• 한국수정란이식학회지
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• 제27권3호
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• pp.183-187
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• 2012

Adult stem cell transplantation has been increased every year, because of the lack of organ donors for regenerative medicine. Therefore, development of reliable and safety cryopreservation and bio-baking method for stem cell therapy is urgently needed. The present study investigated safety of dimethyl sulfoxide (DMSO) such as common cryoprotectant on porcine bone marrow derived mesenchymal stem cells (pBM-MSCs) by evaluating the activation of Caspase-3 and -7, apoptosis related important signal pathway. pBM-MSCs used for the present study were isolated density gradient method by Ficoll-Paque Plus and cultured in A-DMEM supplemented 10% FBS at $38.5^{\circ}C$ in 5% $CO_2$ incubator. pBM-MSCs were cryopreserved in A-DMEM supplemented either with 5%, 10% or 20% DMSO by cooling rate at $-1^{\circ}C$/min in a Kryo 360 (planner 300, Middlesex, UK) and kept into $LN_2$. Survival rate of cells after thawing did not differ between 5% and 10% DMSO but was lowest in 20% DMSO by 0.4% trypan blue exclusion. Activation of Caspase-3 and -7 by Vybrant FAM Caspase-3 and -7 Assay Assay Kit (Molecular probes, Inc.OR, USA) was analyzed with a flow cytometer. Both of cryopreserved and control groups (fresh pBM-MSCs) were observed after the activation of Caspase-3 and -7. The activation did not differ between 5% and 10% DMSO, but was observed highest in 20% DMSO. Therefore 5% DMSO can be possibly used for cell cryopreservation instead of 10% DMSO.

• 대한의생명과학회지
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• 제23권2호
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• pp.49-56
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• 2017

Fibroblast growth factor (FGF)-2 is one of the most effective growth factors to increase the growth rate of mesenchymal stem cells (MSCs). Previously, we reported that low dose of FGF-2 (1 ng/ml) induced proliferation of bone marrow-derived mesenchymal stem cells (BMSCs) through AKT and ERK activation resulting in reduction of autophagy and senescence, but not at a high dose. In this study, we investigated the effects of high dose FGF-2 (10 ng/ml) on proliferation, autophagy and senescence of BMSCs for long term cultures (i.e., 2 months). FGF-2 increased the growth rate of BMSCs in a dose dependent manner for a short term (3 days), while during long term cultures (2 months), population doubling time was increased and accumulated cell number was lower than control in BMSCs when cultured with 10 ng/ml of FGF-2. 10 ng/ml of FGF-2 induced immediate de-phosphorylation of ERK1/2, expression of LC3-II, and increase of senescence associated ${\beta}$-galactosidase (SA-${\beta}$-Gal, senescence marker) expression. In conclusion, we showed that 10 ng/ml of FGF-2 was inadequate for ex vivo expansion of BMSCs because 10 ng/ml of FGF-2 induced growth retardation via ERK1/2 de-phosphorylation and induction of autophagy and senescence in BMSCs.

• Journal of Periodontal and Implant Science
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• 제39권2호
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• pp.119-128
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• 2009

Purpose: The aim of this study was to evaluate the stemness of cells from canine dental tissues and bone marrow. Methods: Canine periodontal ligament stem cells (PDLSC), alveolar bone stem cells (ABSC) and bone marrow stem cells(BMSC) were isolated and cultured. Cell differentiations (osteogenic, adipogenic and chondrogenic) and surface antigens (CD146, STRO-1, CD44, CD90, CD45, CD34) were evaluated in vitro. The cells were transplanted into the subcutaneous space of nude mice to assess capacity for ectopic bone formation at 8 weeks after implantation. Results: PDLSC, ABSC and BMSC differentiated into osteoblasts, adipocytes and chondrocytes under defined condition. The cells expressed the mesenchymal stem cell markers differently. When transplanted into athymic nude mice, these three kinds of cells with hydroxyapatite /${\beta}$- tricalcium phosphate (HA/TCP) carrier showed ectopic bone formation. Conclusions: This study demonstrated that canine dental stem cells have stemness like bone marrow stem cells. Transplantation of these cells might be used as a therapeutic approach for dental stem cell-mediated periodontal tissue regeneration.

• 대한임상검사과학회지
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• 제40권2호
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• pp.106-112
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• 2008

Bone marrow derived mesenchymal stem cells (BMSCs) are largely studied for their potential clinical use. But it is hard to get enough number of those cells for clinical trials and give serious pain to the patients. Adipose tissue is derived from the embryonic mesenchyme and contains a stroma that is easily isolated with large amount. This cell population (adipose derived stem cells: ADSCs) can be isolated from human lipoaspirates and like MSCs, differentiate toward the osteogenic, adipogenic, myogenic and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the ADSCs extracted from omental or subcutaneous fat tissue were expanded during third to fifth passages. The phenotype of the ADSCs was identified by the conventional cell surface markers using flow cytometry: positive for CD29 and CD44, but negative for CD34, CD45, CD117 and HLA-DR that similar to those observed on BMSCs. The ADSCs were able to differentiate into the osteoblast or adipocytes with induction media. Finally, ADACs expressed multiple CD marker antigens similar to those observed on BMSCs and differentiated into osteoblast, adipocyte. With this, human adipotissue contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

• 대한두개안면성형외과학회지
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• 제19권3호
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• pp.181-189
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• 2018

Background: Autogenous bone grafts have several limitations including donor-site problems and insufficient bone volume. To address these limitations, research on bone regeneration is being conducted actively. In this study, we investigate the effects of a three-dimensionally (3D) printed polycaprolactone (PCL)/tricalcium phosphate (TCP) scaffold on the osteogenic differentiation potential of adipose tissue-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMSCs). Methods: We investigated the extent of osteogenic differentiation on the first and tenth day and fourth week after cell culture. Cytotoxicity of the 3D printed $PCL/{\beta}-TCP$ scaffold was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, prior to osteogenic differentiation analysis. ADSCs and BMSCs were divided into three groups: C, only cultured cells; M, cells cultured in the 3D printed $PCL/{\beta}-TCP$ scaffold; D, cells cultured in the 3D printed $PCL/{\beta}-TCP$ scaffold with a bone differentiation medium. Alkaline phosphatase (ALP) activity assay, von Kossa staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting were performed for comparative analysis. Results: ALP assay and von Kossa staining revealed that group M had higher levels of osteogenic differentiation compared to group C. RT-PCR showed that gene expression was higher in group M than in group C, indicating that, compared to group C, osteogenic differentiation was more extensive in group M. Expression levels of proteins involved in ossification were higher in group M, as per the Western blotting results. Conclusion: Osteogenic differentiation was increased in mesenchymal stromal cells (MSCs) cultured in the 3D printed PCL/TCP scaffold compared to the control group. Osteogenic differentiation activity of MSCs cultured in the 3D printed PCL/TCP scaffold was lower than that of cells cultured on the scaffold in bone differentiation medium. Collectively, these results indicate that the 3D printed PCL/TCP scaffold promoted osteogenic differentiation of MSCs and may be widely used for bone tissue engineering.

• 생명과학회지
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• 제24권11호
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• pp.1238-1243
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• 2014

중간엽줄기세포(mesenchymal stem cell, MSC)은 세포치료로 각광받아 널리 사용되고 있다. 이들은 줄기세포의 분화성을 이용하여 많은 만성질환에 연관되어 치료제로 사용되고 있다. 줄기세포는 다른 화학적 치료법에 비해 많은 장점을 가지고 있다. 왜냐하면 줄기세포치료는 자기자신, 혹은 동종의 세포를 이용한 치료이기 때문에 화학 치료에 비해 부작용이나 치료의 위험성이 덜하다. 그리고 마이크로RNA또한 최근 기 존재와 기능이 밝혀져서 연구되고 있는데 특히 항암, 세포생장촉진 등의 기능을 이용해 항암, 만성질환 치료에 접목되어 치료제로의 역할이 기대된다. 마이크로RNA는 대부분의 대사과정이나 항상성조절에 관여되어있다. 따라서 마이크로RNA가 저 발현 혹은 과 발현하게 되면 만성질환으로 이어지게 된다. 하지만 줄기세포와 마이크로RNA의 상호간 보조효과는 잘 연구되어 있지 않다. 따라서 이들 간의 상관관계를 확인하기 위하여 태반유래 줄기세포(PDSC)와 골수줄기세포(BM-MSC), 대조군으로 섬유아세포(Fibroblast, WI-38)을 사용하여 이들이 발현하는 마이크로RNA 발현을 확인해 보았다. 각각의 MSC 세포주에 대하여 특정 마이크로RNA의 발현량을 확인해 보았다. 결과 PDSC의 경우엔 마이크로RNA-34a의 발현이 높았고 BM-MSC의 경우에는 마이크로RNA-27a, 33a, 33b, 211의 발현이 높은 것을 확인할 수 있었다. 따라서 우리는 각각의 MSC세포주와 그들이 발현하는 기능성 마이크로RNA을 연관지어 효과적인 세포치료에 활용될 수 있을 것을 기대한다.

• 대한한방내과학회지
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• 제38권1호
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• pp.1-9
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• 2017

Objective: This study investigated the effect of purified Carthamus tinctorius (C. tinctorius) extracted with a hot water and ethanol method on adipogenic differentiation of mouse bone marrow-derived mesenchymal stromal stem cells (mBMSCs). Methods: The C. tinctorius was extracted using hot water and ethanol. The samples were concentrated by a rotary evaporator and were then dried using a freeze-dryer. The mBMSCs were cultured and maintained in a minimum essential medium eagle alpha (${\alpha}-MEM$) supplemented with 10% FBS and 1% antibiotic antimycotic solution. To induce adipogenic differentiation, the cells were treated with Dulbecco's modified eagle's medium-low glucose (DMEM-LG) containing 1 mg/mL insulin, 1 mM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine. To evaluate the adipogenic differentiation ability, oil-red O staining was performed after adipogenic differentiation for 21 days. The mRNA expression and protein level of adipogenic-related genes were quantified by quantitative real-time PCR and western blotting, respectively. Results: In the results of the MTT assay, no concentrations of C. tinctorius extracts showed toxicity on mBMSCs, so we fixed the treatment concentration of the extract at 100 ng/mL. In oil-red O staining, the water-C. tinctorius extract treatment significantly decreased adipogenic differentiation compared with the control and ethanol extract groups. The water-C. tinctorius extract group in particular showed reduced mRNA and protein expression of Peroxisome proliferator-activated receptor gamma ($Ppar{\gamma}$) and CCAAT/enhancer-binding protein alpha ($C/ebp{\alpha}$), which are adipogenic-related transcription factors. Conclusion: These data suggest that extract of C. tinctorius decreased the adipogenic differentiation of mBMSCs, while only water-C. tinctorius extract had an effect on different adipogenesis in mBMSCs. The C. tinctorius will be a useful therapeutic reagent for the prevention of obesity-related diseases such as diabetes, hyperlipidemia, coronary artery disease, and osteoporosis.

• The Korean Journal of Physiology and Pharmacology
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• 제20권1호
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• pp.63-67
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• 2016

Severe graft-versus-host disease (GVHD) is an often lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). The safety of clinical-grade mesenchymal stem cells (MSCs) has been validated, but mixed results have been obtained due to heterogeneity of the MSCs. In this phase I study, the safety of bone marrow-derived homogeneous clonal MSCs (cMSCs) isolated by a new subfractionation culturing method was evaluated. cMSCs were produced in a GMP facility and intravenously administered to patients who had refractory GVHD to standard treatment resulting after allogeneic HSCT for hematologic malignancies. After administration of a single dose ($1{\times}10^6cells/kg$), 11 patients were evaluated for cMSC treatment safety and efficacy. During the trial, nine patients had 85 total adverse events and the rate of serious adverse events was 27.3% (3/11 patients). The only one adverse drug reaction related to cMSC administration was grade 2 myalgia in one patient. Treatment response was observed in four patients: one with acute GVHD (partial response) and three with chronic GVHD. The other chronic patients maintained stable disease during the observation period. This study demonstrates single cMSC infusion to have an acceptable safety profile and promising efficacy, suggesting that we can proceed with the next stage of the clinical trial.

• IMMUNE NETWORK
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• 제14권2호
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• pp.81-88
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• 2014

Mesenchymal stem cells (MSCs) are present in diverse tissues and organs, including bone marrow, umbilical cord, adipose tissue, and placenta. MSCs can expand easily in vitro and have regenerative stem cell properties and potent immunoregulatory activity. They inhibit the functions of dendritic cells, B cells, and T cells, but enhance those of regulatory T cells by producing immunoregulatory molecules such as transforming growth factor-${\beta}$, hepatic growth factors, prostaglandin $E_2$, interleukin-10, indolamine 2,3-dioxygenase, nitric oxide, heme oxygenase-1, and human leukocyte antigen-G. These properties make MSCs promising therapeutic candidates for the treatment of autoimmune diseases. Here, we review the preclinical studies of MSCs in animal models for systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, and experimental autoimmune encephalomyelitis, and summarize the underlying immunoregulatory mechanisms.

• 생명과학회지
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• 제26권12호
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• pp.1355-1359
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• 2016

인간 중간엽 줄기세포(hMSCs)는 신경세포(neuron-like cells)를 포함한 다양한 세포로 분화할 수 있는 능력을 지닌 성체 줄기세포(adult stem cells)이다. 본 연구에서는 인간의 골수유래-중간엽 줄기세포(bone marrow-mesenchymal stem cells; hBM-MSCs)를 이용한 신경분화에서 신경세포 표지자(neuronal marker)인 NF-M, Tuj-1 뿐만 아니라 성상세포 표지자(glial marker)인 GFAP의 발현 역시 의미 있게 증가함을 real-time PCR, Western blot, and immunocytochemical staining법을 통하여 관찰하였다. 이를 개선하기 위하여, 신경분화에 중요한 신호전달자(signal intermediator)인 PDE4를 억제한 후 신경분화를 유도하였다. PDE4 억제자인 rolipram 혹은 resveratrol를 각각 처리하여 신경분화한 줄기세포(Roli- or RSV-dMSCs)에서 NF-M, Tuj-1의 발현이 증가하였고 반면, GFAP의 발현은 감소함을 real-time PCR, Western blot, and immunocytochemical staining법을 통하여 관찰하였다. 본 연구를 통하여, PDE4를 조절하며 줄기세포의 신경분화를 개선할 수 있음을 보였다.