproductive Medicine
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• v.26 no.1
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• pp.83-87
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• 1999

Since the blastocyst is broken and spreads out on a flat plastic culture dish (two dimensional culture) during in vitro development, it has been difficult to study the implantation process. It also has been difficult to analyse the interactions between endometrial epithelial and stromal cells because of the lack of a long-term in vitro model which can stimulate in vivo characteristics, as these cells eventually fail to proliferate or cease to express differentiated functions. Recently nontransformed cell lines, CUE-P and CUS-V2, derived from rat endometrial epithelium and stroma were reported. In this study, morphology of CUE-P and CUS-V2 was examined and oxytocin gene expression by CUE-P cells was demonstrated by RT-PCR. The CUE-P cells have a cuboidal morphology and CUS-V2 cells resemble fibroblast and exhibit a spindle-like morphology. In RT-PCR, same size of PCR products of oxytocin gene at hypothalamus, uterus and CUE-P cells were demonstrated. These results showed three dimensional culture system could be made by using the new cell lines.

• Journal of dental hygiene science
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• v.21 no.4
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• pp.227-232
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• 2021

Background: Cancer-associated fibroblasts (CAFs) are abundant in tumor microenvironments and interact with cancer cells to promote tumor proliferation in oral squamous cell carcinoma (OSCC). Cathepsin D (CTSD) is a soluble lysosomal aspartic endopeptidase involved in tumor proliferation and angiogenesis. In this preliminary study, we observed CTSD expression in OSCC and CAFs, postulating that CTSD might act as a bridge between OSCC and CAFs. Methods: Human epidermal keratinocytes (HEKs), OSCC, and immortalized human normal oral fibroblasts (hTERT-hNOFs) were used in this study. Additionally, we used hTERT-hNOFs transfected with an empty vector, WT (wild-type)-YAP (Yes-associated protein), and YAPS127A (YAP serine 127 to alanine). YAP127A hTERT-hNOFs activated fibroblasts similar to CAFs. To identify CTSD expression between OSCC and CAFs, conditioned medium (CM) was collected from each cell. Protein expression of CTSD was identified by western blotting. Results: To identify the expression of CTSD in fibroblasts stimulated by OSCC, we treated fibroblasts with CM from HEK and OSCC. Results indicated that hTERT-hNOFs with OSCC CM showed a weakly increased expression of CTSD compared to stimulation by HEK CM. This indicates that CAFs, YAPS127 hTRET-hNOFs, overexpress CTSD protein. HEK cells showed no CTSD expression, regardless of treatment with fibroblast CM, whereas OSCC highly expressed CTSD proteins compared with the CTSD expression in HEK cells. We also found that CTSD expression was unaffected by changes in transforming growth factor-β levels. Conclusion: This study proposes that CTSD might have potential as an interacting executor between OSCC and CAFs. Further studies are needed to investigate the role of CTSD in tumor and stromal cells.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.281-287
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• 2013

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal ${\alpha}$-1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from ${\alpha}$-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% $CO_2$ incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers ($CD45^-$, $29^+$, $90^+$ and $105^+$) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited $CD45^-$, $29^+$, $90^+$ and $105^+$ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at $1{\times}10^3$ cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs($15.0{\pm}0.4{\mu}m$) had a little larger cell size than Wild BM-MSCs ($13.5{\pm}0.3{\mu}m$). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

• Development and Reproduction
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• v.14 no.4
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• pp.233-241
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• 2010

Human adipose stem cells are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue and these cells have characteristics very similar to bone marrow mesenchymal stromal cells (BMMSCs). However, liposuction procedure, donor age, body mass index, and harvesting sites might generate differences in the initial cell population and the preparations are a heterogeneous mixture of precursors with different subsets. Therefore, in this study, we investigated the characteristics of human thigh adipose stem cells and the differentiation potential into mesodermal and endodermal lineage. Thigh adipose stem cells maintained fibroblast-like morphology similar to BM-MSCs and they underwent average 56.5 doublings and produced $5{\times}10^{22}$ cells. These cells expressed SCF, Oct4, nanog, vimentin, CK18, FGF5, NCAM, Pax6, BMP4, HNF4a, nestin, GATA4, HLA-ABC, and HLA-DR genes at p3 and they also expressed Oct4, Thy-1, FSP, vWF, vimentin, desmin, CK18, CD54, CD4, CD106, CD31, a-SMA, HLA-ABC proteins. Moreover, they could differentiate into mesodermal lineage cells such as adipocyte, osteoblast and chondrocyte. In addition, they also differentiated into insulin secreting cells in our culture condition. In conclusion, human thigh adipose stem cells retain proliferative potential and expression patterns similar to BM-MSCs and they also differentiate into various cell types. Thus, human thigh adipose stem cells might be useful alternative cell source for clinical application.

• Journal of Veterinary Clinics
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• v.30 no.3
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• pp.214-217
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• 2013

A 3-year-old intact female hedgehog (Atelerix albiventris) was presented for evaluation of mandibular swelling. Fine needle aspiration of swollen mandibular was performed and smears were stained with Romanowsky type stain for cytological evaluation. Smears were highly cellular with predominance of variably shaped keratinized or non keratinized squamous cells with low numbers of spindloid to abnormally elongated cells. Cytologic impression was squamous cell carcinoma. The mass was surgically removed for histological examination. Microscopically tortuous and anastomosing delicate to broad pegs and nests of neoplastic squamous epithelial cells were supported by a moderate collagenous and spindloid fibroblast stroma. Tumor cells had moderate anisocytosis and mild anisokaryosis and range from moderately to well keratinized, with areas of intratumoral acantholysis accompanied by mixed stromal lymphoplasmacytic, neutrophilic inflammation. The mitotic index is 2-3 per high-power field. Tumor cells were expanding the subcutis subjacent to the layer of skeletal muscle and incorporating the osseous tissue fragments. The final diagnosis was squamous cell carcinoma. The patient survived three months after surgery without any further medical treatments.

• Microbiology and Biotechnology Letters
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• v.38 no.4
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• pp.420-427
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• 2010

A process for manufacturing virally-safe bovine amniotic membrane(BAM) has been developed for biological dressing. BAM was harvested from a healthy bovine placenta, and then the epithelium was removed. The remaining stromal layer was consecutively disinfected with 70% ethanol and 0.05% sodium hypochlorite. The stromal layer was incubated in a decellularization solution containing 0.25%(w/v) trypsin to remove the cellular components. The resulting acelluar BAM was lyophilized to preserve its biochemical and structural integrity. The BAM was packed and exposed to 25 kGy of gamma irradiation for sterilization purpose. Histological, electron microscopical, and biochemical observations showed that the acellualr BAM had intact structural integrity of three dimensional collagen fibers and contained several growth factors, accelerating wound healing, such as EGF (Epidermal growth factor), KGF (Keratinocyte growth factor), and FGF (Fibroblast growth factor). Bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), bovine parainfluenza virus type 3 (BPIV-3), and bovine parvovirus (BPV) were chosen as the biological indicators for validation of viral safety of the acellular BAM. Samples from relevant stages of the production process were spiked with each virus and subjected to viral inactivation processes. Viruses were recovered from the samples and then titrated immediately. All the viruses tested were completely inactivated to undetectable levels within 1 h of 70% ethanol treatment. Enveloped viruses such as BHV, BVDV, and BPIV-3 were more effectively inactivated than BPV by 0.05% sodium hypochlorite treatment. BHV, BVDV, and BPIV-3 were completely inactivated to undetectable levels by 25 kGy of gamma irradiation. Also BPV was effectively inactivated by 25 kGy of gamma irradiation. The cumulative log reduction factors of BHV, BVDV, BPIV-3, and BPV were ${\geq}$13.30, ${\geq}$14.32, ${\geq}$15.22, and ${\geq}$7.57, respectively. These results indicate that the production process for acelluar BAM has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.