Dong Jae Cho

Human amniotic fluid-derived stem cells have characteristics of multipotent stem cells

Abstract.  Objectives: To characterize mesenchymal stem cell‐like cells isolated from human amniotic fluid for a new source of therapeutic cells. Materials: Fibroblastoid‐type cells obtained from amniotic fluid at the time of birth. Methods: The ability of ex vivo expansion was investigated until senescence, and stem cell‐like characteristics were analyzed by examining differentiation potential, messenger RNA expression and immunophenotypes. Results and Conclusions: A morphologically homogenous population of fibroblastoid‐type (HAFFTs) cells, similar to mesenchymal stem cells from bone marrow (BM‐MSCs), was obtained at the third passage. The cells became senescent after 27 passages over a period of 8 months while undergoing 66 population doublings. Under appropriate culture conditions, by the 8th passage they differentiated into adipocytes, osteocytes, chondrocytes and neuronal cells, as revealed by oil red O, von Kossa, Alcian blue and anti‐NeuN antibody staining, respectively. Immunophenotype analyses at the 17th passage demonstrated the presence of TRA‐1–60; SSEA‐3 and‐4; collagen types I, II, III, IV and XII; fibronectin; α‐SMA; vimentin; desmin; CK18; CD44; CD54; CD106; FSP; vWF; CD31; and HLA ABC. Reverse transcriptase–polymerase chain reaction analysis of the HAFFTs from passages 6–20 showed consistent expression of Rex‐1, SCF, GATA‐4, vimentin, CK18, FGF‐5 and HLA ABC genes. Oct‐4 gene expression was observed up to the 19th passage but not at the 20th passage. HAFFTs showed telomerase activity at the 5th passage with a decreased level by the 21st passage. Interestingly, BMP‐4, AFP, nestin and HNF‐4α genes showed differential gene expression during ex vivo expansion. Taken together, these observations suggest that HAFFTs are pluripotent stem cells that are less differentiated than BM‐MSCs, and that their gene expression profiles vary with passage number during ex vivo expansion.

Bone mineral density, arterial stiffness, and coronary atherosclerosis in healthy postmenopausal women.

Objective: The aim of this study was to assess the correlation between bone mineral density (BMD), arterial stiffness, and coronary atherosclerosis in healthy postmenopausal women. Methods: We performed a retrospective review of 152 postmenopausal women who visited the health promotion center for a routine checkup, after excluding participants who had factors affecting BMD and coronary artery disease. BMD was evaluated by dual-energy x-ray absorptiometry in the lumbar spine and femur. Arterial stiffness was measured by brachial-ankle pulse wave velocity (baPWV), and coronary atherosclerosis was assessed by 64-row multidetector computed tomography. Results: Women with osteoporosis had a significantly higher baPWV than those in the osteopenia and normal BMD groups. Higher baPWV was also associated with the presence of atherosclerosis. The baPWV was significantly positively correlated with age, systolic blood pressure, diastolic blood pressure, and neutrophil to lymphocyte ratio and negatively correlated with femur BMD. The optimum predictive value of baPWV in coronary atherosclerosis was estimated using the receiver operating characteristic curve. A cutoff of 1,506 cm/second would give a sensitivity of 83.3% and specificity of 82.9%. A higher overall risk for coronary atherosclerosis emerges with higher baPWV levels (>1,500 cm/s) after controlling for age and cardiovascular risk factors. Conclusions: Arterial stiffness by measuring baPWV can be a useful independent predictor for coronary atherosclerosis. In addition, our results suggest that postmenopausal women with osteoporosis should be considered for further evaluation of coronary atherosclerosis.

Expression of Cyclooxygenase-2 in Eutopic Endometrium and Ovarian Endometriotic Tissue in Women with Severe Endometriosis

Background/Aims: The purpose of this study is to evaluate the level and pattern of cyclooxygenase-2 (Cox-2) expression in endometriosis and to investigate the correlation between the expression of Cox-2 and several clinicosurgical parameters. Methods: Twenty-six patients with endometriosis and 21 patients with other benign gynecological conditions were enrolled. The eutopic endometrium was sampled by pipelle, and fragments of ovarian endometrioma and non-endometriotic ovarian cysts were sampled during surgery. Total RNA isolation and semiquantitative reverse transcriptase polymerase chain reaction was performed. Results: The expression of Cox-2 mRNA (mean ± SEM) in eutopic endometrium and ovarian endometriotic tissue significantly increased in patients with endometriosis compared with the controls. The expression of Cox-2 increased significantly in the proliferative phase in eutopic endometrium and in the secretory phase in ovarian endometriotic tissue of patients with endometriosis compared with the controls. Cox-2 mRNA expression in the endometrium and ovarian lesions significantly correlated with serum CA-125 and the diameter of the endometrioma. Conclusions: Cox-2 expression increased in the eutopic endometrium and ovarian endometriotic tissue of the patients with endometriosis. These findings indicate that Cox-2 may be involved in the pathogenesis and progression of endometriosis.

ORIGINAL ARTICLE: Endometrial Osteopontin mRNA Expression and Plasma Osteopontin Levels are Increased in Patients with Endometriosis

Problem  The aim of this study was to evaluate osteopontin (OPN) mRNA expression in eutopic endometrium and plasma OPN levels in patients with endometriosis.

Implication of ADAM-8, -9, -10, -12, -15, -17, and ADAMTS-1 in Implantational Remodeling of a Mouse Uterus

In the present study, whether the ADAM-8, -9, -10, -12, -15, -17, and ADAMTS-1 proteins might play a role in mouse uterus during periimplantation period was investigated. Immunoblotting analyses demonstrated that all ADAM proteins consistently appeared throughout days 1 to 8 of pregnancy but with a variation depending on the species of ADAM gene, the progression of pregnancy, and the site of the uterus. Immunohistochemical analyses indicated that ADAM proteins were localized in the luminal or glandular epithelial layers with a varying intensity depending on the species of ADAM and the progression of pregnancy. Particularly ADAM-8, -12, and -15, were predominantly located in the implantation site of the uterine tissues, whereas little or no protein was localized in the interimplantation site. Based upon these observations, it is suggested that the ADAMs might play an important role in the remodeling of the mouse uterus during the periimplantation period.

Expression of integrins, cyclooxygenases and matrix metalloproteinases in three-dimensional human endometrial cell culture system

The objective of this investigation was to establish a three-dimensionally cultured human endometrium which could be used as a tissue model for the mechanism study of implantation in vitro. By using human endometrial stromal (ES) and epithelial cells (EE) from hysterectomy specimens, reconstruction of endometrium in culture was established by first layering a collagen gel containing ES cells, then overlaying with the Matrigel containing endometrial epithelial (EE) cells. Ultrastructural examination of the 48 h-endometrial cell culture revealed monolayered columnar EE cells with microvilli on the collagen layer containing ES cells and appearance of the tight junctions and desmosomes between EE cells, a cell layer closely resembling the native endometrium. Immunohistochemical characterization of the reconstructed endometrium showed a strong immunoreactivity for cytokeratin, integrin α1, α4 and β3 subunits, cyclooxygenases-1 and -2, matrix metalloproteinases-1, -2, -3 and -9, and tissue inhibitor of metalloproteinases-1 and -2 in the EE cells comparable to the native endometrial epithelium. ES cells also showed stronger immunoreactivity for cyclooxygenases, integrins and MMPs, but less for cytokeratin. Gelatin zymographic analyses of the media obtained from the reconstructed endometrium model showed gelatinase activity bands at 57, 60, 72, 92 and 97 kDa molecular weight, respectively. The present study provides a possibility that our three-dimensionally cultured endometrium model could mimic the morphological and functional characteristics of the native endometrium. The model could be used to clarify the roles of various molecules involved in the human implantation.

Bovine Follicular Fluid and Serum Share a Unique Isoform of Matrix Metalloproteinase-2 That Is Degraded by the Oviductal Fluid

Abstract Whereas the mammalian fertilization environment consists of possible products of the mutual interaction between oviductal and follicular fluids in addition to both fluid components, little is known regarding the interaction. In the present study, we have demonstrated that a mutual interaction occurs, resulting in the biochemical changes of follicular fluid components. Gelatin zymographic analyses of bovine follicular fluid (bFF) showed consistently a distinct, gelatinolytic activity having a molecular weight of 110 kDa (GA110) in addition to other gelatinases, whereas bovine oviductal fluid (bOF) showed a lack of GA110. Surprisingly, when bFF was mixed with bOF before zymography, the GA110 of bFF mostly disappeared at a 1:1 (v/v) mixture, completely disappeared at a 1:10 mixture, as fast as within 30 min after mixing. Other bFF gelatinase activities were not affected by bOF at 1:1 or 10:1 mixtures. Addition of EDTA or phenanthroline, but not of phenylmethylsulfonyl fluoride or trypsin inhibitor, to the mixture greatly increased the gelatinolytic activity of bFF GA110. The increased activity of bFF GA110 by EDTA was again abolished by subsequent bOF treatment. Addition of aminophenylmercuric acetate to the EDTA-treated bFF also abolished GA110; however, this was accompanied by the disappearance of other gelatinases, except the 62-kDa gelatinase, the activity of which increased as the treatment continued up to 24 h. Addition of EDTA or phenanthroline to the gelatin gel incubation buffer after electrophoresis abolished almost all gelatinases of bFF, except those of 88–84 kDa, demonstrating that they were indeed gelatinases or isoforms. Bovine serum and fetal bovine serum also showed the presence of GA110, the activity of which was increased by EDTA. However, ovarian granulosa cell homogenate did not exhibit GA110. Immunoblot experiments using antibodies against matrix metalloproteinase (MMP)-2 and MMP-9 demonstrated that bFF GA110 was an isoform of MMP-2, and that the 62-kDa form was an active form of MMP-2. Disappearance of immunoreactive GA110 of bFF and serum by bOF was also observed. Based on these observations, we conclude that bFF and bovine serum share a unique isoform of MMP-2, and that bOF can specifically degrade the isoform, suggesting that a mutual interaction between bFF and bOF could occur at the time of ovulation.

Hysteroscopic Endometrial Ablation as a Treatment for Abnormal Uterine Bleeding in Patients with Renal Transplants

STUDY OBJECTIVE To assess the effectiveness and safety of hysteroscopic endometrial ablation as a surgical management of abnormal uterine bleeding that develops in patients with renal transplants. DESIGN Retrospective study (Canadian Task Force classification II-2). SETTING Yonsei University Medical College, Severance Hospital. PATIENTS Sixty-two women with abnormal uterine bleeding who had undergone renal transplantation. INTERVENTION Hysteroscopic endometrial ablation. MEASUREMENTS AND MAIN RESULTS Fifty-four out of 62 patients (87.0%) who had undergone hysteroscopic endometrial ablation reported decreased bleeding (95% CI: 0.76 to 0.94): amenorrhea in 25 (40.3%), spotting in 19 (30.6%), and eumenorrhea in 10 (16.1%). Mean follow-up duration was 6 months. No complications related to the procedure were reported. Levonorgestrel-releasing intrauterine systems (LNG-IUSs) were inserted into eight patients who experienced continuous bleeding, five of whom showed symptomatic improvement: spotting in three (4.9%) and eumenorrhea in two (3.2%). The three patients (4.9%) in whom the LNG-IUS had no effect had hysterectomies, and the resultant pathologic findings were two cases of adenomyosis and one case of simple endometrial hyperplasia without atypia. CONCLUSION Hysteroscopic endometrial ablation as a surgical management of abnormal uterine bleeding that develops in patients with renal transplants is an effective and safe procedure.

ORIGINAL ARTICLE: Endometrial Osteopontin mRNA Expression and Plasma Osteopontin Levels are Increased in Patients with Endometriosis: OSTEOPONTIN IN ENDOMETRIOSIS

Problem  The aim of this study was to evaluate osteopontin (OPN) mRNA expression in eutopic endometrium and plasma OPN levels in patients with endometriosis.