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Won Yun Choi,
Hwang Gyun Jeon,
Young Chung,
Jung Jin Lim,
Dong Hyuk Shin,
Jung Mo Kim,
Byung Sung Ki,
Seung-Hun Song,
Seong-Jun Choi,
Keun-Hong Park, [......],
Ji Suk Moon,
Sung Jun Jung,
Hyun Mi Kang,
Seah Park,
Hyung-Min Chung,
Jung Jae Ko, Kwang Yul Cha,
Tae Ki Yoon,
Haekwon Kim,
Dong Ryul Lee
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ABSTRACT: Human adult stem cells are a readily available multipotent cell source that can be used in regenerative medicine. Despite many advantages, including low tumorigenicity, their rapid senescence and limited plasticity have curtailed their use in cell-based therapies. In this study, we isolated CD34/CD73 double-positive (CD34+/CD73+) testicular stromal cells (HTSCs) and found that the expression of CD34 was closely related to the cells' stemness and proliferation. The CD34+/CD73+ cells grew in vitro for an extended period of time, yielding a multitude of cells (5.6 x 1016 cells) without forming tumors in vivo. They also differentiated into all 3 germ layer lineages both in vitro and in vivo, produced cartilage more efficiently compared to bone marrow stem cells and, importantly, restored erectile function in a cavernous nerve crush injury rat model. Thus, these HTSCs may represent a promising new autologous cell source for clinical use.
Stem cells and development 03/2013; · 4.15 Impact Factor
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ABSTRACT: Human pre-ovulatory follicular fluid (FF) contains a higher concentration of melatonin than serum. The aim of this study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcomes of an in-vitro maturation (IVM) IVF-embryo transfer programme for patients with polycystic ovarian syndrome (PCOS). Melatonin concentrations in the culture media of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes after using IVM media with or without melatonin were analysed. In the culture media of GC or COC, melatonin concentrations gradually increased. When human chorionic gonadotrophin priming protocols were used, implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control group (P<0.05). Pregnancy rates were also higher, although not significantly. The findings suggest that the addition of melatonin to IVM media may improve the cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes. It is speculated that follicular melatonin may be released from luteinizing GC during late folliculogenesis and that melatonin supplementation may be used to improve the clinical outcomes of IVM IVF-embryo transfer. Melatonin is primarily produced by the pineal gland and regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction. Interestingly, human pre-ovulatory follicular fluid contains a higher concentration of melatonin than serum. However, in contrast to animal studies, the direct role of melatonin on oocyte maturation in the human system has not yet been investigated. So, the aim of the study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcome of an in-vitro maturation (IVM) IVF-embryo transfer programme for PCOS patients. The melatonin concentrations in culture medium of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes of IVM IVF-embryo transfer using IVM medium alone or supplemented with melatonin were analysed. In the culture media of GC or COC, the melatonin concentration gradually increased. With human chorionic gonadotrophin priming, the pregnancy and implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control (P<0.05). Our findings suggest that follicular melatonin is released from luteinizing GC during late folliculogenesis and plays a positive role in oocyte maturation. Therefore, addition of melatonin into IVM medium may improve cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes.
Reproductive biomedicine online 10/2012; · 2.04 Impact Factor
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Dohoon Kim,
Chun-Hyung Kim,
Jung-Il Moon,
Young-Gie Chung,
Mi-Yoon Chang,
Baek-Soo Han,
Sanghyeok Ko,
Eungi Yang, Kwang Yul Cha,
Robert Lanza,
Kwang-Soo Kim
Cell stem cell 07/2009; 4(6):472-6. · 23.56 Impact Factor
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ABSTRACT: The Second Biennial International Collaborative Symposium on Stem Cell Research, held in Seoul, Korea, on 18-19 September 2008, showcased talks by a roster of established and emerging leaders in stem cell biology, and demonstrated how far and fast the field has moved in the last 2 years.
Regenerative Medicine 02/2009; 4(1):129-31. · 3.72 Impact Factor
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ABSTRACT: To report the survival rate of oocytes and the rate of successful pregnancies obtained from super-rapid cooling of oocytes using slush nitrogen (SN(2)).
Prospective clinical research.
A university-affiliated hospital.
Twenty-eight infertile women who underwent 30 cycles of IVF-ET using previously vitrified oocytes.
Oocytes were vitrified by super-rapid cooling using SN(2).
Morphological normality of thawed oocytes and clinical outcome.
In 30 cycles of ovarian stimulation for IVF, 364 surplus oocytes from 28 patients were vitrified using SN(2). Three hundred two (85.1% +/- 2.9%) of the oocytes survived after warming. Fertilization and cleavage rates were 77.4% +/- 3.5% (168/218) and 94.3% +/- 2.1% (158/168), respectively. Thirteen pregnancies (43.3%) resulted from 30 uterine transfers of 120 embryos with an implantation rate of 14.2% (17/120). There were no differences between the pregnancy rate after vitrification/warming and that obtained from routine noncryopreserved oocytes.
The present report suggests that super-rapid cooling may improve the clinical efficacy of human oocyte vitrification and may be a valuable tool for human assisted reproductive technologies.
Fertility and sterility 11/2007; 88(4):952-6. · 3.97 Impact Factor
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ABSTRACT: The establishment of new technology for genetic modification in human embryonic stem (ES) cell lines has raised great hopes for achieving new ground in basic and clinical research. Recently, lentiviral vector technology has been shown to be highly effective and therefore could emerge as a popular tool for human ES cell genetic modification. The objectives of this study were to evaluate the efficiency of promoters in lentiviral gene delivery systems in mammalian ES cells, including mouse, monkey, and human, and to construct efficient and optimized conditions for lentivirus-mediated transfection systems. Mammalian ES cells were transfected with self-inactivating (SIN) human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors containing the human polypeptide chain elongation factor-1alpha (EF-1alpha) promoter or cytomegalovirus (CMV) promoter and analyzed by fluorescence-activated cell sorting (FACS) analysis for the expression of the enhanced green fluorescent protein (eGFP) reporter gene. The efficiency of the EF-1alpha promoter was higher than that of the CMV promoter in all ES cells tested. The EF-1alpha promoter efficiently drove gene expression (14.74%) compared with CMV promoter (3.69%) in human ES cells. We generated a stable eGFP+ human ES cell line (CHA3-EGFP human ES cells) that continuously expressed high levels of EGFP ( approximately 95%) from the EF-1alpha promoter and was maintained for up to 60 weeks with undifferentiated proliferation. The established CHA3-EGFP human ES cell lines were characterized as being negative for nondifferentiation markers and teratoma formation. These results imply that genetic modification by lentiviral vectors with specific promoters in ES cells constitute a powerful tool for guided differentiation as well as gene therapy.
Stem Cells and Development 09/2007; 16(4):537-45. · 4.46 Impact Factor
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ABSTRACT: Three typical folate metabolism enzymes-i.e. methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS) and MS reductase (MTRR) in the folate cycle-play a critical role in DNA synthesis and methylation reactions. We evaluated whether polymorphisms of these three enzymes are associated with non-obstructive male infertility.
Three hundred and sixty patients with non-obstructive infertility and 325 fertile men without any chromosomal abnormalities were included in this study. The single-nucleotide polymorphism (SNP) analysis was performed by pyrosequencing and PCR-restriction fragment length polymorphism (RFLP) analysis
The frequencies of MTHFR 677TT and MTRR 66GG genotypes were higher in non-obstructive infertile men compared with those in fertile men. By classifying 360 infertile patients into 174 azoospermia and 186 oligoasthenoteratozoospermia (OAT) subjects, the MTHFR 677TT and MS 2756GG types were significantly associated with the azoospermia group (P = 0.0227 and 0.0063, respectively). The frequency of MTRR 66GG was significant in the OAT group (P = 0.0014 versus fertile males).
By analysis of a large number of subjects and a more specific patient selection, we showed the first genetic evidence that MTHFR C677T, MS A2756G and MTRR A66G genotypes were independently associated with male infertility. Each SNP of the three enzymes may have a different impact on the folate cycle during spermatogenesis.
Human Reproduction 01/2007; 21(12):3162-70. · 4.47 Impact Factor
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ABSTRACT: To improve our understanding of the molecular mechanisms underlying early embryo development, further characterization of gene activity in oocytes and embryos is urgently required. The transition from the two-cell to four-cell stage is particularly important in pre-implantation embryonic development, as it involves transcriptional reprogramming and cellular differentiation. In this study, we used a 7.4 K cDNA microarray to screen mRNA transcript levels in the pre-implantation mouse embryo. Real-time PCR was used to confirm microarray data. We profiled 7,410 genes and identified 4,562 genes that were differentially expressed in the pre-implantation embryo. We selected a total of 248 genes with significant expression changes that are functionally involved in the two-cell and two-cell block embryo. Of these genes, 114 were down-regulated and the remainder (n=134) were up-regulated in the two-cell embryo. This study provides a developmental map of a large number of genes in the pre-implantation mouse embryo with particular emphasis on gene expression in the two-cell embryo and two-cell block embryo. Further investigations based on this data will provide a better understanding of the effects of various external conditions and may facilitate comparative analysis of pre-implantation development in other mammalian species, including human.
Theriogenology 10/2006; 66(4):785-96. · 1.96 Impact Factor
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ABSTRACT: To assess the association between the single nucleotide polymorphism of the insulin receptor (INSR) gene and polycystic ovary syndrome (PCOS) in a Korean population.
Case-control study.
University-based hospital.
One hundred seventy-four patients with PCOS and 93 healthy women as controls.
Frequency of three genotypes for single nucleotide polymorphism found in exon 17 of INSR gene.
The high frequency of the T allele was shown both in patient and control groups. The frequency of C allele, which known as a normal allele, was slightly higher in the patient group than in the control group.
The C/T polymorphism in exon 17 of the INSR gene is not associated with susceptibility of PCOS in a Korean population.
Fertility and sterility 09/2006; 86(2):380-4. · 3.97 Impact Factor
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ABSTRACT: In vitro maturation (IVM) programme has developed a culture technique for supporting the growth and maturation of immature oocytes since late of 1980s and has become important in ART methods. The establishment of an IVM programme can yield many advantages. Patients suffering from congenital or postnatal reproductive disorders, such as premature ovarian failure (POF) or polycystic ovarian syndrome (PCOS), can achieve pregnancies by transfer of viable embryos derived from IVM and in vitro fertilisation (IVF) systems. However, the pregnancy rate of IVM-IVF programme for PCO patients was still low. This disadvantage was caused by poor maturation of human immature oocytes and no synchronisation with implantation windows. Recently, application of FSH/hCG priming or optimal maturation medium for proper cytoplasmic maturation improves the success rate of IVM procedures. Indeed, increasing oocyte viability by employing optimised IVM system would also reduce ethical concerns about the disposal of retrieved oocytes with low developmental competence, and concerns about the abuse of the manipulation of human embryos.
Journal of the Indian Medical Association 09/2006; 104(8):446, 448, 473.
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ABSTRACT: Recurrent spontaneous abortion (RSA), defined as the loss of three or more consecutive pregnancies prior to the 20th week of gestation, affects up to 5% of the child-bearing population. To investigate the proteins associated with RSA, the protein expression in human follicular fluid was analyzed using 2-DE. Follicular fluid contains a variety of biologically important proteins for oocyte fertilization and follicle maturation in the mammalian reproductive process. Therefore, it can be used as a provisional source for identifying proteins involved in RSA. In this study, we identified five aberrantly expressed proteins (complement component C3c chain E, fibrinogen gamma, antithrombin, angiotensinogen, and hemopexin precursor) in follicular fluid from RSA patients with MALDI-TOF-MS and nano-LC MS/MS. Western blot analysis confirmed that the protein expression level of fibrinogen gamma and antithrombin was less in follicular fluid from RSA patients than those from normal controls. Semiquantitative RT-PCR and real-time PCR analyses revealed that mRNA level of these coagulation factors was also decreased significantly in chorionic villi of RSA patients compared with normal samples. Taken all together, it is likely that coagulation factors (fibrinogen gamma and antithrombin) play an important role in maintaining the normal pregnancy.
PROTEOMICS 07/2006; 6(11):3445-54. · 4.51 Impact Factor
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ABSTRACT: Y-chromosome microdeletion in male fetuses conceived by intracytoplasmic sperm injection (ICSI) was screened by polymerase chain reaction for sequence-tagged sites in azoospermia factor (AZF)-b and AZFc regions. Treatment with ICSI may lead to vertical transmission, expansion, and de novo Y-chromosome microdeletion in male fetuses.
Fertility and sterility 06/2006; 85(5):1512-5. · 3.97 Impact Factor
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ABSTRACT: We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues (244KRKR247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236-252 and 249-267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (318KKK320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.
FEBS Letters 04/2006; 580(7):1865-71. · 3.54 Impact Factor
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ABSTRACT: We developed a new and efficient method for osteoblastic differentiation of human embryonic stem cells (hESCs) using primary bone-derived cells (PBDs). Three days after embryoid body (hEB) formation, cells were allowed to adhere to culture surface where PBDs were pre-plated and mitomycin C-treated in DMEM/F12 medium supplemented with 5% knockout serum replacement. As early as 14 days, mineralization and formation of nodule-like structures in cocultured hEBs were prominent by von Kossa and Alizarin S staining, and expressions of osteoblast-specific markers including bone sialoprotein, alkaline phosphates, osteocalcin, collagen 1, and core binding factor alpha1 by RT-PCR. In addition, FACS analysis revealed that over 19% of the differentiated cells expressed osteocalcin. These results suggest that PBDs not only have osteogenic effects releasing osteogenic factors as bone morphogenic protein (BMP) 2 and BMP 4 but also have exerted other effects, whether chemical or physical, for the differentiation of hESCs.
Biochemical and Biophysical Research Communications 03/2006; 340(2):403-8. · 2.48 Impact Factor
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ABSTRACT: The purpose of this study was to establish the culture conditions required to isolate, identify and expand male germ stem cell-like cells (GSC-LC) from the testicular tissue of patients with non-obstructive azoospermia (NOA).
Testicular tissues obtained from patients (two with maturation arrest (MA, n = 2) and Sertoli cell-only syndrome (SCOS, n = 11) were dissociated and plated into gelatin-coated dishes. After 2-4 weeks, cultures from both MA patients (100%) and four SCOS patients (36.3%) exhibited multicellular colonies, which proliferated successfully until passage 10. GSC-LC in the colonies displayed alkaline phosphatase activity, as well as Oct-4 and integrin b1 expression after every passage. After the fifth passage, GSC-LC were differentiated by encapsulation in calcium alginate and further cultivation. At 2 and 6 weeks, cells expressed c-Kit, Scp3, testis-specific histone protein 2B (TH2B), and transition protein (TP)-1. Fluorescence in situ hybridization additionally disclosed a few tetraploid and haploid cells at 6 weeks. Human oocytes were activated in the absence of artificial activation and cleaved after the injection of presumptive spermatids.
Our novel culture system may be useful for diagnosing the existence of germ cells and facilitating the treatment of NOA patients.
Human Reproduction 03/2006; 21(2):471-6. · 4.47 Impact Factor
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American Journal of Medical Genetics Part A 03/2006; 140(5):527-32. · 2.39 Impact Factor
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ABSTRACT: To study global gene expression profiles of early folliculogenesis in primordial, primary, and secondary follicles.
A cDNA microarray study using amplified RNAs from isolated follicles.
Experimental animal study.
Female ICR strain mice (12 days old).
Isolation of follicles at each stage, RNA isolation and amplification, microarray hybridization, and statistical analysis for microarray.
Gene lists of various functional groups with an estimated false discovery rate of 5%. Among them, platelet-derived growth factors (PDGFs) and receptors were localized by immunohistochemistry in mouse ovaries.
We analyzed a list of genes according to function, such as apoptosis, cell cycle, cell proliferation and maintenance, cytoskeleton, extracellular matrix, and signal transduction, as well as according to frequency. Among the list of genes, we found all PDGFs (A, B, C, and D) and receptors (alpha and beta) are expressed with differential expression patterns in the oocytes and ovarian cells according to stage of follicular development.
The present report suggests that genome-wide expression profiling using microarray after RNA amplification may become a useful tool to better understand the molecular mechanism(s) involved in early ovarian folliculogenesis.
Fertility and sterility 02/2006; 85(1):193-203. · 3.97 Impact Factor
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ABSTRACT: To quantify mitochondrial DNA using real-time PCR in women with premature ovarian failure (POF) and a control group.
Prospective study.
Genome Research Center for Reproductive Medicine and Infertility, Korea Ministry of Health & Welfare.
Thrity patients with POF and 30 control individuals.
The mitochondrial DNA content was quantified using real-time PCR. The effectiveness of the assay was determined by relative quantification using the comparative threshold cycle (CT) method.
Relative quantification of mitochondrial DNA content.
The mitochondrial DNA content was significantly lower in the POF group than in the control group (0.58 +/- 0.38 vs. 1.15 +/- 0.67; P < .01). In both groups, there was a significant positive correlation between the mitochondrial DNA/28S rRNA ratio and mitochondrial DNA CT (control group: r = 0.774; P < .001; POF group: r = 0.556; P = .001) and a significant negative correlation between the mitochondrial DNA/28S rRNA ratio and 28S rRNA CT (control group: r = -0.677; P < .001; POF group: r = -0.627; P = .001).
This study has established the clinical feasibility of quantifying amounts of mitochondrial DNA, relative to an internal standard, using real-time PCR. Further studies are warranted to elucidate the roles of apoptosis and mitochondrial function in the pathogenesis of POF.
Fertility and sterility 01/2006; 84(6):1712-8. · 3.97 Impact Factor
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ABSTRACT: Octamer-binding transcription factor-4 (Oct4), a member of the POU domain transcription factors, is crucial for both early embryonic development and the maintenance of stem cell pluripotency. The human Oct4 (hOct4) 5' upstream sequence contains four conserved regions (CR1, 2, 3, 4) that are homologous in the murine. In this study, we constructed a series of deletion mutants of the hOct4 5' upstream region and identified cis-regulatory elements that may be important determinants for the transcriptional activity of the hOct4 promoter. Our studies showed that CR2, 3, and 4 each acted as positive cis-regulatory elements in hOct4 promoter activity. We also newly identified a putative negative cis-acting element located between CR1 and CR2. In addition, the sequence -380/-1 at CR1 that contains a GC box was sufficient to provide the minimal promoter activity. Site-directed mutagenesis and electrophoretic mobility shift assays revealed the GC box located in the -380/-1 region may play a critical role in controlling the transcriptional activity of hOct4 by the direct binding of Sp1 or Sp3 transcription factors to the GC box. An overexpression study showed that Sp1 and Sp3 positively and negatively regulate hOct4 promoter activity. Thus, the hOct4 promoter upstream region contains multiple regulatory elements, one of which, the GC box, may be an important cis-regulatory element that regulates the transcription of the hOct4 promoter by the binding of Sp family transcription factors.
Journal of Cellular Biochemistry 12/2005; 96(4):821-30. · 2.87 Impact Factor
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ABSTRACT: Blepharophimosis-ptosis-epicanthus inversus syndrome type I is an autosomal disorder caused by mutations in FOXL2 gene and associated with premature ovarian failure in women by a dominant inheritance. FOXL2 is a recently identified protein that belongs to forkhead family transcription factor, of which signaling pathways are still unknown. Here, we show that FOXL2 induces apoptosis in both Chinese hamster ovary cells and rat granulosa cells, and it interacts with DP103, a DEAD box-containing protein. Overexpression of DP103 itself did not affect cell viability while its coexpression with FOXL2 led to the potentiation of cell death. Our results present previously undiscovered functions of these proteins, an apoptotic activity of FOXL2 in the ovary and a modulating activity of DP103 by interacting with FOXL2.
Biochemical and Biophysical Research Communications 11/2005; 336(3):876-81. · 2.48 Impact Factor
