Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells

Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and MitoFreSHtracer signals in living stem cells take ~2~3 h, and the fractionation of stem cells into subpopulations on the basis of cellular GSH levels takes 3~4.5 h. This method could be applied to almost every kind of mammalian cell with minor modifications to the protocol described here.

The Therapeutic Effect of Human Embryonic Stem Cell-Derived Multipotent Mesenchymal Stem Cells on Chemical-Induced Cystitis in Rats / 대한배뇨장애요실금학회지

PURPOSE: To evaluate the therapeutic effect of human embryonic stem cell (hESC)-derived multipotent mesenchymal stem cells (M-MSCs) on ketamine-induced cystitis (KC) in rats. METHODS: To induce KC, 10-week-old female rats were injected with 25-mg/kg ketamine hydrochloride twice weekly for 12 weeks. In the sham group, phosphate buffered saline (PBS) was injected instead of ketamine. One week after the final injection of ketamine, the indicated doses (0.25, 0.5, and 1×106 cells) of M-MSCs (KC+M-MSC group) or PBS vehicle (KC group) were directly injected into the bladder wall. One week after M-MSC injection, the therapeutic outcomes were evaluated via cystometry, histological analyses, and measurement of gene expression. Next, we compared the efficacy of M-MSCs at a low dose (1×105 cells) to that of an identical dose of adult bone marrow (BM)-derived MSCs. RESULTS: Rats in the KC group exhibited increased voiding frequency and reduced bladder capacity compared to rats of the sham group. However, these parameters recovered after transplantation of M-MSCs at all doses tested. KC bladders exhibited markedly increased mast cell infiltration, apoptosis, and tissue fibrosis. Administration of M-MSCs significantly reversed these characteristic histological alterations. Gene expression analyses indicated that several genes associated with tissue fibrosis were markedly upregulated in KC bladders. However the expression of these genes was significantly suppressed by the administration of M-MSCs. Importantly, M-MSCs ameliorated bladder deterioration in KC rats after injection of a low dose (1×105) of cells, at which point BM-derived MSCs did not substantially improve bladder function. CONCLUSIONS: This study demonstrates for the first time the therapeutic efficacy of hESC-derived M-MSCs on KC in rats. M-MSCs restored bladder function more effectively than did BM-derived MSCs, protecting against abnormal changes including mast cell infiltration, apoptosis and fibrotic damage.

Adipose stem cell-derived nanovesicles inhibit emphysema primarily via an FGF2-dependent pathway

Cell therapy using stem cells has produced therapeutic benefits in animal models of COPD. Secretory mediators are proposed as one mechanism for stem cell effects because very few stem cells engraft after injection into recipient animals. Recently, nanovesicles that overcome the disadvantages of natural exosomes have been generated artificially from cells. We generated artificial nanovesicles from adipose-derived stem cells (ASCs) using sequential penetration through polycarbonate membranes. ASC-derived artificial nanovesicles displayed a 100 nm-sized spherical shape similar to ASC-derived natural exosomes and expressed both exosomal and stem cell markers. The proliferation rate of lung epithelial cells was increased in cells treated with ASC-derived artificial nanovesicles compared with cells treated with ASC-derived natural exosomes. The lower dose of ASC-derived artificial nanovesicles had similar regenerative capacity compared with a higher dose of ASCs and ASC-derived natural exosomes. In addition, FGF2 levels in the lungs of mice treated with ASC-derived artificial nanovesicles were increased. The uptake of ASC-derived artificial nanovesicles was inhibited by heparin, which is a competitive inhibitor of heparan sulfate proteoglycan that is associated with FGF2 signaling. Taken together, the data indicate that lower doses of ASC-derived artificial nanovesicles may have beneficial effects similar to higher doses of ASCs or ASC-derived natural exosomes in an animal model with emphysema, suggesting that artificial nanovesicles may have economic advantages that warrant future clinical studies.

Tracking Intravenous Adipose-Derived Mesenchymal Stem Cells in a Model of Elastase-Induced Emphysema / 결핵및호흡기질환

BACKGROUND: Mesenchymal stem cells (MSCs) obtained from bone marrow or adipose tissue can successfully repair emphysematous animal lungs, which is a characteristic of chronic obstructive pulmonary disease. Here, we describe the cellular distribution of MSCs that were intravenously injected into mice with elastase-induced emphysema. The distributions were also compared to the distributions in control mice without emphysema. METHODS: We used fluorescence optical imaging with quantum dots (QDs) to track intravenously injected MSCs. In addition, we used a human Alu sequence-based real-time polymerase chain reaction method to assess the lungs, liver, kidney, and spleen in mice with elastase-induced emphysema and control mice at 1, 4, 24, 72, and 168 hours after MSCs injection. RESULTS: The injected MSCs were detected with QD fluorescence at 1- and 4-hour postinjection, and the human Alu sequence was detected at 1-, 4- and 24-hour postinjection in control mice (lungs only). Injected MSCs remained more in mice with elastase-induced emphysema at 1, 4, and 24 hours after MSCs injection than the control lungs without emphysema. CONCLUSION: In conclusion, our results show that injected MSCs were observed at 1 and 4 hours post injection and more MSCs remain in lungs with emphysema.

The Molecular Nature of Very Small Embryonic-Like Stem Cells in Adult Tissues

Pluripotent stem cells (PSCs) have been considered as the most important cells in regenerative medicine as they are able to differentiate into all types of cells in the human body. PSCs have been established from several sources of embryo tissue or by reprogramming of terminally differentiated adult tissue by transduction of so-called Yamanaka factors (Oct4, Sox2, Klf4, and cMyc). Interestingly, accumulating evidence has demonstrated the residence of PSCs in adult tissue and with the ability to differentiate into multiple types of tissue-committed stem cells (TCSCs). We also recently demonstrated that a population of pluripotent Oct4(+) SSEA-1(+)Sca-1(+)Lin-CD45(-) very small embryonic-like stem cells (VSELs) resides in the adult murine bone marrow (BM) and in other murine tissue. These very small (~3-6 microm) cells express pluripotent markers such as Oct4, Nanog, and SSEA-1. VSELs could be specified into several tissue-residing TCSCs in response to tissue/organ injury, and thus suggesting that these cells have a physiological role in the rejuvenation of a pool of TCSCs under steady-state conditions. In this review article, we discuss the molecular nature of the rare population of VSELs which have a crucial role in regulating the pluripotency, proliferation, differentiation, and aging of these cells.

Very small embryonic-like stem-cell optimization of isolation protocols: an update of molecular signatures and a review of current in vivo applications

As the theory of stem cell plasticity was first proposed, we have explored an alternative hypothesis for this phenomenon: namely that adult bone marrow (BM) and umbilical cord blood (UCB) contain more developmentally primitive cells than hematopoietic stem cells (HSCs). In support of this notion, using multiparameter sorting we were able to isolate small Sca1+Lin-CD45- cells and CD133+Lin-CD45- cells from murine BM and human UCB, respectively, which were further enriched for the detection of various early developmental markers such as the SSEA antigen on the surface and the Oct4 and Nanog transcription factors in the nucleus. Similar populations of cells have been found in various organs by our team and others, including the heart, brain and gonads. Owing to their primitive cellular features, such as the high nuclear/cytoplasm ratio and the presence of euchromatin, they are called very small embryonic-like stem cells (VSELs). In the appropriate in vivo models, VSELs differentiate into long-term repopulating HSCs, mesenchymal stem cells (MSCs), lung epithelial cells, cardiomyocytes and gametes. In this review, we discuss the most recent data from our laboratory and other groups regarding the optimal isolation procedures and describe the updated molecular characteristics of VSELs.

Differential alternative splicing of human transglutaminase 4 in benign prostate hyperplasia and prostate cancer

Transglutaminase 4 is a member of enzyme family that catalyzes calcium-dependent posttranslational modification of proteins. Although transglutaminase 4 has been shown to have prostate-restricted expression pattern, little is known about the biological function of transglutaminase 4 in human. To gain insight into its role in prostate, we analyzed the expression status of human transglutaminase 4 in benign prostate hyperplasia (BPH) and prostate cancer (PCa). Unexpectedly, RT-PCR and nucleotide sequence analysis showed four alternative splicing variants of transglutaminase 4: transglutaminase 4-L, -M (-M1 and -M2) and -S. The difference between transglutaminase 4-M1 and -M2 is attributed to splicing sites, but not nucleotide size. The deduced amino acid sequences showed that transglutaminase 4-L, -M1 and -M2 have correct open reading frames, whereas transglutaminase 4-S has a truncated reading frame. RT-PCR analysis of clinical samples revealed that transglutaminase 4-M and -S were detected in all tested prostate tissue (80 BPH and 48 PCa). Interestingly, transglutaminase 4-L was found in 56% of BPH (45 out of 80) and only in 15% of PCa (7 out of 48). However, transglutaminase 4-L expression did not correlate with serum prostate-specific antigen (PSA) level, prostate volumes or PSA densities. These results will provide a clue to future investigation aiming at delineating physiological and pathological roles of human transglutaminase 4.

Transglutaminase 2 inhibits apoptosis induced by calciumoverload through down-regulation of Bax

An abrupt increase of intracellular Ca2+ is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca2+ triggers apoptotic cell death through mitochondrial swelling and activation of Ca2+-dependent enzymes. Transglutaminase 2 (TG2) is a Ca2+-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca2+-overload. Overexpression of TG2 inhibited the A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca2+-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca2+-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.

Clinical and sonographic predictors for successful labor induction in preterm women / 대한산부인과학회지

OBJECTIVE: To identify the clinical and sonographic parameters which predict the likelihood of successful labor induction in preterm women. METHODS: This prospective observational study enrolled 103 consecutive preterm women (<37 weeks gestation) with singleton gestations scheduled for induction of labor. Transvaginal ultrasound for measurement of the cervical length was performed and the Bishop score was determined by digital examination. The parameters studied were gestational age at induction, parity, maternal age, Bishop score, sonographic cervical length, and current body mass index (BMI). Univariate and multivariate statistical methods were used for data analysis. RESULTS: Successful induction of labor occurred in 45 (44%) of women. Multiple logistic regression analysis identified parity, maternal BMI, and gestational age at induction as the independent predictors of successful labor induction in preterm women, although gestational age did not reach statistical significance (P=0.056). However, maternal age, sonographic cervical length, and Bishop score did not provide independent contribution to success of induction. CONCLUSION: In preterm women undergoing induction of labor parity, maternal BMI, and gestational age at induction were independent parameters in predicting successful induction of labor. However, sonographic cervical length and Bishop score had poor predictive values for success of labor induction.

Reference range for the sonographically estimated fetal weights in twin gestations / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to establish the reference range for the sonographically estimated fetal weights (EFW) in twin gestations at 26 to 38 weeks' gestation. METHODS: This is a retrospective study of EFW that were obtained from electronic medical records of live births of all twins delivered between June 2003 through August 2007 at Seoul National University Bundang hospital. EFW was calculated using the mathematical model of Hadlock and only one measurement from each twin was used. Linear regression and Kolmogorov-Smirnov test were used for statistical analysis. RESULTS: Three hundred and forty-three twin pregnancies (686 fetuses) were included in this study. Reference range for EFW in twin gestations was presented as mean, standard deviation and percentiles. Scatterplots of EFW against gestational age (GA) were created with regression line of best fit and a linear growth function was observed between GA and EFW between 26 and 38 weeks' gestation (EFW=161.807 X GA - 3349.735 (r2=0.861, p<0.001). CONCLUSIONS: We established the reference range for EFW in twin gestations at 26 to 38 weeks' gestation. These results can be used as reference guidelines in the assessment of fetal growth restriction in Korean twin pregnancies.

A prospective longitudinal study of normal reference ranges for the length of fetal limb bones by ultrasound examination / 대한산부인과학회지

OBJECTIVE: To establish the reference ranges for the length of fetal limb bones (femur, tibia, fibula, humerus, ulna, radius, foot) at 12 to 38 weeks' gestation. METHODS: The candidates of this prospective longitudinal study were 43 consecutive women with uncomplicated singleton gestations who initially had antenatal care from the first trimester of pregnancy and follow-up regularly. We performed serial measurements of each limb bone and foot bone length by ultrasound examination every 4 weeks until 28 weeks, and then by 2 weeks until 36 weeks, and weekly thereafter. RESULTS: A total of 2,633 and 321 of fetal limb and foot bone lengths were measured. The reference ranges for the length of limb bones and foot length were presented as mean, 95% confidence interval of the means and in percentile. CONCLUSIONS: Our study results are represented as percentile tables for each of the limb bone length. These results can be provided as standardized data of the fetal limb bone length to distinguish from skeletal dysplasia and may well be suggested as the reference guideline for normal fetal limb bone length in Korea.

Immunocytochemical detection of HPV16 E7 in cervical smear

Cervical cancer is characterized by a long period of preclinical dysplasia or carcinoma in situ progressing into invasive cancer. Although Papanicolaou (Pap) smear test has contributed significantly to the early detection of precursor lesions, the cytological screening has inherent problems that produce considerable false negative/positive results. Since the infection of high-risk type of human papillomavirus (HPV) is strongly associated with cervical cancer, we investigated the feasibility of an immunostaining test to detect cells infected by HPV in cervical smear. We produced monoclonal antibodies against HPV16 E7 in mice by repeated injections with the recombinant HPV16 E7. Western blot analysis and immunocytochemical assay demonstrated that the selected monoclonal antibody, mAb (130-9-7), reacts specifically with cultured cervical cancer cell lines infected by HPV16. Specific staining was observable with the HPV16-positive smear specimens obtained from the cervical cancer patients, whereas no staining was detected with the HPV-negative smear specimens. To achieve the desired sensitivity, specificity and reproducibility, we modified and optimized the conventional immunocytochemical procedure for cervical smear specimens. Our results suggest that this immunostaining method for detecting high-risk HPV in cervical smear may be used as a strategy to distinguish a high-risk group, especially those patients with low grade cytological abnormality.

Clinical significance of anti-filaggrin antibody recognizing uncitrullinated filaggrin in rheumatoid arthritis

Filaggrin is expressed in the cornified layer of epidermis and known to be one of the antigenic targets in rheumatoid arthritis. Although the citrulline residue in filaggrin is thought to be an antigenic determinant recognized by autoantibodies, the diagnostic sensitivity of synthetic citrullinated peptide is variable. To investigate the implication of anti-filaggrin antibodies recognizing uncitrullinated filaggrin in rheumatoid arthritis, we assayed antibody titers using unmodified recombinant filaggrin in the sera from 73 patients with rheumatoid arthritis, 150 patients with other connective tissue diseases and 70 normal controls. We also performed the correlation analysis between antibody titers and the clinical variables in patients with rheumatoid arthritis. Titers of IgG anti-filaggrin antibodies were significantly higher in rheumatoid arthritis patients compared to normal controls (P=0.02), but not in patients with osteoarthritis, ankylosing spondylitis or systemic lupus erythematosus. IgG anti-filaggrin antibodies were more frequently found in patients with rheumatoid arthritis compared to normal controls (12.3% vs 1.4% respectively, P=0.04). An anti-filaggrin antibody titer was correlated with visual analogue scale of pain, tender joint count, Ritchie articular index or C-reactive protein, but not with anti-nuclear antibody or rheumatoid factor. These results suggest that anti-filaggrin antibody recognizes the uncitrullinated filaggrin as an antigen and its titer correlates with clinical parameters, explaining the variable sensitivity of anti-filaggrin antibody test.

Factors influencing pregnancy rates in intrauterine insemination / 대한산부인과학회지

OBJECTIVE: We have studied the factors that influence the pregnancy rate in ovarian hyperstimulation and intrauterine insemination (IUI) in infertility patients. METHODS: Seventy two patients who visited the infertility clinic in Dongguk University Kyong-ju hospital from January 2002 to December 2003 underwent IUI after 99 cycles of ovarian hyperstimulation. We administered clomiphene with exogenous gonadotropin from cycle day 3 and then IUI was performed. The variables selected for retrospective analysis were patient's age (or=2), number of follicles (>or=2), size of dominant follicle (>or=18 mm), total sperm counts (>or=10 X 10(6)). Chi-square test, Fisher's exact test and a multiple logistic regression analysis were used to detect differences between groups in each variable. RESULTS: The pregnancy rate per patient was 29.2% (21/72) and 21.2% (21/99) per cycle. Factors that influenced pregnancy rate were age, number of treatment cycles, number of preovulatory follicles (>or=16 mm), and total inseminated motile sperm counts, all of which showed significant differences (por=35), number of treatment cycles (2 cycle vs. 1 cycle), number of preovulatory follicles (>or=16 mm) (>or=3 vs. 1), and total inseminated motile sperm counts (>or=40 X 10(6) vs. <10 X 10(6)). The odds ratio of each of these variables were 12.6, 3.37, 11.64, and 10.59. CONCLUSION: For successful pregnancy rates in IUI after ovarian hyperstimulation, the patient's age, number of treatment cycles, number of preovulatory follicles, and total inseminated motile sperm counts should be considered.

Different inhibition characteristics of intracellular transglutaminase activity by cystamine and cysteamine

The treatment of cystamine, a transglutaminase (TGase) inhibitor, has beneficial effects in several diseases including CAG-expansion disorders and cataract. We compared the inhibition characteristics of cystamine with those of cysteamine, a reduced form of cystamine expected to be present inside cells. Cystamine is a more potent inhibitor for TGase than cysteamine with different kinetics pattern in a non- reducing condition. By contrast, under reducing conditions, the inhibitory effect of cystamine was comparable with that of cysteamine. However, cystamine inhibited intracellular TGase activity more strongly than cysteamine despite of cytoplasmic reducing environment, suggesting that cystamine itself inhibits in situ TGase activity by forming mixed disulfides.

A Case of Pseudomyxoma Peritonei Associated with Borderline Mucinous Tumor of Both Ovaries and Appendix / 대한산부인과학회잡지

Pseudomyxoma peritonei is a rare condition characterized by mucinous ascites associated with peritoneal and omental implants. This is most commonly originated from mucinous tumor of the ovary or appendix. We have experienced a case of pseudomyxoma peritonei associated with borderline mucinous tumor of both ovaries and appendix. We concluded that the appendix is the primary lesion of pseudomyxoma peritonei by various immunohistochemical stains and therefore reporting this case with the brief review of literatures.

Improved immunodetection of human papillomavirus E7

Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell line which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.

A case of Patent Urachus / 대한비뇨기과학회지

The patent urachus is the failure of complete urachal lumen closure results in a free communication between the bladder and the umbilicus. The urachus remains patent and urine leaks from the umbilicus. A 53 day old female infant was admitted to Seoul Red Cross Hospital due to urine leakage accompanied by varing purulent discharge from the enlarged protruding umbilicus, measuring 2 Cm x 2 Cm x 2 Cm, since birth. The fistulogram showed a long fistulous tract communicate with bladder and umbilicus. After complete resection of the urachus and umbilicus with a bladder cuff, the patient had no urine leakage from umbilicus. We report a case of patent urachus with some literature.

Comparison Between the Results of Transurethral Resection and Open Prostatectomy for the Benign Prostatic Hypertrophy of 20 gm or More / 대한비뇨기과학회지

Among the 65 patients undergoing transurethral resection of the prostate from 1985 to 1988, the complication incidence was significantly higher in 25 patients whose weight of resected prostate was above 20 gm than in 40 patients below 20 gm (p<0.05). So to choose mote proper surgical method, the results of operation, motality and morbity of these 25 TURP patients were compared with 35 open prostatectomy patients whose weight of resected prostate was between 20 gm and 60 gm during same period. The mean incidence of complication for open prostatectomy (60%) was nearly twice that of the TURP (32%) (p<0.05). The age of the patient had no significant influence on the incidence of complication in both groups. In case of the weight of resected prostate below 40 gm complication incidence for the TURP (26.3%) was significantly lower compared with open prostatectomy(66.7%) (p<0.05), but in case of above 40 gm there was no significant difference in both groups. In case of the length of resection below 120 min complication incidence was significantly low in TURP (21.4%) compared with open prostatectomy (61.5%) (p<0.05), but in case of above 120 min. there was no significant difference in both groups. In the open prostatectomy there was no increase in complication incidence by the weight of the gland and the operation time, but in TURP, complicat,40 gm incidence was definitely increased when either weight of the resected prostate was above 40 gm or length of resection was above 120 minutes. Therefore, it seems that TURP is preferable surgical method for benign prostatic hypertrophy unless open surgery is necessarily indicated.

Four Cases of the Fournier's Gangrene / 대한비뇨기과학회지

Fournier described five patients with gangrene of male external genitalia in 1883 and emphasized three characteristics: (1) abrupt onset in young healthy male, (2) rapid progression to gangrene,(3) absence of discernible cause. But more recent reports described genital gangrene as occurring in any age group and 4 patients in our cases, the mean age was 46 years with an age range of 31 to 59 years. Predisposing causes were as follows: case 1. prostatic calculi, Buerger's disease, case 2, perianal abscess, case 3, diabetes mellitus, case 4, tuberculous spondylitis accompanied by paraplegia and bed sores, liver cirrhosis. The duration of symptoms prior to the development of gangrene varied between 4 to 10 days. The cultured organisms were as follows : case 1. Alpha-hemolytic streptococcus, case 2. E. coli, Alpha-hemolytic streptococcus case 3. Alpha-hemolytic streptococcus, case 4, Mycobacterium tuberculosis, proteus species. Reconstructive surgery of defected scrotum was performed postoperative 8 to 41 days (mean 28.7) and total admission period was 25 to 83 days (mean 46 days).