Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

Bone marrow (BM) cell harvesting is a crucial element in the isolation of mesenchymal stem cells (MSCs). A simple method for harvesting cat BM cells is described. The results show that a large number of BM cells can rapidly be harvested from the cat by this simple procedure. MSCs prepared by density-gradient method were spindle-shaped morphology with bipolar or polygonal cell bodies and strongly positive for CD9 and CD44 and negative for CD18 and CD45-like. They were capable of differentiation to adipocytic and osteocytic phenotypes when exposed to appropriate induction media. The advantages of this method are its rapidity, simplicity, low invasiveness, and low donor attrition and good outcome.

Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal and differentiation into a variety of cell types. They represent an attractive source of cells for gene and cell therapy. The purpose of this study is to direct the specific expression of the DsRed reporter gene in Sca-1+ BMMSCs differentiated into a cardiomyogenic lineage. We constructed the prMLC-2v-DsRed vector expressing DsRed under the control of the 309 tp fragment of the rat MLC-2v 5'-flanking region. The specific expression of the DsRed reporter gene under the transcriptional control of the 309 bp fragment of the rat MLC-2v promoter was tested in 5-azacytidine healed-Sca-1+ BMMSCs over 2 weeks after the prMLC-2v-DsRed transfection. The prMLC-2v-DsRed was specifically expressed in the Sca-1+ BMMSCs with cardiomyogenic lineage differentiation and it demonstrates that the 309 bp sequences of the rat MLC-2v 5'-flanking region is sufficient to confer cardiac specific expression on a DsRed reporter gene. The cardiac-specific promoter-driven reporter vector provides an important tool for the study of stem cell differentiation and cell replacement therapy in ischemic cardiomyopathy.

시험물질 산삼배양추출물의유전독성 평가를 위해 수컷 ICR 마우스 골수세포를 이용한 소핵시험을 실시하였다. 1회 투여 최고량은 예비 시험에서 결정하였다. 약 7주령의 수컷 마우스에 시험물질 0, 500, 1,000 및 2,000 mg/kg의 용량을 1일 1회 2일간 복강내 투여하고, 최종 투여로부터 약 24시간 후에 골수세포를 수거하여 소핵 유발과 세포독성을 평가하였다. 개체당 2,000개의 다염성적혈구(michromatic crythrocyte, PCE)중에 나타나는 소핵을 가진 다염성 적혈구(micronucleated polychromatic erythrocyte, MNPCE)의 수를 게수한 결과, 모든 시험물질 투여군은 음성 대조군에 비해 통계학적으로 유의한 증가는 나타나지 않았으며 일반 증상에서도 모든 시험군은 투여로 인한 것으로 판단되는 증상은 관찰되지 않았다. 부검시 체중에 있어서는 시험물질 최고 용량군에서 유의한 감소가 관찰되었다. 따라서 산삼배양추출물은 위 시험 조건에서, 본 시험에 사용한 마우스 골수세포에 소핵을 유발하지 않는 것으로 사료된다.

Mesenchymal stem cells (MSCs) are considered to be attractive approaching in gene or drug delivery for cancer therapeutic strategies. In this study, the ability and feasibility of human bone marrow derived MSCs expressing the cytosine deaminase (CD)/5-Fluorocytosin (5-FC) prodrug was evaluated to target human osteosarcoma cell line Cal-72. At first, the fibroblast-like cells were successfully obtained from human bone marrow and demonstrated that they contained full of stem characteristics by the ability of differentiation into adipocyte/osteocyte and expression of typical mesenchymal markers CD90, CD44, while negative for CD34 and CD133 markers. We established the stable CD-expressing MSCs cell line (CD-MSCs) by transfection of pEGFP-C3 containing cytosine deaminase::uracil phos-phoribosyltransferase (CD::UPRT) gene into MSCs, and confirmed that the manipulated MSCs still remained full characteristics of multipotent cells and shown migration toward human osteosarcoma cancer cells Cal-72 as high as origin MSCs. Based on bystander effect, the therapeutic CD-MSCs significantly augmented the cytotoxicity on cancer cell Cal72 in either direct co-culture or conditioned medium in the presence of 5-FC. Moreover, in osteosarcoma cancer- bearing mice, the therapeutic CD/5-FC MSCs showed the inhibition of tumor growth compared with control mice which was s.c injected with only Cal72. Our findings suggest that these therapeutic CD-MSCs may be suitable and viable cellular vehicles for targeting human osteosarcoma cancer.

Hematopoietic stem cells (HSCs) can self-renew and can differentiate to a variety of specialized blood cells). The proliferation and homing of HSCs are strictly regulated both in the system level and local level. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, potent species-specific stimulator of granulocyte-macrophage, eosinophil, megakaryocyte and erythroid progenitors. In clinical purpose GM-CSF has been used as hematopoietic growth factor. It can promote the mobilization of HSC from bone marrow to peripheral blood. The number of HSCs mobilized into blood can be modulated by the kinds of cytolines. However, the information for the cytokine which promote mobilization is limited. Basic fibroblast growth factor (bFGF or FGF-2) induces the change of niche and affects the maintenance and differentiation of HSCs. FGF-2 positively regulates hematopoiesis, by acting on stroma cells, on early and committed hematopoietic progenitors, and possibly on some mature blood cells. In this study, we investigated the effect of FGF-2 on HSCs mobilization and proliferation compare to GM-CSF. GM-CSF and FGF-2 were injected for 2 or 5 days into peritoneum of CD-1 mice (6～8 wks old) and sampling the bone marrow and peripheral blood. The bone marrow cells and peripheral blood were analyzed using FACS. In GM-CSF group, the number of HSCs was significantly increased by 2 days of injection but was significantly decreased in 5 days of injection. On the other hand the number of HSCs was significantly increased by the administration of FGF2 both in 2 days and 5 days. GM-CSF and FGF-2 are all increased the number of HSC both in bone marrow and peripheral blood. From these results, it is revealed that chronic administration of GM-CSF does not cause of the increase the number of HSCs. On the other hand, FGF2 can stimulate the proliferation of HSC without inhibition by the treatment period. It is suggested that GM-CSF and FGF2 may use different mechanisms to stimulate the HSCs proliferation. IP: 220.149.***.