• 대한수의학회지
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• 제58권4호
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• pp.201-209
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• 2018

Canine mammary tumors are among the most frequently observed cutaneous tumors in female dogs. Cancer stem cells (CSCs), referred to as tumor-initiating cells, are thought to have properties similar to normal stem cells such as the ability to self-renewal and to differentiate into various cell types. Biological understanding of CSCs and the critical pathways involved in their maintenance are important in research and therapy for mammary tumors. We conducted the present study on sphere formation from REM134 cells by using methylcellulose to produce tumorspheres on a large scale and compared the specific markers of the spheres-formed and plating-cultured REM134 cells. The results revealed that the tumorspheres cultured in methylcellulose had higher seeding density and improved morphology compared to those produced in normal sphere formation medium. Expression levels of stemness markers and CSC-related markers were higher in tumorsphere-forming cells than in plating-cultured cells. Subsequently, we transplanted the tumorsphere-forming and plating-cultured cells into female nude mice to examine their tumorigenic potential. Tumor volume increased rapidly in mice transplanted with tumorsphere-derived cells compared to plating-cultured cells. We observed a novel sphere-forming condition for REM134 cells and showed that REM134 cell tumorspheres can exhibit improved CSC properties.

• 한국동물생명공학회지
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• 제38권2호
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• pp.54-61
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• 2023

Background: Germ cells undergo towards male or female pathways to produce spermatozoa or oocyte respectively which is essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the potential of trans-differentiation to the multiple cell lineages. Methods: Herein, rat MSCs were isolated from bone marrow and characterized by their morphological features, expression of MSC surface markers, and in vitro differentiation capability. Results: Thereafter, we induced these cells only by retinoic acid supplementation in MSC medium and, could able to show that bone marrow derived MSCs are capable to trans-differentiate into male germ cell-like cells in vitro. We characterized these cells by morphological changes, the expressions of germ cell specific markers by immunophenotyping and molecular biology tools. Further, we quantified these differentiated cells. Conclusions: This study suggests that only Retinoic acid in culture medium could induce bone marrow MSCs to differentiate germ cell-like cells in vitro. This basic method of germ cell generation might be helpful in the prospective applications of this technology.

• Asian Pacific Journal of Cancer Prevention
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• 제15권5호
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• pp.2013-2020
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• 2014

Background: The aldehyde dehydrogenase 1 family member A1 (ALDH1A1) is one of the promising markers for identifying cancer stem cells in many cancer types, along with other markers including CD44. The aim of the present study was to evaluate the expression and clinical significance of putative cancer stem cell markers, CD44 and ALDH1A1, in a series of urothelial carcinomas of urinary bladder (UCUB) by tissue microarray (TMA). Materials and Methods: A total of 159 Urothelial Carcinomas (UC) including 96 (60%) low grade and 63 (40%) high grade carcinomas were immunohistochemically examined for the expression of CD44 and ALDH1A1. Correlations of the relative expression of these markers with clinicopathological parameters were also assessed. Results: High level expression of ALDH1A1 was found in 16% (25/159) of bladder UC which was significantly correlated with increased tumor size (p value=0.002), high grade (p value<0.001), pathologic stage (T1, p value=0.007 and T2, p value<0.001) and increased rate of recurrence (p value=0.013). A high level of CD44 expression was found in 43% (68/159) of cases, being positively correlated with histologic grade (p value=0.032) and recurrence (p value=0.039). Conclusions: Taken together, our results showed that ALDH1 was concurrently expressed in a fraction of CD44+ tumors and its expression correlated with poor prognosis in UCs. ALDH1A1 could be an ideal marker for targeted therapy of UCs in combination with conventional therapies, particularly in patients with high grade carcinomas. These findings indicate that cells expressing ALDH1A1 along with CD44 can be a potential therapeutic target in bladder carcinomas.

• Molecules and Cells
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• 제23권1호
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• pp.49-56
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• 2007

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential.

• Archives of Plastic Surgery
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• 제39권6호
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• pp.593-599
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• 2012

Background This study aimed to investigate the possibility of isolating mesenchymal stem cells (MSCs) from human thigh adipose tissue and the ability of human thigh adipose stem cells (HTASCs) to differentiate into hepatocytes. Methods The adipose-derived stem cells (ADSCs) were isolated from thigh adipose tissue. Growth factors, cytokines, and hormones were added to the collagen coated dishes to induce the undifferentiated HTASCs to differentiate into hepatocyte-like cells. To confirm the experimental results, the expression of hepatocyte-specific markers on undifferentiated and differentiated HTASCs was analyzed using reverse transcription polymerase chain reaction and immunocytochemical staining. Differentiation efficiency was evaluated using functional tests such as periodic acid schiff (PAS) staining and detection of the albumin secretion level using enzyme-linked immunosorbent assay (ELISA). Results The majority of the undifferentiated HTASCs were changed into a more polygonal shape showing tight interactions between the cells. The differentiated HTASCs up-regulated mRNA of hepatocyte markers. Immunocytochemical analysis showed that they were intensely stained with anti-albumin antibody compared with undifferentiated HTASCs. PAS staining showed that HTASCs submitted to the hepatocyte differentiation protocol were able to more specifically store glycogen than undifferentiated HTASCs, displaying a purple color in the cytoplasm of the differentiated HTASCs. ELISA analyses showed that differentiated HTASCs could secrete albumin, which is one of the hepatocyte markers. Conclusions MSCs were islolated from human thigh adipose tissue differentiate to heapatocytes. The source of ADSCs is not only abundant abdominal adipose tissue, but also thigh adipose tissue for cell therapy in liver regeneration and tissue regeneration.

• BMB Reports
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• 제55권3호
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• pp.142-147
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• 2022

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.

• Asian-Australasian Journal of Animal Sciences
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• 제24권1호
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• pp.37-44
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• 2011

Parthenogenetic embryonic stem (pES) cells could provide a valuable model for research into genomic imprinting and X-linked diseases. In this study, pES cell lines were established from oocytes of hybrid offspring of Kunming and 129/Sv mice, and pluripotency of pES cells was evaluated. The pES cells maintained in the undifferentiated state for more than 50 passages had normal karyotypes with XX sex chromosomes and exhibited high activities of alkaline phosphatase (AKP) and telomerase. Meanwhile, these cells expressed ES cell molecular markers SSEA-1, Oct-4, Nanog, and GDF3 but not SSEA-3 detected by immunohistochemistry and RT-PCR. The pES cells could be differentiated into various types of cells from three germ layers in vitro by analysis of embryoid bodies (EBs) with immunohistochemistry and RT-PCR, and in vivo by observation of pES cell-derived teratoma sections. Therefore, the established pES cell lines contained all features of mouse ES cells. This work provides a new strategy for isolating pES cells from Kunming mice, and the pES cell lines could be applied as the cell model in research into genomic imprinting and epigenetic regulation of Kunming mice.

• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.1023-1035
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• 2016

The purpose of this study was to investigate the role of colon cancer stem cells (CSCs) during chemically-induced rat multi-step colon carcinogenesis with or without the treatment with a specific cyclooxygenase-2 inhibitor drug (celecoxib). Two experiments were performed, the first, a short term 12 week colon carcinogenesis bioassay in which only surrogate markers for colon cancer, aberrant crypt foci (ACF) lesions, were formed. The other experiment was a medium term colon cancer rat assay in which tumors had developed after 32 weeks. Treatment with celecoxib lowered the numbers of ACF, as well as the tumor volumes and multiplicities after 32 weeks. Immunohistochemical proliferating cell nuclear antigen (PCNA) labeling indexes LI (%) were downregulated after treatment by celecoxib. Also different cell surface antigens known to associate with CSCs such as the epithelial cell adhesion molecule (EpCAM), CD44 and CD133 were compared between the two experiments and showed differential expression patterns depending on the stage of carcinogenesis and treatment with celecoxib. Flow cytometric analysis demonstrated that the numbers of CD133 cells were increased in the colonic epithelium after 12 weeks while those of CD44 but not CD133 cells were increased after 32 weeks. Moreover, aldehyde dehydrogenase-1 activity levels in the colonic epithelium (a known CSC marker) detected by ELISA assay were found down-regulated after 12 weeks, but were up-regulated after 32 weeks. The data have also shown that the protective effect of celecoxib on these specific markers and populations of CSCs and on other molecular processes such as apoptosis targeted by this drug may vary depending on the genetic and phenotypic stages of carcinogenesis. Therefore, uncovering these distinction roles of CSCs during different phases of carcinogenesis and during specific treatment could be useful for targeted therapy.

• Journal of Korean Neurosurgical Society
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• 제62권2호
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• pp.153-165
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• 2019

Objective : Spinal cord injury (SCI) is a very serious health problem, usually caused by a trauma and accompanied by elevated levels of inflammation indicators. Stem cell-based therapy is promising some valuable strategies for its functional recovery. Nestin-positive progenitor and/or stem cells (SC) isolated from pancreatic islets (PI) show mesenchymal stem cell (MSC) characteristics. For this reason, we aimed to analyze the effects of rat pancreatic islet derived stem cell (rPI-SC) delivery on functional recovery, as well as the levels of inflammation factors following SCI. Methods : rPI-SCs were isolated, cultured and their MSC characteristics were determined through flow cytometry and immunofluorescence analysis. The experimental rat population was divided into three groups : 1) laminectomy & trauma, 2) laminectomy & trauma & phosphate-buffered saline (PBS), and 3) laminectomy+trauma+SCs. Green fluorescent protein (GFP) labelled rPI-SCs were transplanted into the injured rat spinal cord. Their motilities were evaluated with Basso, Beattie and Bresnahan (BBB) Score. After 4-weeks, spinal cord sections were analyzed for GFP labeled SCs and stained for vimentin, $S100{\beta}$, brain derived neurotrophic factor (BDNF), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), vascular endothelial growth factor (VEGF) and proinflammatory (interleukin [IL]-6, transforming growth factor $[TGF]-{\beta}$, macrophage inflammatory protein [MIP]-2, myeloperoxidase [MPO]) and anti-inflammatory (IL-1 receptor antagonis) factors. Results : rPI-SCs were revealed to display MSC characteristics and express neural and glial cell markers including BDNF, glial fibrillary acidic protein (GFAP), fibronectin, microtubule associated protein-2a,b (MAP2a,b), ${\beta}3$-tubulin and nestin as well as anti-inflammatory prostaglandin E2 receptor, EP3. The BBB scores showed significant motor recovery in group 3. GFP-labelled cells were localized on the injury site. In addition, decreased proinflammatory factor levels and increased intensity of anti-inflammatory factors were determined. Conclusion : Transplantation of PI-SCs might be an effective strategy to improve functional recovery following spinal cord trauma.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.1-6
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• 2010

Mesenchymal stem cells (MSCs) have the multipotent capacity and this potential can be applied for obtaining valuable cell types which can use for cell therapy on various regenerative diseases. However, insufficient availability of cellular source is the major problem in cell therapy field using adult stem cell sources. Recently, human embryonic stem cells (hESCs) have been highlighted to overcome a limitation of adult cellular sources because they retain unlimited proliferation capacity and pluripotency. To use of hESCs in cell therapy, above all, animal pathogen free culture system and purification of a specific target cell population to avoid teratoma formation are required. In this study, we describe the differentiation of a mesenchymal stem cell-like cells population from feeder-free cultured hESCs(hESC-MSCs) using direct induction system. hESC-MSCs revealed characteristics similar to MSCs derived from bone marrow, and undifferentiated cell markers were extremely low in hESC-MSCs in RT-PCR, immunostaining and FACS analyses. Thus, this study proffer a basis of effective generation of specialized human mesenchymal stem cell types which can use for further clinical applications, from xenofree cultured hESCs using direct induction system.