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  • Title/Summary/Keyword: Embryonic developmental

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Improvement of Motor Behavior of Parkinson′s Disease Animal Model by Nurr1-Transfected Human Embryonic Stem Cells.

  • Lee, Chang-Hyun;Cho, Hwang-Yoon;Kil, Kwang-Soo;Lee, Gun-Soup;Yoon, Ji-Yeon;Lee, Young-Jae;Kim, Eun-Young;Park, Se-Pill;Lim, Jin-Ho
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.103-103
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    • 2003
  • 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 14.8±33.9 (P<0.05) of the number before transplantation, however, the ratio increased slightly to 13.6±56.3 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.

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Human Embryonic Stem Cells Experience a Typical Apoptotic Process upon Oxidative Stress

  • Lee, Gun-Soup;Lee, Young-Jae;Kim, Eun-Young;Park, Se-Pill;Lim, Jin-Ho
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.97-97
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    • 2003
  • 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 H2O2. 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 H2O2. 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 H2O2, 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.

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Optimal Conditions for Artificial Fertilization, Embryonic Development, and Larval Growth of the Purple Clam, Saxidomus purpuratus from Southern Coast of Korea

  • Choi, Jin-Woo;Kim, Su-Kyoung;Choi, Yong-Suk;Lee, Chang-Hoon;Lee, Woo-Jin;Ryu, Tae-Kwon
    • The Korean Journal of Malacology
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    • v.19 no.1
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    • pp.33-40
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    • 2003
  • To obtain the basic information on culture conditions for the larvae of Saxidomus purpuratus, experiments were conducted on the population from southern coast for (1) the success in fertilization and development from artificial fertilization among different months of a year, (2) the viability of sperms after exposure to seawater, (3) and the effects of temperature, salinity, and food organism on the survival and growth of larvae. Gametes obtained from dissection showed high rate of fertilization at all months. But the rate of development was higher only May-July. Developmental success seemed to be related with the quality of eggs at the time of fertilization. Developmental times for 2-cell, 4-cell, 8-cell, blastula, trochophore larva, and veliger larva at 20C were 1.5, 2, 4, 18, 24, and 32 hr, respectively. Sperms could survive for more than 8 hr, however, actively swimming sperms could be found within 1 hr after exposure to seawater. It is recommended that sperms should be used for fertilization as soon as possible when they are exposed to seawater. At temperature of 35C, all the larvae died during 48 hr. Larval survival decreased when salinity was either lower than 20 psu or higher than 40 psu, and was 0% when salinity was 10 psu. Optimal range of temperature and salinity for rearing larvae of S. purpuratus were 20-25C and 20-40 psu, respectively. Larvae grew from 111.5 to 235.3 μm during 21 days. Larvae fed mixed diets grew faster than unialgal diets. The fastest growth was observed when larvae were fed on the mixture of Isochrysis galbana and Nannochloris oculata.

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Maintained MPF Level after Oocyte Vitrification Improves Embryonic Development after IVF, but not after Somatic Cell Nuclear Transfer

  • Baek, Ji I;Seol, Dong-Won;Lee, Ah-Reum;Lee, Woo Sik;Yoon, Sook-Young;Lee, Dong Ryul
    • Molecules and Cells
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    • v.40 no.11
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    • pp.871-879
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    • 2017
  • Levels of maturation-promoting factor (MPF) in oocytes decline after vitrification, and this decline has been suggested as one of the main causes of low developmental competence resulting from cryoinjury. Here, we evaluated MPF activity in vitrified mouse eggs following treatment with caffeine, a known stimulator of MPF activity, and/or the proteasome inhibitor MG132. Collected MII oocytes were vitrified and divided into four groups: untreated, 10 mM caffeine (CA), 10μM MG132 (MG), and 10 mM caffeine + 10μM MG132 (CA+MG). After warming, the MPF activity of oocytes and their blastocyst formation and implantation rates in the CA, MG, and CA+MG groups were much higher than those in the untreated group. However, the cell numbers in blastocysts did not differ among groups. Analysis of the effectiveness of caffeine and MG132 for improving somatic cell nuclear transfer (SCNT) technology using cryopreserved eggs showed that supplementation did not improve the blastocyst formation rate of cloned mouse eggs. These results suggest that maintaining MPF activity after cryopreservation may have a positive effect on further embryonic development, but is unable to fully overcome cryoinjury. Thus, intrinsic factors governing the developmental potential that diminish during oocyte cryopreservation should be explored.

Self-Reprogramming of Spermatogonial Stem Cells into Pluripotent Stem Cells without Microenvironment of Feeder Cells

  • Lee, Seung-Won;Wu, Guangming;Choi, Na Young;Lee, Hye Jeong;Bang, Jin Seok;Lee, Yukyeong;Lee, Minseong;Ko, Kisung;Scholer, Hans R.;Ko, Kinarm
    • Molecules and Cells
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    • v.41 no.7
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    • pp.631-638
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    • 2018
  • Spermatogonial stem cells (SSCs) derived from mouse testis are unipotent in regard of spermatogenesis. Our previous study demonstrated that SSCs can be fully reprogrammed into pluripotent stem cells, so called germline-derived pluripotent stem cells (gPS cells), on feeder cells (mouse embryonic fibroblasts), which supports SSC proliferation and induction of pluripotency. Because of an uncontrollable microenvironment caused by interactions with feeder cells, feeder-based SSC reprogramming is not suitable for elucidation of the self-reprogramming mechanism by which SSCs are converted into pluripotent stem cells. Recently, we have established a Matrigel-based SSC expansion culture system that allows longterm SSC proliferation without mouse embryonic fibroblast support. In this study, we developed a new feeder-free SSC self-reprogramming protocol based on the Matrigel-based culture system. The gPS cells generated using a feeder-free reprogramming system showed pluripotency at the molecular and cellular levels. The differentiation potential of gPS cells was confirmed in vitro and in vivo. Our study shows for the first time that the induction of SSC pluripotency can be achieved without feeder cells. The newly developed feeder-free self-reprogramming system could be a useful tool to reveal the mechanism by which unipotent cells are self-reprogrammed into pluripotent stem cells.

Optimal Conditions for the Embryonic Development of Sea Urchin, Strongylocentrotus intermedius for Using the Bioassay (북쪽말똥성게, Strongylcentrotus intermedius를 이용한 생물검정 최적 발생조건)

  • Ryu, Tae-Kwon;Sung, Chan-Gyoung;Han, Gi-Myung;Hwang, In-Young;Lee, Taek-Kyun;Lee, Chang-Hoon
    • Environmental Analysis Health and Toxicology
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    • v.22 no.3
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    • pp.211-218
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    • 2007
  • Even though some standard developmental bioassay protocols for environmental assessment using sea urchins have already been described, there have not been many attempts to apply and modify these protocols with Korean species. Therefore, there is a strong need to establish standard bioassay protocols using sea urchins commonly found in Korea. Prior to developing a new protocol, it is essential to know the optimal conditions for the bioassay procedures. We investigated the optimal conditions (temperature, salinity, and embryo density) of the sea urchin Strongylocentrotus intermedius. The ideal temperature for developmental bioassay of S. intermedius was determined to be 15C and the time required for the embryo to become pluteus larva was 72 hr. The optimal range of salinity for the embryo toxicity using S. intermedius was between 30 to 32 psu, which is similar to the range found in the natural habitats of adult populations. The optimum density of embryos at the beginning of bioassays was 100 embryos/mL. When the assays were carried out at higher densities, the proportion of normally developed larvae decreased significantly.

Embryonic Stem Cells Lacking DNA Methyltransferases Differentiate into Neural Stem Cells that Are Defective in Self-Renewal

  • Bong Jong Seo;Tae Kyung Hong;Sang Hoon Yoon;Jae Hoon Song;Sang Jun Uhm;Hyuk Song;Kwonho Hong;Hans Robert Scholer;Jeong Tae Do
    • International Journal of Stem Cells
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    • v.16 no.1
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    • pp.44-51
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    • 2023
  • Background and Objectives: DNA methyltransferases (Dnmts) play an important role in regulating DNA methylation during early developmental processes and cellular differentiation. In this study, we aimed to investigate the role of Dnmts in neural differentiation of embryonic stem cells (ESCs) and in maintenance of the resulting neural stem cells (NSCs). Methods and Results: We used three types of Dnmt knockout (KO) ESCs, including Dnmt1 KO, Dnmt3a/3b double KO (Dnmt3 DKO), and Dnmt1/3a/3b triple KO (Dnmt TKO), to investigate the role of Dnmts in neural differentiation of ESCs. All three types of Dnmt KO ESCs could form neural rosette and differentiate into NSCs in vitro. Interestingly, however, after passage three, Dnmt KO ESC-derived NSCs could not maintain their self-renewal and differentiated into neurons and glial cells. Conclusions: Taken together, the data suggested that, although deficiency of Dnmts had no effect on the differentiation of ESCs into NSCs, the latter had defective maintenance, thereby indicating that Dnmts are crucial for self-renewal of NSCs.

In Vitro Development and the Improving Effects of Bovine Embryos in Simple Media (소 초기배의 단순배양액에서의 체외발생 및 개선효과)

  • 이홍준;서승운;이상호;송해범
    • Journal of Embryo Transfer
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    • v.10 no.3
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    • pp.251-256
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    • 1995
  • This study was experimented that developmental effects of bovine in vitro fertilized embryos by coculture system and supplementation of energy materials into simple media. With the ovaries from slaughter house in vitro maturation by 24h, in vitro fertilization was performed with sperms collected by Percoll gradient method. Fertilized embryos were cocultured in 15% FCS+CZB medium with BOEC(bovine oviductal epithelial cell), GCM (granulosa cell monolayer) and MEFC(mouse embryonic fihrohlast cell). And also in this study, there was trying to improve the early developmental rate of embryos by addition of concentration-controlled Na-pyruvate, D-glucose which were used as energy sources into CZB medium. In vitro developmental rate was confirmed by the cleavage rate of 48h post-IVF and the embryo development rate at 240h culture. In the coculture system BOEC had 20.0% of blastocysts rate, which was higher than that of other coculture systems. To determine the optimum concentration for early embryo developmental rate rapidly, through the gradient of concentrations of Na-pyruvate and D-glucose, we focused on the cleavage rate at 48h and blastocysts rate at 240h. In case of Na-pyruvate, cleavage rate and developmental rate over 3-cell were lower at the concentration of 1.OOrnM than the other treatment concentrations, otherwise the blastocysts rate was higher as 23.2% than the others. That result showed that as like reported group which had higher develop-mental rate over 3-cell was also higher to the blastocysts rate. In case of D-glucose, there was no effects through the concentration changes. It was the result of this study for which the use of BOEC coculture system and 1.OOmM Na-pyruvate as an energy source had an effect upon embryo development.

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Different Potential of Hematopoietic Differentiation in Two Distinct Mouse Embryonic Stem Cells (두 개의 다른 마우스 배아줄기세포의 차별적인 조혈세포 분화능)

  • Kim, Jin-Sook;Kang, Ho-Bum;Song, Jee-Yeon;Oh, Goo-Taeg;Nam, Ki-Hoan;Lee, Young-Hee
    • Development and Reproduction
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    • v.9 no.2
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    • pp.105-114
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    • 2005
  • Embryonic stem(ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decision and to develop methods for getting enough cell numbers for clinical applications. Hematopoiesis has been widely studied, and hematopoietic differentiation from ES cells is a good model to study lineage commitment. In this study, we investigated stemness and compared the efficiency of hematopoietic differentiation using two different mouse embryonic stem cell lines TC-1 and B6-1. Although the two cell lines showed known stem cell properties with minor differences, the embryoid body formation efficiency in methylcellulose was much higher in TC-1 than B6-1. When measured potentials of hematopoietic differentiation using functional(colony-forming cell) and phenotypic(specific marker expression) assays, we found that TC-1 can differentiate into hematopoietic cells in methylcellulose culture but B6-1 cannot. These results imply that we can improve the efficiency of hematopoietic cell differentiation by selection of proper cell lines and this may be also applied in the differentiation of human embryonic stem cells.

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Proteome Analysis of Chicken Embryonic Gonads: Identification of Major Proteins from Cultured Gonadal Primordial Germ Cells

  • Lee, Sang-In;Han, Beom-Ku;Park, Sang-Hyun;Kim, Tae-Min;Sin, Sang-Soo;Lee, Young-Mok;Kim, Hee-Bal;Lim, Jeong-Mook;Han, Jae-Yong
    • Proceedings of the Korea Society of Poultry Science Conference
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    • 2005.11a
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    • pp.66-67
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    • 2005
  • The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGC) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 day of incubation, and the gPGC were cultured in vitro until colony formed. After 7-10 days in cultured gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of thistype will serve as an important reference for germ cell biology and transgenic research.

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