• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.247-265
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• 2008

Objectives: Many types of liver diseases can damage regenerative potential of mature hepatocytes, hepatic progenitor cells or oval cells. In such cases, a stem cell-based therapy can be an alternative therapeutic option. We examined whether human amnion-derived mesenchymal stem cells (HAM) and human umbilical cord-derived stem cells (HUC) could differentiate into hepatocyte-like cells as therapeutic cells for the liver diseases. Methods: HAM and HUC were isolated from the amnion and umbilical cord of the volunteers after a caesarean section with informed consent. In order to differentiate these cells into hepatocyte-like cells, cells were cultivated in hepatogenic medium using culture plates coated with fibronectin. Effects of hepatocyte growth factor, L-ascorbic acid 2-phosphate, insulin premixture fibroblast growth gactor 4, dimethylsulfoxide, oncostatin M and/or dexamethasone were examined on the hepatic differentiation. After differentiation, the cells were analyzed by RT-PCR, immunocytochemistry, immunoblotting, albumin ELISA, urea assay and periodic acid-schiffs staining. Results: Initial fibroblast-like appearance of HAM and HUC changed to a round shape during culture in the hepatogenic medium. However, in all hepatogenic conditions examined, HUC secreted more amounts of albumin or urea into medium than HAM. Expression of some of hepatocyte-specific genes increased and expression of new genes were observed in HUC following cultivation in hepatogenic medium. Results of immunocytochemistry and immunoblotting analyses demonstrated that HUC secreted albumin into the culture medium. PAS staining further demonstrated that HUC could store glycogen inside of the cells. Conclusions: Both HUC and HAM could differentiate into albumin-secreting, hepatocyte-like cells. Under the same hepatogenic conditions examined, HUC more efficiently differentiated into hepatocyte-like cells compared with the HAM. The results suggest that HUC and HAM could be used as sources of stem cells for the cell-based therapeutics such as in liver diseases.

• Korean Journal of Plant Resources
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• v.36 no.4
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• pp.402-412
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• 2023

Peucedanum japonicum Thunberg is a biennial plant that usually flowers and dies after seed setting at 2^nd year. We inspected the flowering and fertilization characteristics to establish the breeding system. The plant forms complex umbel inflorescences consisting of multiple umbellets and numerous flowers. Each flower is bisexual but allogamous because of protandry. A flower bloomed for three days with matured pollen and no stigma appearance. Inspection of pollen tube growth indicates that the pollen viability can be maintained for up to 3~7 days after first flowering (DAF). In a umbellet, flowers bloomed during 1~5 DAF and the pollen maintained viability during 3~10 DAF, and the pistil developed and maintained fertility during 8~12 DAF. Theoretically, self-pollination can be possible during 8~10 DAF in the same umbellet. Approximately 4.4% of seeds were developed by self-pollination among flowers in an enveloping solitary umbel. There is at least a 9~10 days difference for the development of an umbel with another umbel at the next branch, which indicates pollination can be permitted between different branches in an individual plant. Finally, we have established a rapid breeding method to shorten the breeding cycle to one year from two years by integrating the flowering and pollination characteristics.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.34 no.s02
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• pp.45-65
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• 1989

The Korean peninsular having the complexity of the photography and variability of climate is located within passing area of a lots of typhoon occurring from the southern islands of Philippines. So, there are various patterns of wind and flooding damages in paddy field occuring by the strong wind and the heavy rain concentrated during the summer season of rice growing period in Korea. The wind damages to rice plants in Korea were mainly caused by saline wind, dry wind and strong wind when typhoon occurred. The saline wind damage having symptom of white head or dried leaves occurred by 1.1 to 17.2 mg of salt per dry weight stuck on the plant which was located at 2. 5km away from seashore of southern coastal area during the period(from 27th to 29th, August, 1986) of typhoon &Vera& accompanying 62-96％ of relative humidity, more than 6 m per second of wind velocity and 22.5 to 26.4$^{\circ}C$ of air temperature without rain. Most of the typhoons accompanying 4.0 to 8. 5m per second of wind and low humidity (lesp an 60％) with high temperature in the east coastal area and southen area of Korea. were changed to dry and hot wind by the foehn phenomenon. The dry wind damages with the symptom of the white head or the discolored brownish grain occurred at the rice heading stage. The strong wind caused the severe damages such as the broken leaves, cut-leaves and dried leaves before heading stage, lodging and shattering of grain at ripening stage mechanically during typhoon. To reduce the wind damages to rice plant, cultivation of resistant varieties to wind damages such as Sangpoongbyeo and Cheongcheongbyeo and the escape of heading stage during period of typhoon by accelerating of heading within 15th, August are effective. Though the flood disasters to rice plant such as earring away of field, burying of field, submerging and lodging damage are getting low by the construction of dam for multiple purpose and river bank, they are occasionally occurred by the regional heavy rain and water filled out in bank around the river. Paddy field were submerged for 2 to 4 days when typhoon and heavy rain occurred about the end of August. At this time, the rice plants that was in younger growing stage in the late transplanting field of southern area of Korea had the severe damages. Although panicles of rice plant which was in the meiotic growing stage and heading stage were died when flooded, they had 66％ of yield compensating ability by the upper tilling panicle produced from tiller with dead panicle in ordinary transplanting paddy field. It is effective for reduction of flooding damages to cultivate the resistant variety to flooding having the resistance to bacterial leaf blight, lodging and small brown planthopper simultaneously. Especially, Tongil type rice varieties are relatively resistant to flooding, compared to Japonica rice varieties. Tongil type rice varieties had high survivals, low elongation ability of leaf sheath and blade, high recovering ability by the high root activity and photosynthesis and high yield compensating ability by the upper tillering panicle when flooded. To minimize the flooding and wind damage to rice plants in future, following research have to be carried out; 1. Data analysis by telemetering and computerization of climate, actual conditions and growing diagnosis of crops damaged by disasters. 2. Development of tolerant varieties to poor natural conditions related to flooding and wind damages. 3. Improvement of the reasonable cropping system by introduction of other crops compensating the loss of the damaged rice. 4. Increament of utilization of rice plant which was damaged.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.25-33
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• 2006

Objective: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. Methods: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific genes expressions with immunocytochemistry and RT-PCR. Results: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cell specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. Conclusion: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.311-319
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• 2009

Objective: Human embryonic stem cells (hESCs) have the capacity to differentiate into all of the cell types and therefore hold promise for cell therapeutic applications. In order to utilize this important potential of hESCs, enhancement of currently used technologies for handling and manipulating the cells is required. The cryopreservation of hESC colonies was successfully performed using the vitrification and slow freezing-rapid thawing method. However, most of the hESC colonies were showed extremely spontaneous differentiation after freezing and thawing. In this study, we were performed to rapidly collect of early passage hESCs, which was thawed and had high rate of spontaneously differentiation of SNUhES11 cell line. Methods: Four days after plating, partially spontaneously differentiated parts of hESC colony were cut off using finely drawn-out dissecting pipette, which is mechanical separation method. Results: After separating of spontaneously differentiated cells, we observed that removed parts were recovered by undifferentiated cells. Furthermore, mechanical separation method was more efficient for hESCs expansion after thawing when we repeated this method. The recovery rate after removing differentiated parts of hESC colonies were 55.0%, 74.5%, and 71.1% when we have applied this method to three passages. Conclusion: Mechanical separation method is highly effective for rapidly collecting and large volumes of undifferentiated cells after thawing of cryopreserved early passage hESCs.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.283-292
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• 2009

Objective: Human embryonic stem cells (hESCs) can proliferate indefinitely and differentiate into all kinds of cell types in vitro. Therefore, hESCs can be used as a cell source for cell-based therapy. Transduction of foreign genes to hESCs could be useful for tracing differentiation processes of hESCs and elucidation of gene function. Thus, we tried to introduce enhanced green fluorescent protein (eGFP) gene to hESCs, XX and XY cell lines in this study. Methods: Lentivirus containing eGFP was packaged in 293T cells and applied to hESCs to transduce eGFP. Expression of transduced eGFP was evaluated under the fluorescence microscope and eGFP positive population was analyzed by FACS. Expression of undifferentiation state markers such as Oct4, Nanog, SSEA4 and Tra-1-81 was examined by RT-PCR and/or immunofluorescence in eGFP-hESCs after transduction. In addition, the ability of eGFP-hESCs to form embryoid bodies (EBs) was tested. Results: eGFP was successfully transduced to hESCs by lentivirus. eGFP expression was stably maintained up to more than 40 passages. eGFP-hESCs retained expression patterns of undifferentiation state markers after transduction. Interestingly, disappearance of transduced eGFP was notably observed during spontaneous differentiation of eGFP-hESCs. Conclusion: We established eGFP expressing hESC lines using lentivirus and showed the maintenance of undifferentiation characteristics of these eGFP-hESCs. This reporter-containing hESCs could be useful for tracing the processes of differentiation of hESCs and other studies.

• Development and Reproduction
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• v.10 no.1
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• pp.63-73
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• 2006

This study aimed to find out suitable culture conditions for the differentiation of human amniotic membrane-derived stem cells(HAM) into hepatocyte-like cells. Almost homogenous population of fibroblast-like cells was successfully isolated from the amniotic membrane. In comparison to the non-coated plates and in the absence of insulin/transferrin/selenium(ITS), HAM cultured on the fibronectin-coated plates and in the presence of ITS showed the more intense immunocytochemical staining against the albumin. Addition of both fibroblast growth factor(FGF)-1 and -2 to the differentiation medium gave stronger staining compared to the treatment with FGF-1 or -2 alone. Periodic acid Schiff's base staining of glycogen and morphological turnover of fibroblast-like appearance of HAM into round shape matched the results of immunocytochemical studies. When the efficiency of two-step culture method was examined on the differentiation of HAM into hepatocyte-like cells, all of the results of immunocytochemical staining, periodic acid Schiff's staining and morphological change exhibited effective hepatic differentiation of HAM compared to the continuous culture method. Immunoblot analyses of HAM- conditioned media against the albumin showed that the culture of HAM in the presence of both ITS and fibronectin always gave a stronger staining intensity than those in the absence of them, and that the addition of ether mixture of FGF-4 and either FGF-2 or transforming growth $factor(TGF)-{\alpha}$ to the culture medium significantly enhanced the albumin secretion by HAM. Based on these observations, it is suggested that HAM could differentiate into hepatocyte-like cells under a culture condition consisting of fibronectin and ITS, and addition of FGF-4 with either one of FGF-2 or $TGF-{\alpha}$ could enhance the hepatic differentiation of HAM.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.3
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• pp.171-178
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• 2006

Objective: Human embryonic stem (ES) cells have a great potential in regenerative medicine and tissue engineering. The human ES cells could be differentiated into specific cell types by treatments of growth factors and alterations of gene expressions. However, the efficacy of guided differentiation and isolation of specific cells are still low. In this study, we characterized isolated cells from differentiated human ES cells by magnetic activated cell sorting (MACS) system using specific antibodies to cell surface markers. Methods: The undifferentiated hES cells (Miz-hESC4) were sub-cultured by mechanical isolation of colonies and embryoid bodies were spontaneously differentiated with DMEM containing 10% FBS for 2 weeks. The differentiated cells were isolated to positive and negative cells with MACS system using CD34, human epithelial antigen (HEA) and human fibroblast (HFB) antibodies, respectively. Observation of morphological changes and analysis of marker genes expression were performed during further culture of MACS isolated cells for 4 weeks. Results: Morphology of the CD34 positive cells was firstly round, and then it was changed to small polygonal shape after further culture. The HEA positive cells showed large polygonal, and the HFB positive spindle shape. In RT-PCR analysis of marker genes, the CD34 and HFB positive cells expressed endodermal and mesodermal genes, and HEA positive cells expressed ectodermal genes such as NESTIN and NF68KD. The marker genes expression pattern of CD34 positive cells changed during the extension of culture time. Conclusion: Our results showed the possibility of successful isolation of specific cells by MACS system from undirected differentiated human ES cells. Thus, MACS system and marker antibodies for specific cell types might be useful for guided differentiation and isolation of specific cells from human ES cells.

• Development and Reproduction
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• v.12 no.1
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• pp.19-30
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• 2008

Proper development of fertilized oocyte to blastocyst is a key step in mammalian development to implantation. During development of preimplantation embryos, the mammalian embryo needs supply the energy substrate for keep viability. Usually mammalian oocyte get substrate especially energy substrate from oviduct and uterus, because it does not store much substrate into cytoplasm during oogenesis. Carbohydrates are known as a main energy substrate for preimplantation stage embryos. Glucose, lactate and pyruvate are essential component in preimplantation embryo culture media and there are stage specific preferences to them. Glucose transporter and $H^+$-monocarboxylate cotransporter are a main mediator for carbohydrate transport and those expression levels are primarily under the control of intrinsic or extrinsic factors like insulin and glucose. Other organic substances, amino acids, lipids and nucleotides are used as energy substance and cellular regulation factor. Though since 1960s, successful development of fertilized embryo to blastocyst has been accomplished with chemically defined medium for example BWW and give rise to normal offspring in mammals, the role of metabolites and the regulation of intermediary metabolism are still poorly understood. Glucose may permit expression of metabolic enzymes and transporters in compacting morula, capable of generating the energy required for blastocyst formation. In addition, it has been suggested that the cytokines can modulate the metabolic rate of carbohydrate in embryos and regulate the preimplantation embryonic development through control the metabolic rate. Recently we showed that lactate can be used as a mediator for preimplantation embryonic development. Those observations indicate that metabolites of carbohydrate are required by the early embryo, not only as an energy source, but also as a key substrate for other regulatory and biosynthetic pathways. In addition metabolites of carbohydrate may involve in cellular activity during development of preimplantation embryos. It is suggested that through these regulation and with other regulation mechanisms, embryo and uterus can prepare the embryo implantation and further development, properly.

• Development and Reproduction
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• v.14 no.2
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• pp.143-154
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• 2010

This article analyzes two leading Korean cases which led to opposite conclusions: the Boramae Hospital Case (Korean Supreme Court 2002 Do 995) and the Shinchon Severance Hospital Case (Korean Supreme Court 2009 Da 17471). In doing so, it pays particular attention to the acceptance, modification, and rejection of paternalism, specifically 'physician paternalism' and 'familial paternalism', both of which have long and strongly influenced the Korean medical environment. In Boramae Hospital, the Court emphasized the obligation of the physician in terms of the life of the patient (eg: protecting and preserving the life and welfare of the patient). Its position seemed to be based on the traditional physician paternalism which presupposes the ability of physicians to identify right and wrong choices according to natural laws. However, the Court saw itself as the final arbiter of who identifies and determines the real world content and consequences of that natural law. In short, the Court elevated itself to the supreme guardian of the patient, and held that its decision cannot be overruled by that of the patient's family. So without specifically referring to the importance of the family and the role of familial decisions, both long-observed traditions in medical decision-making in Korea, the Court shifted away from familial paternalism. In Shinchon Severance Hospital, the Court explained the meaning of the patient's powers of self-rulemore concretely, explaining its scope and substance in greater detail. The Court held that one can exercise the right of self-rule, even over issues such as death, in the form of 'previous medical directions'. However, this case does not represent a wholesale acceptance of medical autonomy (ie: it does not accept self-rule unconditionally). Rather, the Court accepted the importance of the opinions and decision of physicians and of the Hospital Ethics Commission, and the Court still retained to itself the authority to review and make alterations to 'material' decision. The Court did not overlook the importance of the decision of the patient's family, but it also did not relinquish its status as supreme guardian, emphasizing the 'objective' nature of a decision from the court.