• IMMUNE NETWORK
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• 제1권1호
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• pp.87-94
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• 2001

목적 : 제대혈의 조혈모세포 체외확장 시 조혈세포 증폭과 더불어 조혈미세환경의 변화가 일어난다. 이때 제대혈 $CD34^+$ 세포에서 유래되는 지지세포의 계열 분석조혈성장인자 분비능력을 알아보고 지지세포 증식 조건을 확립하여 효과적인 제대혈의 체외증폭을 제시하고자 하였다. 방법 : 제대혈부터 $CD34^+$ 세포를 분리하여 실험에 사용하였다. 무혈청배지에서 각종 조혈성장인자를 다양한 조합으로 첨가하여 배양하였고 증식정도는 현미경으로 관찰하여 배양용기를 점유한 면적 비율로 계산하였다. 세포외간질 단백의 효과를 분석하기 위하여 collagen S, fibronectin, laminin 및 poly-L-ly sine를 미리 coating한 용기에 배양하여 분석하였다. 제대혈 $CD34^+$ 세포를 조혈성장인자의 첨가 없이 3주간 액체배양하였다. 배양 시, 1주, 2주 및 3주에 상층액을 얻어 $-80^{\circ}C$에 보관하였다가 한꺼번에 IL-3, IL-6, GM-CSF, IL-$1{\beta}$ 및 TNF-$\alpha$등을 ELISA 방법으로 내부적으로 분비되는 량을 측정하였다. 분화된 지지세포의 계열을 분석하기 위해 E-selectin, VCAM-1, ICAM-1, PECAM-1, vWF, vimentin 및 CD 14 항체를 이용하여 면역화학염색 후 형광현미경으로 관찰하였다. 결과 : 제대혈 $CD34^+$ 세포 체외증폭시키는 과정에서 배양 4일에 지지세포가 출현하기 시작하여 7-10일이 지나면서 증식하기 시작하였고 14-2 1일 경에 서로 뭉치는 양상을 보여주었다. 제대혈 $CD34^+$ 세포 배양하면서 내부적으로 분비되는 GM-CSF, IL-6의 측정치는 시간이 지남에 따라 증가되었다. 제대혈 $CD34^+$ 세포 체외확장 시 지지세포의 증식 정도는 TPO+FL+SCF+LIF의 조합의 조혈성장인자가 첨가되었을 때 그리고 세포외간질 단백 성분 중 1% poly-L-lysine으로 처리한 경우 가장 효과적이었다. 결론 : 체외 증폭시 제대혈 $CD34^+$ 세포로부터 지지세포가 나타났으며 적절한 조혈성장인자의 첨가나 세포외간질 단백의 첨가에 의해 증폭될 수 있다.

• Animal cells and systems
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• 제2권1호
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• pp.1-8
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• 1998

Hepatocyte growth factor (HGF), originally discovered and cloned as a powerful mitogen for hepatocytes, is a four kringle-containing growth factor which specifically binds to membrane-spanning tyrosine kinase, c-Met/HGF receptor. HGF has mitogenic, motogenic (enhancement of cell movement), morphogenic (e.g., induction of branching tubulogenesis), and anti-apoptotic activities for a wide variety of cells. During embryogenesis, HGF supports organogenesis and morphogenesis of various tissues, including liver, kidney, lung, gut, mammary gland, and tooth. In adult tissues HGF elicits an organotrophic function which supports regeneration of organs such as liver, kidney, lung, and vascular tissues. HGF is also a novel member of neurotrophic factor in nervous systems. Together with the preferential expression of HGF in mesenchymal or stromal cells, and c-Met/HGF receptor In epithelial or endothelial cells, the HGF-Met coupling seems to orchestrate dynamic morphogenic processes through epithelial-mesenchymal (or-stromal) interactions for organogenesis and organ regeneration. HGF or HGF gene may well become unique therapeutic tools for treatment of patients with various organ failure, through its actions to reconstruct organized tissue architectures. This review focuses on recently characterized biological and physiological functions integrated by HGF-Met coupling during organogenesis and organ regeneration.

• Asian-Australasian Journal of Animal Sciences
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• 제24권1호
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• pp.110-124
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• 2011

Anatomically separate fat depots differ in size, function, and contribution to pathological states such as the metabolic syndrome. We isolated pre-adipocytes from different adipose depots, omental, subcutaneous and intramuscular, of beef cattle, and cultured in vitro to determine the basis for the variations and attribute these variations to the inherent properties of adipocyte progenitors. The proliferating cells from all depots before the confluence were harvested and the proteome was analyzed by a functional proteomic approach, involving 2-DE and MALDI-TOF/TOF. More than 252 protein spots were identified, selected and analyzed by Image Master (ver 7.0) and MALDI-TOF/TOF. Further, our analysis showed that there were specific differences in proteome expression patterns among proliferating precursor cells from the three depots. Sixteen proteins were found to be differentially expressed and these were identified as proteins involved in cellular processes, heat shock/chaperones, redox proteins, cytoskeletal proteins and metabolic enzymes. The results also enabled us to understand the basic roles of these proteins in different inherent properties exhibited by adipose tissue depots.

• Asian-Australasian Journal of Animal Sciences
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• 제13권2호
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• pp.161-166
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• 2000

This study was designed to determine the effects of the insulin-like growth factor (IGF-1) and estradiol $17-{\beta}$ on the in vitro proliferation of stromal vascular cell from Hanwoo omental, subcutaneous, intermuscular and intramuscular adipose tissues. Cells were cultured in M199+20% newborn calf serum and the proliferation of cells was measured by direct microscopic cell counting and change of genomic DNA amount. Cell numbers increased slightly over the first 72 hour of culture and then increased greatly, regardless of adipose tissue depots. In IGF-1 treatment, the number of omental preadipocytes maintained highest level from the beginning to the 20th day of culture. However, in estradiol-$17{\beta}$ treatment, those tended to be lower than the control from the beginning of culture and significantly lower at the 24th day. When IGF-1 was added to subcutaneous preadipocytes, the numbers of cells were higher from 11th day than those from other treatments, although there was no statistical significance. For intermuscular preadipocytes treated with IGF-1, its numbers were significantly (p<0.05) higher at 11th day, and in the other days it showed a similar tendency to those of the subcutaneous tissue. In this experiment, preadipocytes were taken from 24 month old fully matured steers and the highest proliferation rate was shown in intramuscular tissue followed by those of subcutaneous preadipocytes. Addition of $5{\mu}M$ estradiol-$17{\beta}$ to the growth medium failed to promote the replication of Hanwoo preadipocytes, as indicated by direct cell counts and total genomic DNA content. As the culture period proceeded, the amounts of DNA were increased, but the patterns of increment were not consistent with the results of cell numbers.

• Molecules and Cells
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• 제37권6호
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• pp.487-496
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• 2014

Angiotensinogen (AGT), the precursor of angiotensin I, is known to be involved in tumor angiogenesis and associated with the pathogenesis of coronary atherosclerosis. This study was undertaken to determine the role played by AGT in endothelial progenitor cells (EPCs) in tumor progression and metastasis. It was found that the number of EPC colonies formed by AGT heterozygous knockout ($AGT^{+/-}$) cells was less than that formed by wild-type (WT) cells, and that the migration and tube formation abilities of $AGT^{+/-}$ EPCs were significantly lower than those of WT EPCs. In addition, the gene expressions of vascular endothelial growth factor (VEGF), Flk1, angiopoietin (Ang)-1, Ang-2, Tie-2, stromal derived factor (SDF)-1, C-X-C chemokine receptor type 4 (CXCR4), and of endothelial nitric oxide synthase (eNOS) were suppressed in $AGT^{+/-}$ EPCs. Furthermore, the expressions of hypoxia-inducible factor (HIF)-$1{\alpha}$and $-2{\alpha}$ were downregulated in $AGT^{+/-}$ early EPCs under hypoxic conditions, suggesting a blunting of response to hypoxia. Moreover, the activation of Akt/eNOS signaling pathways induced by VEGF, epithelial growth factor (EGF), or SDF-$1{\alpha}$ were suppressed in $AGT^{+/-}$ EPCs. In $AGT^{+/-}$ mice, the incorporation of EPCs into the tumor vasculature was significantly reduced, and lung tumor growth and melanoma metastasis were attenuated. In conclusion, AGT is required for hypoxia-induced vasculogenesis.

• Clinical and Experimental Otorhinolaryngology
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• 제11권4호
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• pp.281-287
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• 2018

Objectives. Regenerative treatment using stem cells may serve as treatment option for empty nose syndrome (ENS), which is caused by the lack of turbinate tissue and deranged nervous system in the nasal cavity. We aimed to assess the efficacy and safety of the autologous stromal vascular fraction (SVF) in the treatment of ENS. Methods. In this prospective observational clinical study, we enrolled 10 ENS patients who volunteered to undergo treatment of ENS through the injection of autologous SVF. Data, including demographic data, pre- and postoperative Sino-Nasal Outcome Test-25 (SNOT-25) scores, overall patient satisfaction, and postoperative complications, were prospectively collected. Nasal secretion was assessed using the polyurethane foam absorption method, and the levels of biological markers were analyzed in both ENS group and control group using enzyme-linked immunosorbent assay. The SVF extracted from abdominal fat was diluted and injected into both inferior turbinates. Results. Among the 10 initial patients, one was excluded from the study. Subjective satisfaction was rated as "much improved" in two and "no change" in seven. Among the improved patients, the mean preinjection SNOT-25 score was 55.0 and the score at 6 months after injection was 19.5. However, the average SNOT-25 score of nine participants at 6 months after injection (mean${\pm}$standard deviation, $62.4{\pm}35.8$) did not differ significantly from the baseline SNOT-25 score ($70.1{\pm}24.7$, P>0.05, respectively). Among the various inflammatory markers assessed, the levels of interleukin $(IL)-1{\beta}$, IL-8, and calcitonin gene-related peptide were significantly higher in ENS patients. Compared with preinjection secretion level, the nasal secretions from SVF-treated patients showed decreased expressions of $IL-1{\beta}$ and IL-8 after injection. Conclusion. Although SVF treatment appears to decrease the inflammatory cytokine levels in the nasal mucosa, a single SVF injection was not effective in terms of symptom improvement and patient satisfaction. Further trials are needed to identify a more practical and useful regenerative treatment modality for patients with ENS.

• Journal of Animal Science and Technology
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• 제63권2호
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• pp.426-439
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• 2021

It is already well known that castration improves marbling quality but exact timing of castration is still highly debated in beef cattle production industry. After castration, blood hormonal changes occur in steer and objective of this study was to investigate the effects of growth hormone (GH) levels on adipocyte differentiation in stromal vascular cells (SVCs) and transdifferentiation into adipocytes in C2C12 myoblasts. Total GH concentrations were measured via enzyme-linked immunosorbent assay (ELISA) in 24 male calves and 4 female calves. Cell proliferation, cellular triglyceride (TG) accumulation, and the cell's lipolytic capability were measured in C2C12 myoblasts and SVCs. Myogenic, adipogenic, and brown adipocyte-specific gene expression was measured via real-time polymerase chain reaction (PCR) using SYBR green. Serum GH levels were the highest in late-castrated calves. Treatment with 5 ng/mL GH resulted in greater TG accumulation as well as increased CCAAT-enhancer-binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ expression compared to that after treatment with 15 ng/mL GH. Treatment with 5 ng/mL GH also resulted in lower myogenin (myo)G and myoD expression compared to that after treatment with 15 ng/mL GH. The expression of bone morphogenetic protein (BMP) 7 after treatment with 5 ng/mL GH was higher than that after treatment with 15 ng/mL GH. But carcass characteristics data showed no significant difference between early and late castrated steers. Therefore, our results indicate that castration timing does not seem to be inevitable determinate of carcass qualities, particularly carcass weight and marbling score in Hanwoo beef cattle.

• Journal of Veterinary Science
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• 제24권3호
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• pp.37.1-37.14
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• 2023

Background: Toll-like receptor (TLR) agonists have been used as adjuvants to modulate immune responses in both animals and humans. Objectives: The objective of this study was to evaluate the combined effects of the TLR 4 agonist monophosphoryl lipid A (MPL) and the TLR 3 agonist polyinosinic:polycytidylic acid (Poly I:C) on equine peripheral blood mononuclear cells (PBMCs), monocyte-derived dendritic cells (MoDCs), and bone marrow-derived mesenchymal stromal cells (BM-MSCs). Methods: The PBMCs, MoDCs, and BM-MSCs collected from three mixed breed horses were treated with MPL, Poly I:C, and their combination. The mRNA expression of interferon gamma (IFN-γ), interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-12p40, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) was determined using real-time polymerase chain reaction. Results: The combination of MPL and Poly I:C significantly upregulated immunomodulatory responses in equine cells/ without cytotoxicity. The combination induced greater mRNA expression of pro-inflammatory cytokines IFN-γ and IL-6 than MPL or Poly I:C stimulation alone in PBMCs. In addition, the combination induced significantly higher mRNA expression of IL-1β, IL-6, and IL-12p40 in MoDCs, and IL-8, MCP-1, and VEGF in BM-MSCs compared to stimulation with a single TLR agonist. Conclusions: The combination of MPL and Poly I:C can be used as a potential adjuvant candidate for vaccines to aid in preventing infectious diseases in horses.

• BMB Reports
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• 제53권2호
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• pp.118-123
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• 2020

Cardiac regeneration with adult stem-cell (ASC) therapy is a promising field to address advanced cardiovascular diseases. In addition, extracellular vesicles (EVs) from ASCs have been implicated in acting as paracrine factors to improve cardiac functions in ASC therapy. In our work, we isolated human cardiac mesenchymal stromal cells (h-CMSCs) by means of three-dimensional organ culture (3D culture) during ex vivo expansion of cardiac tissue, to compare the functional efficacy with human bone-marrow derived mesenchymal stem cells (h-BM-MSCs), one of the actively studied ASCs. We characterized the h-CMSCs as CD90^low, c-kit^negative, CD105^positive phenotype and these cells express NANOG, SOX2, and GATA4. To identify the more effective type of EVs for angiogenesis among the different sources of ASCs, we isolated EVs which were derived from CMSCs with either normoxic or hypoxic condition and BM-MSCs. Our in vitro tube-formation results demonstrated that the angiogenic effects of EVs from hypoxia-treated CMSCs (CMSC-Hpx EVs) were greater than the well-known effects of EVs from BM-MSCs (BM-MSC EVs), and these were even comparable to human vascular endothelial growth factor (hVEGF), a potent angiogenic factor. Therefore, we present here that CD90^lowc-kit^negativeCD105^positive CMSCs under hypoxic conditions secrete functionally superior EVs for in vitro angiogenesis. Our findings will allow more insights on understanding myocardial repair.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제36권3호
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• pp.85-93
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• 2014

Purpose: Nodal metastasis is the main prognostic factor in the patients with oral squamous cell carcinoma (OSCC). We investigated the association between tumor-associated lymphatics and OSCC characteristics. Methods: Thirty-four specimens were used for the immunohistochemical staining with the antibody for vascular endothelial growth factor (VEGF)-C, VEGF-D, VEGF receptor (VEGFR)-3, phosphorylated VEGFR-3, D2-40, and matrix metallproteinases (MMPs). We observed the distribution of the lymphangiogenic factors and quantified the degree of expression. We determined lymphatic vessel density (LVD) and lymphatic vessel dilatation with D2-40 immunostaining. We assessed the association of LVD or lymphatic vessel dilatation with tumor progression or tumor differentiation. Results: OSCC cells expressed lymphangiogenic ligands. Lymphangiogenic receptor, VEGFR-3, was expressed and activated in some tumor cells as well as in tumor-associated endothelial cells. LVD was not associated with tumor size or nodal status, but lymphatic vessel dilatation was higher in tumors with nodal metastasis, and also higher in poorly differentiated tumors. In stromal area of OSCC, MMP-1 and MMP-10 were up-regulated and the basement membrane of tumor-associated endothelial cells was destroyed by these collagenases. Conclusion: In the primary tumors with nodal metastasis, especially in poorly differentiated OSCC, tumor cells invaded the dilated lymphatic vessels via ruptured sites. MMP-1 and MMP-10 are important in the lysis of the glycocalyx inside the tumor-associated lymphatic endothelial cells.