배반포 단계의 난자에서 차이 나게 발현하는 유전자의 발굴을 통해 초기 동물 발생과 분화에 관한 기전을 알 수 있다. 본 연구에서는 새로운 차별발현 역전사효소중합법, 이름하여 에닐링 콘트롤 프라이머(ACP) 방법에 의해 생쥐 배반포에서 차이 나게 발현하는 유전자를 줄기세포와 비교하여 발굴하였다. 총 100개의 ACP를 사용하여 26개의 유전자 단편을 확인하였고, BLAST 탐색에 의해 유전자 정보 은행(GeneBank/EMBL)에 저장된 유전자와 동일하다

As an effort to direct differentiation of human embryonic stem (hES, MB03) cells to dopamine-producing neuronal cells, Nurr1 was transfected using conventional transfection protocol into MB03 and examined the expression of tyrosine hydroylase (TH) after differentiation induced by retinoic acid (RA) and ascorbic acid (AA). Experimentally, cells were transfected with linearized Nurr1 cDNA in pcDNA3.1 (+)-hygovernight followed by selection in medium containing hygromycin-B (150 /ml). Expression of Nurr1 mRNA was confirmed by RT-PCR and protein by immunocytochemistry in the drug resistant clones. In order to study the effect of Nurr1 protein on the differentiation pattern of ES cells, one of the positive clones (MBNr24) was allowed to form embryoid body (EB) for 2 days and were induced to differentiate for another 4 days using RA (1 ) and AA (50 mM) (2-/4+ protocol) followed by selection in N2 medium for 10 or 20 days. After 10 days in N2 medium, cells immunoreactive to anti-GFAP, anti-TH, or anti-NF200 antibodies were 38.8%, 11%, and 20.5%, respectively. After 20 days in N2 medium, cells expressing GFAP, TH, or NF200 were 28%, 15% and 44.8%, respectively but approximately 9% of MB03 expressed TH protein when the cells were induced to differentiate using a similar prorocol, These results suggest that ectopic expression of Nurr1 enhances generation of TH+ cells as well as neuronal cells when hES cells were differentiated by 2-/4+ protocol.

The purpose of this study is to evaluate an efficacy of in vitro differentiated human embryonic stem (hES, MB03) cells expressing Nurr1 in relief of symptomatic motor behavior of Parkinson's disease (PD) animal models MB03 was genetically modified to express Nurr1 protein and was induced to differentiate according to 2-/4+ protocol using retinoic acid and ascorbic acid. The differentiation-induced cells were selected for 10 to 20 days thereafter in N2 medium. Upon selection, cells expressing GFAP, TH, or NF200 were 38.8%, 11%, and 20.5%, respectively. in order to examine therapeutic effects of the differentiated cells in PD animal model, rats were unilaterally lesioned by administration of 6-kydroxydopamine HCI (6-OHDA) into medial forebrain region (MFB, AP -4.4 mm, ML 1.2 mm, DV 78 mm with incision bar set at -2.4 mm), as a reference to bregma and the surface of the skull. Confirmation of successful lesion by apomorphine-induced rotational behavior, differentiated cells were transplanted into the striatum (AP 1.0, ML 3.5, DV -5.0; AP 0.6, ML 2.5, DV -4.5). Improvements of asymmetric motor behavior by the transplantation were examined every two weeks after the surgery. In two weeks, numbers of rotation by the experimental rats were (P<0.05) of the number before transplantation, however, the ratio increased slightly to in six weeks. In contrast, the ratio of sham-grafted animals ranged from 112.3+8.5% to 139.2+28.9% during the examination. Immunohistochemical studies further confirmed the presence, survival, migration, and expression of TH of the transplanted human cells.

Embryonic stem (ES) cells proliferate extensively in the undifferentiated state and have the potential to differentiate into a variety of cell types in response to various environmental cues. The generation of functional dopaminergic neurons from ES cells is promising for cell replacement therapy to treat Parkinson's disease. We compared the in vitro differentiation potential of pluripotent human embryonic stem (hES, MB03) cells induced with basic fibroblast growth factor (bFGF) or retinoic acid (RA). Both types of treatment resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)- during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression, compared to control (P<0.05). In contrast, no effect was observed on the rate of mature or glutamic acid decarboxylase-positive neurons. Immunostaining and HPLC analyses revealed the higher levels of TH (20.3%) and dopamine in bFGF and TGF- treated hES cells than in RA or BDNF treated hES cells. The results indicate that TGF- may be successfully used in the bFGF induction protocol to yield higher numbers of functional dopaminergic neurons from hES cells.

Main strategy for a treatment of Parkinson's disease (PD), due to a progressive degeneration of dopaminergic neurons, is a pharmaceutical supplement of dopamine derivatives or ceil replacement therapy. Both of these protocols have pros and cons; former exhibiting a dramatic relief but causing a severe side effects on long-term prescription and latter also having a proven effectiveness but having availability and ethical problems Embryonic stem (ES) cells have several characteristics suitable for this purpose. To investigate a possibility of using ES cells as a carrier of therapeutic gene(s), human ES (hES, MB03) cells were transfected with cDNAs coding for tyrosine hydroxylase (TH) in pcDNA3.1 (+) and the transfectants were selected using neomycin (250 ). Expression of TH being confirmed, two of the positive clone (MBTH2 & 8) were second transfected with GTP cyclohydrolase 1 (GTPCH 1) in pcDNA3.1 (+)-hyg followed by selection with hygromycin-B (150 ) and RT-PCR confirmation. By immune-cytochemistry, these genetically modified but undifferentiated dual drug-resistant cells were found to express few of the neuronal markers, such as NF200, -tubulin, and MAP2 as well as astroglial marker GFAP. This results suggest that over-production of BH4 by ectopically expressed GTPCH I may be involved in the induction of those markers. Transplantation of the cells into striatum of 6-OHDA- denervated PD animal model relieved symptomatic rotational behaviors of the animals. Immunohistochemical analyses showed the presence of human cells within the striatum of the recipients. These results suggest a possibility of using hES cells as a carrier of therapeutic gene(s).

Pluripotent embryonic stem (ES) cells differentiate spontaneously into beating cardiomyocytes via embryo-like aggregates. We describe the use of mouse embryonic stem (mES03) cells as a reproducible differentiation system for cardiomyocyte. To induce cardiomyocytic differentiation, mES03 cells were dissociated and allowed to aggregate (EB formation) at the presence of 0 75% dimethyl sulfoxide (DMSO) for 4 days and then another 4 days without DMSO (4+/4-). Thus treated EBs were plated onto gelatin-coated dish for differentiation. Spontaneously contracting colonies which appeared in approximately 4-5 days upon differentiation. Expression of cardiac-specific genes were determined by RT-PCR. Rebust expression of myosin light chain (MLC-2V), cardiac myosin heavy chain , cardiac muscle heavy polypeptide 7 -MHC), cardiac transcription factor GATA4 and skeletal muscle-specific -subunit of the L-type calcium channel () were detected as early as 8 days after EB formation, but message of cardiac muscle-specific -subunit of the L-type calcium channel (CaCh) were revealed at a low level. Strikingly, the expression of atrial natriuretic factor (ANF) was not detected. When spontaneous contracting cell masses were examined their electrophysiological features by patch-clamp technique, it showed ventricle-like action potential 17 days after the EB formation. This study indicates that mES03 cell-derived cardiomyocytes displayed biochemical and electrophysiological properties of cardiomyocytes and DMSO enhanced development of cardiomyocytes in 4+/4- method.

Recently, human embryonic stem (hES) cells have become very important resources for ES cell basic research, cell replacement therapy, and other medical applications; thus, efficient cryopreservation methods for these cells are needed. This study examined whether a newly developed minimum volume cooling (MVC) vitrification method, which was tested through cryopreservation of sensitive bovine oocytes, can be used for freezing hES cells. Feeder-free cultured hES cell (MB03) colonies were mechanically dissected into several small clumps following enzymatic treatment. We compared the freezing efficiency of a slow-cooling method using a cryo-module (0.4-0.6C/min, 20-30 clumps/vial) and MVC vitrification using a modified 0.5-ml French mini-straw designated as a MVC straw (>/min, 10 clumps/straw) After thawing, in vitro survival of hES cell clumps was higher for MVC-vitrified cells (80.8%, 97/120) than for slow-cooled cells (38.2%, 39/102). Further, the proliferation rate of surviving MVC-vitrified cells was similar to that of control hES cells from 2 weeks after thawing. In addition, vitrified-thawed hES cells demonstrated a normal karyotype, were positively immunostained for surface marker antibodies (AP, SSEA-4 and TRA-1-60) and the Oct-4 antibody, and could differentiate into all three embryonic germ layer cells in vitro. This result demonstrates that hES cell clumps can be successfully cryopreserved by a newly developed MVC vitrification method without loss of human cell characteristics.

Embryonic stem (ES) cells, derived from preimplantation embryos, are able to differentiate into various types of cells consisting the whole body, or pluripotency. In addition to the plasticity, ES cells are expected to be different from terminally differentiated cells in very many ways, such as patterns of gene expressions, ability and response of the cells in confronting environmental stimulations, metabolism, and growth rate. As a model system to differentiate these two types of cells, human ES (hES, MB03) cells and terminally differentiated cells (HeLa), we examined the ability of these two types of cells in confronting a severe oxidative insult, that is . Ratio of dying cells as determined by the relative amount of dye neutral red entrapped within the cells after the exposures. Cell death rates were not significantly different when either MB03 or HeLa were exposed up to 0.4 mM . However, relative amount of dye entrapped within the cells sharply decreased down to 0.12% in HeLa cells when the cells were exposed to 0.8 mM , while it was approximately 54% in MB03. Pretreatment of cells with BSO (GSH chelator) and measurement of GSH content results suggest that cellular GSH is the major defensive mechanism of hES cells. Induction of apoptosis in hES cell was confirmed by DNA laddering, induction of Bax, and chromatin condensation. In summary, hES cells 1) are extremely resistant to oxidative stress, 2) utilize GSH as a major defensive mechanism. and 3) experience apoptosis upon exposure to oxidative stress.

Adult stem cells can make identical copies of themselves for long periods of time. They also give rise to many differentiated mature cell types that have characteristic morphology and specialized function. Human adult stem cells are the attractive raw materials for the cell/tissue therapy, however, it is not easy to get from the adult tissues. In the present study, we tried to isolate a cell population derived from human umbilical cord vein which has been discarded after birth. The cells were isolated after treatment of the umbilical vein with collagenase or trypsin. After 3 days of culture, two kinds of cell populations were found consisting of adherent cells with endothelial cell-like and fibroblast-like morphology, respectively. When these cells were subcultured 12 times over a period of 3 months, almost cells appeared uniformly to exhibit fibroblastoid morphology which was different from that of mesenchymal stem cells obtained from human bone marrow The results of RT-PCR analyses showed distinct expression of BMP-4, oct-4, and SCF genes but not of GATA, PAX-6 and Brachyury genes. On immunohistochemical staining, the cells were negative for the von Willebrand factor(vWF), alpha-smooth muscle actin and placental alkaline phosphatase. From these observations, it is suggested that stem-like cells might be present in human umbilical cord vein.

Coculture of HSC with bone marrow-derived mesenchymal stem cells (BM-MSCs) is one of used methods to increase cell numbers before transplant to the patients. However, because of difficulties to purify HSCs after coculture with BM-MSCs, it needs to develop a method to overcome the problem. In the present study, we have examined whether a culture insert placed over a feeder layer might support the expansion of HSCs within the insert. cells isolated from the umbilical cord blood by using midiMACS were divided into three groups. A group of 1 cells were grown on a culture insert without feeder layer (Direct). The same number of HSCs was directly cocultured with BM-MSCs (Contact). The third group was placed onto an insert below which BM-MSCs were grown (Insert). To distinguish feeder cells from HSCs, BM-MSCs was pre-labeled fluorescently with PKH26 and 1 cells were seeded in the culture dishes. After culture for 13 days, the expansion factor (x) of HSCs that were grown without feeder layer (Direct) was In contrast, the number of HSCs directly cocultured with feeder layer was 59.6 0.5 and that of HSCs cultured onto an insert was The percentage of BM-MSCs cells remained being fluorescent was after culture. Immune-phenotypically large proportion of cultured cells were founded to be differentiated into myeloid/monocyte progenitor cells. The ability of BM-MSCs, fetal lung, cartilage and brain tissue cells to support ex vivo expansion of HSCs was also examined using the insert. After 11 days of coculture with each of these cells, the expansion factor of HSCs was 15.0, 39.0, 32.0 and 24.0, respectively. Based upon these observations, it is concluded that the coculture method using insert is very effective to support ex vivo expansion of HSCs and to eliminate the contamination of other cells used to coculture wth HSCs.

It was found that the purified extract from A. gigas Nakai (polysaccharide, M.W., 25 kD) controled differentiating human ES cells. Its optimal supplementation concentration was decided as 0.8 (μg/ml) to efficiently control the differentiation. It also enhanced the cell growth, compared to the control. However, most widely used and commercially available differentiating agent, Leukemia Inhibitory Factor (LIF) negatively affected on the cell growth even though it controls the differentiation of ES cells, down to 40-50 % based on morphological observation and telomerase activity. It was presumed that the extract first affected on cell membrane and resulted in controlling signal system, then amplify gene expression of telomere, which enhanced the telomerase activity up to three times compared to the control. LIF only increased the enzyme activity up to two times. It was confirmed that the extract from A. gigas Nakai could be used for substituting currently used differentiation controlling agent, LIF from animal resources as a cheap plant resource and not affecting the cell growth. It can broaden the application of the plants not only to functional foods and their substitutes but also to fine chemicals and most cutting-edge biopharmaceutical medicine.

배아줄기세포(embryonic stem cell, ESC)는 미분화상태로 지속적인 계대가 가능하며, 정상 핵형과 전 분화능(pluripotency)을 가져 생체내-외에서 분화 유도시 삼배엽성의 모든 세포로 분화 가능하다. ESC를 feeder 세포 없이 부유배양하면 배아체(embryoid body, EB)를 형성하고, 초기 배아 발생과 유사한 분화 양상을 갖는다. ESC의 분화 유도가 초기배아 발생처럼 생식호르몬(GTH: FSH, LH; steroids