• Development and Reproduction
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• v.11 no.3
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• pp.167-177
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• 2007

In the present study, we isolated three human adult stem cells including adipose tissue-derived stem cells(HAD), umbilical cord-derived stem cells(HUC), and amnion-derived stem cells(HAM) and analysed their characteristics. And we examined whether HAD could be used as therapeutical cells for the heart diseases. Both HAM and HUC appeared very similar morphology but HAD was different. Doubling time of HUC was most fast, but total doubling numbers of HUC was same with HAM. Total doubling numbers of HAD was much more than others. Expression patterns of genes and proteins of three human adult stem cells were very similar. Also they were differentiated into adipocytes, osteocytes, and chondrocytes. In addition, they expressed many cardiomyocyte-related genes. But expression pattern of genes is a little different. When HAD were cultivated in the presence or absence of various combinations of BMP and FGF after 5-azacytidine expose for 24 h, expression of Cmlc-1, and ${\alpha}1c$ genes was significantly increased. However, expression of troponin T, troponin I and Kv4.3 genes was not changed. Based on these observations, it is suggested that HAD, HUC, and HAM might be used as potentially therapeutical cells for clinical application.

• Journal of Physiology & Pathology in Korean Medicine
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• v.31 no.6
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• pp.374-379
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• 2017

Hair loss affects interpersonal relationships and causes psychological stress. In this study, we investigated the antioxidant activity of Cynanchi Wilfordii Radix (CWR) and its effects on dermal papilla (DP) cells. Antioxidant efficacy was examined by ABTS assay. To confirm the effect on cell activity, MTS assay was performed and cell count was directly measured by hemocytometer. The mRNA expression of genes involved in hair formation and hair loss formation was confirmed by quantitative RT-PCR. CWR has a strong antioxidant activity. Cell viability of DP cells was increased to 118.5% by treatment of 0.5 mg/ml CWR for 24 hours, but the effect on the cell number was insignificant. These results suggest that CWR increases mitochondrial activity without promoting cell proliferation. Treatment of DP cells with 0.5 mg/ml CWR resulted in 48.5% reduction of mRNA expression of type 2 $5{\alpha}$-reductase, a major cause of male hair loss. In addition, mRNA expression of bone morphogenetic pretein (BMP), fibroblast growth factor (FGF)7, and FGF10, which are closely related to hair growth, was also decreased. Reactive oxygen species (ROS) acts as a cause of hair loss. The excellent antioxidant efficacy of CWR is thought to be able to effectively remove ROS. The dihydrotestosterone produced by type 2 $5{\alpha}$-reductase in DP cells is a potent inducer of male pattern hair loss. The inhibitory effect of type 2 $5{\alpha}$-reductase mRNA on DP cells induced by CWR may induce a positive therapeutic effect of male pattern hair loss.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.263-267
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• 2008

This study was conducted to compare the expression pattern of the specific factors associated with pregnancy and angiogenesis during early pregnancy in Hanwoo. Synchronized female Hanwoo ($4{\sim}6$ year-old) were inseminated artificially. After 10 weeks after artificial insemination (AI), the pregnancy was tested by rectal palpation method. Three pregnant and non-pregnant Hanwoo were used in this experiment, respectively. The plasma progesterone level was measured by ELISA. Western blot analysis was performed to detect the expression of pregnancy associated glycoprotein (PAG) or angiogenic factors (VEGF, B-FGF, ANP-1, and TIE-2). The plasma P4 level was increase gradually in pregnant group and maintained high level. The concentration of PAG was significantly higher from $5^{th}$ weeks in pregnant group compared to that of non-pregnant group (p<0.05). The concentrations of the VEGF (p<0.05), B-FGF (p<0.05), and ANP-1 (p<0.05) were significantly increased from $6^{th}\;or\;7^{th}$ week after AI in pregnant group, respectively. And the intensity of TIE-2, ANP-1 receptor, was well matched with ANP-1 (p<0.05). Taken together, it can be postulated that the blood vessels connected with fetus and dam were formed dramatically around 40 days after AI, because the expression levels of the angiogenic factors were increased significantly from this time in pregnant Hanwoo.

• Development and Reproduction
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• v.12 no.2
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• pp.159-168
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• 2008

Nanog is a newly identified member of the homeobox family of DNA binding transcription factors that functions to maintain the undifferentiated state of stem cells. However, molecular mechanisms underlying the function of Nanog remain largely unknown. To elucidate the regulatory roles of Nanog involved in maintenance of P19 embryonal carcinoma (EC) stem cells, we transfected three small interfering RNA (siRNA) duplexes targeted against different regions of the Nanog gene into P19 cells. The Nanog siRNA-100 duplexes effectively decreased the expression of Nanog up to 30.7% compared to other two Nanog siRNAs, the Nanog siRNA-400 (67.9 %) and -793 (53.0%). When examined by RT-PCR and real-time PCR, the expression of markers for pluripotency such as Fgf4, Oct3/4, Rex1, Sox1 and Yes was downregulated at 48 h after transfection with Nanog siRNA-100. Furthermore, expression of the ectodermal markers, Fgf5 and Isl1 was reduced by Nanog knockdown. By contrast, the expression of other markers for pluripotency such as Cripto, Sox2 and Zfp57 was not affected by Nanog knockdown at this time. On the other hand, the expression of Lif/Stat3 pathway molecules and of the endoderm markers including Dab2, Gata4, Gata6 and the germ cell nuclear factor was not changed by Nanog knockdown. The results of this study demonstrated that the knockdown of Nanog expression by RNA interference in P19 cells was sufficient to modulate the expression of pluripotent markers involved in the self-renewal of EC stem cells. These results provide the valuable information on potential downstream targets of Nanog and add to our understanding of the function of Nanog in P19 EC stem cells.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.255-260
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• 1994

원시생식세포(primordial germ cell; PGC)는 성성숙 이후에 기능을 갖는 생식세포의 근원이 되는 세포로서, 다능성을 갖고 있는 것으로 알려져 있다. 그러므로 chimera 및 유전자 변환동물 생산을 위해 널리 사용되어 온 배아주(embrynic stem; ES)세포를 대신할 다른 세포계라고 생각되어져 많은 연구가 진행되고 있다. 본 실험은 체외배양을 통하여 원시생식세포의 증식과 확립을 위해 배양조건을 구명하고, 또한 성장인자의 효과를 검증하기 위하여 실시되었다. 원시생식세포는 12.5일째의 ICR 생쥐태아의 원시생식선 융기조직으로부터 추출하였으며, DMEM + 20% FCS + nucleosides + antibiotics로 조성된 sDMEM 배양액을 사용하여 mitomycin C로 전처리한 되먹임세포단층(feeder layer)위에서 체외배양하였다. bFGF 및 LIF를 20, 40ng/ml농도로 각각 또는 함께 첨가하여 성장인자의 효과를 검토하였다. 원시생식세포는 성에 따라 유의적인 colony 형성율을 보였고(♂:1.9 colonies / genital ridge, ♀:1.3 colonies / genital ridge), bFGF 및 LIF의 첨가 및 첨가농도에 따라서도 유의성 있는 결과를 보였다(0.3~1.9 colonies / genital riege). 그러나 3회 이상 계대배양을 할 경우, 원시생식세포의 colony를 4% prarformaldehyde로 20분간 고정한 후, tris-maleate buffer(pH 9.0)로 10분간 3회 세정하였다. Fast Red로 염색을 실시한 결과, 대부분의 colony가 염색반응을 보여 다능성을 갖는 원시생식세포의 colony임이 입증되었다. 그러나 대부분의 colony가 3회 이상의 계대배양시 생종율이 급격히 떨어지는 것을 감안하면, 또 다른 미지의 성장인자나 보다 적절한 배양조건이 요구된다고 생각된다.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.19-19
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• 2002

This study was to examine whether the in vitro differentiated neural cells derived from human embryonic stem (hES, MB03) cells can be survived and expressed tyrosin hydroxylase(TH) in grafted normal or PD rat brain. To differentiate in vitro into neural cells, embryoid bodies (EB: for 5 days, without mitogen) were formed from hES cells, neural progenitor cells(neurosphere, for 7-10 days, 20 ng/㎖ of bFGF added N2 medium) were produced from EB, and then finally neurospheres were differentiated into mature neuron cells in N2 medium(without bFGF) for 2 weeks. In normal rat brain, neural progenitor cells or mature neuron cells (1×10/sup 7/ cells/㎖) were grafted to the striatum of normal rats. After 2 weeks, when the survival of grafted hES cells was examined by immunohistochemical analysis, the neural progenitor cell group indicated higher BrdU, NeuN＋, MAP2＋ and GFAP＋ than mature neuron cell group in grafted sites of normal rats. This result demonstrated that the in vivo differentiation of grafted hES cells be increased simultaneously in both of neuronal and glial cell type. Also, neural progenitor cell grafted normal rats expressed more TH pattern than mature neuron cells. Based on this data, as a preliminary test, when the neural progenitor cells were grafted into the striatum of 6-hydroxydopamine lesioned PD rats, we confirmed the cell survival (by double staining of Nissl and NeuN) and TH expression. This result suggested that in vitro differentiated neural progenitor cells derived from hES cells are more usable than mature neuron cells for the neural cell grafting in animal model and those grafted cells were survived and expressed TH in normal or PD rat brain.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.80-80
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• 2003

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms, accompanied by marked cell death in the striatum and cortex. Stereotaxic injection of quinolinic acid (QA) into striatum results in a degeneration of GABAergic neurons and exhibits abnormal motor behaviors typical of the illness. The objective of this study was carried out to obtain basic information about whether parthenogenetic mouse embryonic stem (PmES) cells are suitable for cell replacement therapy of HD. To establish PmES cell lines, hybrid F1 (C57BL/6xCBA/N) mouse oocytes were treated with 7% ethanol for 5 min and cytochalasin-B for 4 hr to initiate spontaneous cleavage. Thus established PmES cells were induced to differentiate using bFGF (20ng/ml) followed by selection of neuronal precursor cells for 8 days in N2 medium. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days, then a final differentiation step in N2 medium for 7 days. To establish recipient animal models of HD, young adult mice (7 weeks age ICR mice) were lesioned unilaterally with a stereotaxic injection of QA (60 nM) into the striatum and the rotational behavior of the animals was tested using apomorphine (0.1mg/kg, IP) 7 days after the induction of lesion. Animals rotating more than 120 turns per hour were selected and the differentiated PmES cells (1$\times$10$^4$cells/ul) were implanted into striatum. Four weeks after the graft, immunohistochemical studies revealed the presence of cells reactive to anti-NeuN antibody. However, only a slight improvement of motor behavior was observed. By Nissl staining, cell mass resembling tumor was found at the graft site and near cortex which may explain the slight behavioral improvement. Detailed experiment on cell viability, differentiation and migration explanted in vivo is currently being studied.

• Archives of Plastic Surgery
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• v.33 no.1
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• pp.1-4
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• 2006

Cryopreserved fibroblast implants represent a major advancement for healing of chronic wounds. Bone marrow stromal cells, which include the mesenchymal stem cells, have a low immunity-assisted rejection and are capable of expanding profoundly in a culture media. Therefore, they have several advantages over fibroblasts in clinical use. The ultimate goal of this study was to compare the wound healing accelerating growth factor secretion of the bone marrow stromal cells with that of the fibroblasts and this pilot study particularly focuses on the growth factor secretion to accelerate wound healing. Bone marrow stromal cells and fibroblasts were isolated from the same patients and grown in culture. At 1, 3, and 5 days post-incubating, secretion of basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta(TGF-${\beta}$) were compared. In TGF-${\beta}$ secretion fibroblasts showed 12~21% superior results than bone marrow stromal cells. In contrast, bFGF levels in the bone marrow stromal cells were 47~89% greater than that in fibroblasts. The VEGF levels of the bone marrow stromal cells was 7~12 fold greater than that of the fibroblasts. Our results suggest that the bone marrow stromal cells have great potential for wound healing accelerating growth factor secretion.

• Biomolecules & Therapeutics
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• v.26 no.5
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• pp.474-480
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• 2018

Most angiogenesis assays are performed using endothelial cells. However, blood vessels are composed of two cell types: endothelial cells and pericytes. Thus, co-culture of two vascular cells should be employed to evaluate angiogenic properties. Here, we developed an in vitro 3-dimensional angiogenesis assay system using spheroids formed by two human vascular precursors: endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs). ECFCs, MSCs, or ECFCs+MSCs were cultured to form spheroids. Sprout formation from each spheroid was observed for 24 h by real-time cell recorder. Sprout number and length were higher in ECFC+MSC spheroids than ECFC-only spheroids. No sprouts were observed in MSC-only spheroids. Sprout formation by ECFC spheroids was increased by treatment with vascular endothelial growth factor (VEGF) or combination of VEGF and fibroblast growth factor-2 (FGF-2). Interestingly, there was no further increase in sprout formation by ECFC+MSC spheroids in response to VEGF or VEGF+FGF-2, suggesting that MSCs stimulate sprout formation by ECFCs. Immuno-fluorescent labeling technique revealed that MSCs surrounded ECFC-mediated sprout structures. We tested vatalanib, VEGF inhibitor, using ECFC and ECFC+MSC spheroids. Vatalanib significantly inhibited sprout formation in both spheroids. Of note, the $IC_{50}$ of vatalanib in ECFC+MSC spheroids at 24 h was $4.0{\pm}0.40{\mu}M$, which are more correlated with the data of previous animal studies when compared with ECFC spheroids ($0.2{\pm}0.03{\mu}M$). These results suggest that ECFC+MSC spheroids generate physiologically relevant sprout structures composed of two types of vascular cells, and will be an effective pre-clinical in vitro assay model to evaluate pro- or anti-angiogenic property.

• Biomolecules & Therapeutics
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• v.27 no.1
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• pp.117-125
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• 2019

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has recently been noted as a repositioning candidate for angiogenesis inhibition and cancer therapy. However, the definite anti-angiogenic mechanism of MBZ remains unclear. In this study, we explored the inhibitory mechanism of MBZ in endothelial cells (ECs) and developed a novel strategy to improve its anti-angiogenic therapy. Treatment of ECs with MBZ led to inhibition of EC proliferation in a dose-dependent manner in several culture conditions in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or FBS, without selectivity of growth factors, although MBZ is known to inhibit VEGF receptor 2 kinase. Furthermore, MBZ inhibited EC migration and tube formation induced by either VEGF or bFGF. However, unexpectedly, treatment of MBZ did not affect FAK and ERK1/2 phosphorylation induced by these factors. Treatment with MBZ induced shrinking of ECs and caused G2-M arrest and apoptosis with an increased Sub-G1 fraction. In addition, increased levels of nuclear fragmentation, p53 expression, and active form of caspase 3 were observed. The marked induction of autophagy by MBZ was also noted. Interestingly, inhibition of autophagy through knocking down of Beclin1 or ATG5/7, or treatment with autophagy inhibitors such as 3-methyladenine and chloroquine resulted in marked enhancement of anti-proliferative and pro-apoptotic effects of MBZ in ECs. Consequently, we suggest that MBZ induces autophagy in ECs and that protective autophagy can be a novel target for enhancing the anti-angiogenic efficacy of MBZ in cancer treatment.