• 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.

• Journal of Korean Neurosurgical Society
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• 제64권5호
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• pp.705-715
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• 2021

Objective : Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3-asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. Methods : Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9-10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×10⁵ cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. Results : Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. Conclusion : Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

• 대한임상검사과학회지
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• 제52권2호
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• pp.112-118
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• 2020

이 연구의 목표는 엔지니어링 중간엽 줄기세포에 의해 발현된 SDF-1의 효과를 규명하고 신경인성방광 랫 모델에서 관련 메커니즘을 조사하는 것이다. Sprague-Dawley 랫(N=48)을 대조군, 신경인성방광군, 신경인성방광군+imMSC군 및 신경 인성방광군+SDF-1 eMSC 군으로 무작위 선정하였다. 신경인성방광 랫 모델은 양측 골반 신경 손상으로 유도하였으며 골수 유래 중간엽 줄기세포를 immortalized한 MSC (empty vector)와 upregulated SDF-1한 MSC ( immortalized+SDF-1 치료유전차 발현)로 엔지니어링 하였다. 엔지니어링 중간엽줄기세포를 양측 골반 신경 손상부위와 방광에 주사하여 생착시켰다. 주사 4주 후 치료 효과를 양측골반신경 및 방광 조직을 마손 삼색 염색 및 면역 염색으로 분석하였다. 신경인성방광군+SDF-1 eMSC 군에서 방광 평활근이 유의하게 증가하였다(P<0.05). 신경마커 베타-III 튜불린 및 SDF-1 발현 또한 유의하게 증가하였으며(P<0.05), 이를 통해 손상된 신경을 복구하고, 신경인성 방광 랫 모델의 방광조직을 회복시켰다.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2011년도 제40회 동계학술대회 초록집
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• pp.20-20
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• 2011

It has been known that, under certain conditions, application of low-temperature atmospheric-pressure plasmas can enhance proliferation of cells. In this study, conditions for optimal cell proliferation were examined for various cells relevant for orthopaedic applications. Plasmas used in our experiments were generated by dielectric barrier discharge (DBD) with a helium flow (of approximately 3 litter/min) into ambient air at atmospheric pressure by a 10 kV~20 kHz power supply. Such plasmas were directly applied to a medium, in which cells of interest were cultured. The cells examined in this study were human synoviocytes, rat mesenchymal stem cells derived from bone marrow or adipose tissue, a mouse osteoblastic cell line (MC3T3-E1), a mouse embryonic mesenchymal cell line (C3H-10T1/2), human osteosarcoma cells (HOS), a mouse myoblast cell line (C2C12), and rat Schwann cells. Since cell proliferation can be enhanced even if the cells are not directly exposed to plasmas but cultured in a medium that is pre-treated by plasma application, it is surmised that long-life free radicals generated in the medium by plasma application stimulate cell proliferation if their densities are appropriate. The level of free radical generation in the medium was examined by dROMs tests and correlation between cell proliferation and oxidative stress was observed. Other applications of plasma medicine in orthopaedics, such as plasma modification of artificial bones and wound healing effects by direct plasma application for mouse models, will be also discussed. The work has been done in collaboration with Prof. H. Yoshikawa and his group members at the School of Medicine, Osaka University.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제28권2호
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• pp.111-117
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• 2006

An area of current research is investigating the app1ication of human mesenchymal stem cells or hMSCs as a cell-based regenerative therapy. In order to achieve effective bone regeneration, appropriate matrices functioning as cell-carriers must be identified and optimized in terms of function, efficacy and biocompatibility. Two methods of approaching optimization of matrices are to facilitate adhesion of the donor hMSCs and furthermore to facilitate recruitment of host progenitor cells to osteoblastic differentiation. Pleiotrophin is an extracellular matrix protein that was first identified in developing rat brains and believed to be associated with developing neuronal pathways. A recent publication by Imai and colleagues demonstrated that transgenic mice with upregulated pleiotrophin expression developed a greater volume of cortical as well as cancellous bone. The proposed mechanism of action of pleiotrophin is demonstrated here. Through either environmental stresses and/or intracellular regulation, there is an increase in pleiotrophin production. The pleiotrophin is released extracellularly into areas requiring bone deposition. A receptor-mediated process recruits host osteoprogenitor cells into these areas. Therefore, the aim of our study was to investigate the osteoconductive properties of pleiotrophin. We wanted to determine if pleiotrophin coating facilitates cellular adhesion and furthermore if this has any effect on hMSCs derived bone formation in an animal model. The results showed a dose dependent response of cellular adhesion in fibronectin samples, and cellular adhesion was facilitated with increasing pleiotrophin concentrations. Histologic findings taken after 5 weeks implantation in SCID mouse showed no presence of bone formation with only a dense fibrous connective tissue. Possible explanations for the results of the osteogenesis assay include inappropriate cell loading.

• Nuclear Medicine and Molecular Imaging
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• 제42권5호
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• pp.394-400
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• 2008

목적: 줄기 세포에서 렌티바이러스와 아데노바이러스를 이용해 유전자를 전달하였을 때 유전자 발현 정도를 정량적으로 비교하는 연구는 보고되지 않았다. 저자들은 쥐의 중간엽줄기세포(이하 rMSC)에 사람 나트륨/옥소 공동수송체 (이하 hNIS) 유전자를 렌티바이러스와 아데노바이러스를 통하여 전달하고 발현 정도를 정량적으로 비교해 보았다. 대상 및 방법: hNIS를 발현하는 rMSE (lenti-hNIS-rMSC)의 생산은 hNIS유전자를 렌티바이러스 벡터인 pLenti/UbC/V5-DEST (Invitrogen)에 클로닝하여 pLenti-hNIS를 얻은 후 rMSC에 감염시켜서 3주간 blasticidin으로 선별하였다. hNIS를 발현하는 재조합 아데노바이러스(Rad-hNIS)는 homologous recombination 방법에 의하여 생산하였으며 Rad-hNIS의 rMSC에 대한 transduction efficiency는 Rad-GFP를 사용하여 MOI 1, 5, 20, 고리고 100에서 평가하였고 그 결과는 각각 $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, 그리고 $98.4{\pm}1.3%$이었다. 두 바이러스 전달 시스템에서 hNIS의 발현정도를 ummunocytochemistry, western blot 그리고 방사성옥소 섭취실험을 통해 평가하였다. 결과: Immunocytochemistry와 western blot으로 hNIS 단백질의 양을 비교하였을 때 lenti-hNIS-rMSC에서 발현하는 hNIS 단백질의 양은 Rad-hNIS를 rMSC에 MOI 20으로 감염시킨 경우보다는 많았으나 MOI 50 보다는 작았다. 그러나 방사성옥소 섭취실험에서 lenti-hNIS-rMSC ($29,704{\pm}6,659\;picomole/10^6\;cells$)는 MOI 100의 Rad-hNIS를 rMSE에 감염시킨 경우($6,168{\pm}2,134\;picomole/10^6\;cells)$ 보다도 높은 섭취율을 보였다. 결론: 렌티바이러스를 통하여 hNIS를 rMSC에서 발현하도록 했을 때 아데노바이러스를 전달 벡터로 사용한 경우에 비하여 단백질 발현 양은 상대적으로 적었으나 hNIS의 기능은 더 우수하였다. hNIS를 리포터 유전자로 사용한 줄기세포추적은 벡터에 따른 유전자 발현효율의 차이를 고려하고 시행되어야 할 것으로 생각한다.

• Journal of Korean Neurosurgical Society
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• 제58권2호
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• pp.101-106
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• 2015

Objective : The aim of this study was to explore the immunity in rats transplanted with adipose-derived mesenchymal stem cells (ADSCs) and acellular nerve (ACN) for repairing sciatic nerve defects. Methods : ADSCs were isolated from the adipose tissues of Wistar rats. Sprague-Dawley rats were used to establish a sciatic nerve defect model and then divided into four groups, according to the following methods : Group A, allogenic nerve graft; Group B, allograft with ACN; Group C, allograft ADSCs+ACN, and Group D, nerve autograft. Results : At the day before transplantation and 3, 7, 14, and 28 days after transplantation, orbital venous blood of the Sprague-Dawley rats in each group was collected to detect the proportion of $CD3^+$, $CD4^+$, and $CD8^+$ subsets using flow cytometry and to determine the serum concentration of interleukin-2 (IL-2), tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$) and $interferon-{\gamma}$ ($IFN-{\gamma}$) using enzyme-linked immunosorbent assay (ELISA). At each postoperative time point, the proportion of $CD3^+$, $CD4^+$, and $CD8^+$ subsets and the serum concentration of IL-2, $TNF-{\alpha}$, and $IFN-{\gamma}$ in group C were all near to those in group B and group D, in which no statistically significant difference was observed. As compared with group A, the proportion of $CD3^+$, $CD4^+$, and $CD8^+$ subsets and the serum concentration of IL-2, $TNF-{\alpha}$, and $IFN-{\gamma}$ were significantly reduced in group C (p<0.05). Conclusion : The artificial nerve established with ADSCs and ACN has no obvious allograft rejection for repairing rat nerve defects.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8271-8279
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• 2016

Background: Hepato-carcinogenesis is multifaceted in its molecular aspects. Among the interplaying agents are altered gap junctions, the proteasome/autophagy system, and mitochondria. The present experimental study was designed to outline the roles of these players and to investigate the tumor suppressive effects of curcumin with or without mesenchymal stem cells (MSCs) in hepatocellular carcinoma (HCC). Materials and Methods: Adult female albino rats were divided into normal controls and animals with HCC induced by diethyl-nitrosamine (DENA) and $CCl_4$. Additional groups treated after HCC induction were: Cur/HCC which received curcumin; MSCs/HCC which received MSCs; and Cur+MSCs/HCC which received both curcumin and MSCs. For all groups there were histopathological examination and assessment of gene expression of connexin43 (Cx43), ubiquitin ligase-E3 (UCP-3), the autophagy marker LC3 and coenzyme-Q10 (Mito.Q10) mRNA by real time, reverse transcription-polymerase chain reaction, along with measurement of LC3II/LC3I ratio for estimation of autophagosome formation in the rat liver tissue. In addition, the serum levels of ALT, AST and alpha fetoprotein (AFP), together with the proinflammatory cytokines $TNF{\alpha}$ and IL-6, were determined in all groups. Results: Histopathological examination of liver tissue from animals which received DENA-$CCl_4$ only revealed the presence of anaplastic carcinoma cells and macro-regenerative nodules. Administration of curcumin, MSCs; each alone or combined into rats after induction of HCC improved the histopathological picture. This was accompanied by significant reduction in ${\alpha}$-fetoprotein together with proinflammatory cytokines and significant decrease of various liver enzymes, in addition to upregulation of Cx43, UCP-3, LC3 and Mito.Q10 mRNA. Conclusions: Improvement of Cx43 expression, nonapoptotic cell death and mitochondrial function can repress tumor growth in HCC. Administration of curcumin and/or MSCs have tumor suppressive effects as they can target these mechanisms. However, further research is still needed to verify their effectiveness.

• 생명과학회지
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• 제31권8호
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• pp.710-718
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• 2021

태반유래 중간엽줄기세포(PD-MSCs)는 재생의학에서 세포기반치료제로 잘 알려진 세포군이다. PD-MSCs의 손상된 부위로의 이동과 호밍 기능은 MSC 생착의 중요한 특성이다. miRNA는 최근 MSC의 증식, 생존 이동과 같은 중요한 기능을 조절하는 것으로 알려져 있다. 본 연구의 목적은 담관결찰(BDL) 쥐 모델에서 PD-MSCs 호밍에 관련된 miRNA 및 표적 유전자를 동정하는 것으로, 마이크로어레이 분석을 이용하여 PD-MSCs 호밍에 관여하는 유전자 표적 miRNA를 선별하였다. BDL 쥐모델에 PD-MSCs을 이식한 일주일 후 간 조직에서 PD-MSCs 생착여 부는 면역형광분석법과 qRT-PCR에 의한 인간 Alu유전자 발현으로 확인되었다. 저산소 및 정상조건(Hyp/Nor)에서 이동한 PD-MSC에 비하여, PD-MSCs 이식한 BDL군 간 조직에서 miRNAs 발현의 차이가 크게 나타났으며, PD-MSCs 호밍 관련 miRNA와 표적유전자를 검증하였다. miR199a-5p 및 miR-148a-3p에 대한 표적 유전자 인테그린 α4 (ITGA4)와 α5 (ITGA5)의 발현은 이식(Tx)그룹에서(p<0.05) 유의하게 상향 조절되었다. 또한 인테그린 β1 (ITGB1)과 β8 (ITGB8)의 발현은 miR-183-5p 및 miR-145-5p억제에 의하여 크게 증가되었다. 따라서 이러한 결과는 BDL에 의해 손상된 쥐간에서 PD-MSCs가 호밍효과을 위해 인테그린 그룹과 관련된 miRNA 발현 조절에 관여함을 나타내었다. 본 연구결과는 miRNA에 의한 인테그린 그룹 조절기능이 BDL에 의해 유도된 간섬유증 쥐모델에서 PD-MSCs의 치료효과에 기여할 수 있음을 시사한다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.71-71
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• 2003

Human umbilical cord blood cells(HUCBC) are rich in mesenchymal progenitor cells, endothelial cell precursors and hematopoietic cells. HUCBC have been used as a source of transplantable stem and progenitor cells. However, little is known about survival and development of HUCBC transplantation in the CNS. Estrogen has a neuroprotective potential against oxidative stress-induced cell death so has an effect on reducing infarct size of ischemic brain. We investigated the potential use of HUCBC as donor cells and tested whether estrogen mediates intravenously infused HUCBC enter and survive in ischemic brain. PKH26 labeled mononuclear fraction of HUCBC were injected into the tail vein of ischemic OVX rat brain with or without $17\beta$-estradiol valerate(EV). Under fluorescence microscopy, labeled cells were observed in the brain section. Significantly more cells were found in the ischemic brain than in the non-ischemic brain. HUCBC transplanted into ischemic brain could migrate and survive. Some of cells have shown neuronal like cells in hippocampus, striatum and cortex tissues. These result suggest that estrogen reduces ischemic damage and increases the migration of human umbilical cord blood cells. This Study was supported by the Korea Science and Engineering Foundation(KOSEF) though the Biohealth Products Research Center(BPRC), Inje University, Korea.