Training ultrasound-guided percutaneous nephrostomy technique with porcine model.

PURPOSE: There is increasing interest in the use of ultrasound for endoscopic and percutaneous procedures. Access can be achieved without radiation exposure under ultrasound guidance. Our aim was to develop a porcine-based training model for ultrasound-guided percutaneous renal access that could also be personalized to a specific patient. MATERIALS AND METHODS: The Institutional Animal Care and Use Committee of Severance Hospital approved the study protocol. An anesthetized pig was placed in the dorsal lithotomy position. For the nephrostomy puncture, a Chiba biopsy needle with an echo tip was used under ultrasound guidance. Eight residents and three consultants in urology participated. Puncture time was defined as the nephrostomy time to confirm the flow of irrigation via the needle. After training, satisfaction survey results for clinical usability and procedural difficulty were evaluated. RESULTS: The 5-point Likert scale satisfaction survey for clinical usability and procedural difficulty found mean results of 4.64 and 4.09 points, respectively. There were no differences between residents and consultants for either variable. For all participants combined, there was a significant difference for nephrostomy time between the first and second trials (278.8±70.6 s vs. 244.5±47.0 s; p=0.007). The between-trial difference was greater for residents (291.5±71.2 s vs. 259.1±41.9 s; p=0.039). The difference for the consultant was not significant (245.0±69.4 s vs. 205.7±42.5 s; p=0.250). CONCLUSIONS: We developed a porcine-based ultrasound-guided nephrostomy puncture training model. Satisfaction survey results indicated high clinical usability and procedural difficulty. For nephrostomy time, the model was more effective for urology residents than for consultants.

The Consecutive 200 Cases of Endoscopic-Combined Intrarenal Surgery: Comparison between Standard and Miniature Surgeries.

Background and Objectives: Percutaneous nephrolithotomy (PCNL) is still the gold-standard treatment for large and/or complex renal stones. Endoscopic combined intrarenal surgery (ECIRS) was developed with the goal of minimizing the number of access tracts of PCNL while simultaneously improving the one-step stone-free rate (SFR). The aim of this study was to share the experience of the consecutive 200 cases of ECIRS in one institute and analyze surgical outcomes of mini-ECIRS and standard ECIRS. Materials and Methods: We performed ECIRS for 200 adult patients between July 2017 and January 2020. An ECIRS was performed with the patient under general anesthesia in the intermediate-supine position. Surgeries were finished using a tubeless technique with a simple ureteral stent insertion. Results: There were significant differences in the mean maximal stone length (MSL), the variation coefficient of stone density (VCSD), the linear calculus density (LCD), the Seoul National University Renal Stone Complexity (S-ReSC), and the modified S-ReSC scores in stone characteristics, and estimated blood loss (EBL) and operation time in peri-operative outcomes between conventional and mini-ECIRS. After propensity-score matching, there was only a difference in EBL between the two groups. In logistic regression models, MSL [odds ratio (OR) 0.953; 95% confidence interval (CI) 0.926-0.979; p < 0.001], LCD (OR 4.702; 95% CI 1.613-18.655; p = 0.013) were significant factors for the success rate after ECIRS. Conclusions: In patients who underwent a mini-ECIRS, the stones were relatively smaller and less complex, and the operation time was shorter. However, if the size of stones was similar, there was no difference in the success rate, but EBL was lower in mini-ECIRS than in standard surgery.

Impact of Preoperative Ureteral Stenting in Retrograde Intrarenal Surgery for Urolithiasis.

Background and Objectives: Ureteral stent insertion passively dilates the ureter. Therefore, it is sometimes used preoperatively before flexible ureterorenoscopy to make the ureter more accessible and facilitate urolithiasis passage, especially when ureteroscopic access has failed or when the ureter is expected to be tight. However, it may cause stent-related discomfort and complications. This study aimed to assess the effect of ureteral stenting prior to retrograde intrarenal surgery (RIRS). Materials and Methods: Data from patients who underwent unilateral RIRS for renal stone with the use of a ureteral access sheath from January 2016 to May 2019 were retrospectively analyzed. Patient characteristics, including age, sex, BMI, presence of hydronephrosis, and treated side, were recorded. Stone characteristics in terms of maximal stone length, modified Seoul National University Renal Stone Complexity score, and stone composition were evaluated. Surgical outcomes, including operative time, complication rate, and stone-free rate, were compared between two groups divided by whether preoperative stenting was performed. Results: Of the 260 patients enrolled in this study, 106 patients had no preoperative stenting (stentless group), and 154 patients had stenting (stenting group). Patient characteristics except for the presence of hydronephrosis and stone composition were not statistically different between the two groups. In surgical outcomes, the stone-free rate was not statistically different between the two groups (p = 0.901); however, the operation time for the stenting group was longer than that of the stentless group (44.8 ± 24.2 vs. 36.1 ± 17.6 min; p = 0.001). There were no differences in the complication rate between the two groups (p = 0.523). Conclusions: Among surgical outcomes for RIRS with a ureteral access sheath, preoperative ureteral stenting does not provide a significant advantage over non-stenting with respect to the stone-free rate and complication rate.

A New Parameter for Calcium Oxalate Stones: Impact of Linear Calculus Density on Non-Contrast Computed Tomography.

Background and Objectives: Non-contrast computed tomography (NCCT) is widely used to evaluate urolithiasis. The NCCT attenuation, measured in Hounsfield units (HU), has been evaluated to predict stone characteristics. We propose a novel parameter, linear calculus density (LCD), and analyze variables from NCCT imaging to predict calcium oxalate (CaOx) stones, which are common and challenging to fragment. Materials and Methods: We retrospectively reviewed the medical records of patients with urolithiasis between 2014 and 2017. Among those, 790 patients were included. Based on the NCCT pre-treatment, the maximal stone length (MSL), mean stone density (MSD), and stone heterogeneity index (SHI) were obtained. In addition, the variation coefficient of stone density (VCSD = SHI/MSD × 100) and linear calculus density (LCD = VCSD/MSL) were calculated. In accordance with the stone analysis, the patients were divided into two groups (CaOx and non-CaOx groups). The logistic regression model and receiver operating characteristic (ROC) curve were used for predictive modeling. Results: In the CaOx group, the SHI, VCSD, and LCD were more significant than in the non-CaOx group (all p < 0.001). SHI (OR 1.002, 95% CI 1.001-1.004, p < 0.001), VCSD (OR 1.028, 95% CI 1.016-1.041, p < 0.001), and LCD (OR 1.352, 95% CI 1.270-1.444, p < 0.001) were significant independent factors for CaOx stones in the logistic regression models. The areas under the ROC curve for predicting CaOx stones were 0.586 for SHI, 0.66 for VCSD, and 0.739 for LCD, with a cut-point of 2.25. Conclusions: LCD can be a useful new parameter to provide additional information to help discriminate CaOx stones before treatment.

Silodosin versus Tamsulosin for Medical Expulsive Therapy of Ureteral Stones: An Updated Systematic Review and Meta-Analysis of Randomized Controlled Trials.

Background and Objectives: This systematic review and meta-analysis of randomized controlled trials was performed to compare the therapeutic effects and safety profiles of silodosin and tamsulosin for medical expulsive therapy (MET) of ureteral stones. Materials and Methods: We searched PubMed, EMBASE, the Cochrane Library, and Web of Science to identify articles published before July 2022 that described randomized controlled trials comparing silodosin and tamsulosin for MET of ureteral stones. Endpoints were stone expulsion rate, stone expulsion time, and total complication rate. Results: In total, 14 studies were included in our analysis. The size of ureteral stones was <1 cm. Compared with tamsulosin, silodosin resulted in a significantly higher stone expulsion rate (p < 0.01, odds ratio (OR) = 2.42, 95% confidence interval (CI) = 1.91 to 3.06, I2 = 0%) and significantly shorter stone expulsion time (p < 0.01, mean difference = −3.04, 95% CI = −4.46 to −1.63, I2 = 89%). The total complication rate did not significantly differ between silodosin and tamsulosin (p = 0.33, OR = 1.15, 95% CI = 0.87 to 1.52, I2 = 7%). Conclusions: Compared with tamsulosin, silodosin resulted in significantly better expulsion of ureteral stones <1 cm. The total complication rate did not significantly differ between silodosin and tamsulosin. Thus, silodosin may be superior to tamsulosin for MET of ureter stones <1 cm.

Surveillance versus Adjuvant Treatment with Chemotherapy or Radiotherapy for Stage I Seminoma: A Systematic Review and Meta-Analysis According to EAU COVID-19 Recommendations.

Background and Objectives: During the coronavirus disease 2019 (COVID-19) outbreak, the European Association of Urology (EAU) Guidelines Office Rapid Reaction Group (GORRG) recommended that patients with clinical stage I (CSI) seminoma be offered active surveillance (AS). This meta-analysis aimed to evaluate the efficacy of AS versus adjuvant treatment with chemotherapy or radiotherapy for improving the overall survival (OS) of CSI seminoma patients. Materials and Methods: A systematic review was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The PubMed/Medline, EMBASE, and Cochrane Library databases were searched. The primary outcome was 5-year OS, and the secondary outcome was the 5-year relapse-free survival (RFS). The outcomes were analyzed as odds ratios (ORs) and 95% confidence intervals (CIs). Results: A total of 14 studies were included. Overall, the quality scores were relatively high, and little publication bias was noted. In terms of the 5-year OS, 7 studies were analyzed; there was no significant difference between AS and adjuvant treatment (OR, 0.99; 95% CI, 0.41−2.39; p = 0.97). In terms of 5-year RFS, 12 studies were analyzed. Adjuvant treatment reduced the risk of 5-year recurrence by 85% compared with AS (OR, 0.15; 95% CI, 0.08−0.26; p < 0.001). Conclusions: In terms of the OS in CSI seminoma patients, no intergroup difference was noted, so it is reasonable to offer AS, as recommended by the EAU GORRG until the end of the COVID-19 pandemic. However, since there is a large intergroup difference in the recurrence rate, further research on the long-term (>5 years) outcomes is warranted.

In Vivo Feasibility Test of a New Flexible Ureteroscopic Robotic System, easyUretero, for Renal Stone Retrieval in a Porcine Model.

PURPOSE: Using a new robotic endoscopic platform system developed for retrograde intrarenal surgery (RIRS) called easyUretero (ROEN Surgical Inc.), we evaluated the feasibility and safety of renal stone retrieval in a porcine model. MATERIALS AND METHODS: Six female pigs were used for our in vivo study. First, 0.3-cm-sized phantom stones were inserted into the kidneys of each pig via the ureteral access sheath. Next, renal stone retrieval was attempted using manual RIRS in three pigs and robotic RIRS in three pigs. Three surgeons performed extraction of 10 stones in each session. RESULTS: The mean stone retrieval time by manual RIRS was significantly shorter than that by robotic RIRS (399.9±185.4 sec vs. 1127.6±374.5 sec, p=0.001). In contrast, the questionnaire regarding usability showed high satisfaction in the surgeons' fatigue category for surgeons using robotic RIRS. The radiation exposure dose was also lower in robotic RIRS than in manual RIRS (0.14 µSv vs. 45.5 µSv). Postoperative ureteral injury assessment revealed Grade 0 in manual RIRS cases and Grades 0, 1, and 2 in robotic RIRS cases. CONCLUSION: The easyUretero system is a new robotic RIRS system that was developed in Korea. The results of the present study suggest that using easyUretero for stone retrieval during RIRS is safe and ergonomic.

Comparison of Surgical Outcomes between Single-Use and Reusable Flexible Ureteroscopes for Renal Stone Management: A Systematic Review and Meta-Analysis.

Background and Objectives: Disposable flexible ureteroscopes have been widely used because of their cost-effectiveness and higher sterility potential compared with reusable flexible ureteroscopes. This study aimed to compare the surgical outcomes and complication rates in patients who undergo reusable or disposable flexible ureteroscopic stone surgeries (fURS) for urinary stone disease. Materials and Methods: A systematic review and meta-analysis were conducted under the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. This systematic review was registered with PROSPERO (CRD42022331291). Clinical trials comparing reusable and disposable fURS for stone disease were found from PubMed, EMBASE, Cochrane Library, and the Web of Science up to March 2022. Participants were patients with upper urinary tract stones; the interventions were reusable or disposable fURS. Outcomes, including stone-free rate, operation time, length of hospital stay, and complication rate, were compared for analysis. Results: Overall, 111 studies were identified, but after removing duplicate studies, 75 studies remained. Thirty-two of these studies were excluded. Of the 43 screened studies, 11 met the eligibility criteria. There was no difference in the stone-free rate (SFR) between disposable and reusable fURS (p = 0.14; OR = 1.36; 95% CI, 0.9 to 2.04). For operation time, no difference was identified between reusable and disposable fURS groups (p = 0.12; MD = -5.31; 95% CI, -12.08 to 1.46). For hospital stay, there was also no difference between the two groups (p = 0.61; MD = -0.03; 95% CI, -0.17 to 0.10). There was no significant difference in complication rate between the two groups (p = 0.85; OR = 0.95; 95% CI, 0.56 to 1.61). Conclusions: There were no differences in the SFR, operation time, length of hospital stay, and complication rate between reusable and disposable fURS. Disposable fURS may be a comparable alternative to reusable fURS.

Effects of intravesical BCG maintenance therapy duration on recurrence rate in high-risk non-muscle invasive bladder cancer (NMIBC): Systematic review and network meta-analysis according to EAU COVID-19 recommendations.

PURPOSE: During the coronavirus disease 2019 (COVID-19) pandemic, the European Association of Urology (EAU) recommended that courses of intravesical bacillus Calmette-Guérin (BCG) therapy lasting more than 1 year could be safely terminated for patients with high-risk non-muscle-invasive bladder cancer (NMIBC). Thus, we conducted a systematic review and network meta-analysis according to EAU's COVID-19 recommendations. MATERIALS AND METHODS: A systematic review was performed following the Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines. We conducted a network meta-analysis of recurrence rate in patients with NMIBC receiving induction therapy (M0) and those receiving maintenance therapy lasting 1 year (M1) and more than 1 year (M2). RESULTS: Nineteen studies of 3,957 patients were included for the network meta-analysis. In a node-split forest plot using Bayesian Markov Chain Monte Carlo (MCMC) modeling, there were no differences between the M1 and M2 groups in recurrence rate [odds ratio (OR) 0.95 (0.73-1.2)]. However, recurrence rate in the M0 group was higher than that in the M1 [OR 1.9 (1.5-2.5)] and M2 [OR 2.0 (1.7-2.4)] groups. P-score tests using frequentist inference to rank the treatments in the network demonstrated that the therapy used in the M2 group (P-score 0.8701) was superior to that used in the M1 (P-score 0.6299) and M0 groups (P-score 0). In rank-probability tests using MCMC modeling, the M2 group showed the highest rank, followed by the M1 and M0 groups. CONCLUSION: In the network meta-analysis, there were no differences between those receiving BCG maintenance therapies in terms of recurrence rate. In the rank tests, therapy lasting more than 1-year appears to be most effective. During the COVID-19 pandemic, 1-year maintenance therapy can be used, but after the COVID-19 pandemic, therapy lasting more than 1-year could be beneficial.

Feasibility of Laser Lithotripsy for Midsize Stones Using Robotic Retrograde Intrarenal Surgery System easyUretero in a Porcine Model.

Purpose: To test the safety and feasibility of laser lithotripsy for midsize renal stones using a newly developed robotic retrograde intrarenal surgery (RIRS) system (easyUretero) in a porcine model. Materials and Methods: Three urologic surgeons representing three different RIRS experience levels (beginner, intermediate, and expert) participated. Four female pigs (aged 6 months) underwent manual or robotic RIRS. Under general anesthesia, a nephrostomy tract was created ventrally, and calcium stones (diameter, 1.0-1.5 cm) were inserted at renal calices. For manual RIRS, surgeons operated a flexible ureteroscope. For robotic RIRS, the ureteroscope was attached to the robotic slave device. The Auriga XL™ Holmium laser was used for lithotripsy. Lasering and stone retrieval time were measured. Kidneys and ureters were inspected for injury at the end of each session. Results: For the expert, both lasering and stone retrieval by manual RIRS were quicker than by robotic RIRS (22.8 ± 11.0 s/stone vs 234.5 ± 102.5 s/stone, p = 0.02; 41.5 ± 0.5 s/stone vs 79.3 ± 8.1 s/stone, p = 0.02). For the intermediate and beginner, lasering and stone retrieval times were not significantly different between manual and robotic procedures (127.8 ± 93.2 s/stone vs 284.8 ± 112.3 s/stone, p = 0.08; 86.0 ± 30.5 s/stone vs 84.1 ± 21.4 s/stone, p = 0.92). All stones were removed. Grade 1 ureteral and renal injuries occurred in both manual RIRS and robotic RIRS. Conclusions: The laser lithotripsy using the easyUretero robotic system is safe and feasible in a porcine model, even for less-experienced surgeons.

The First 100 Cases of Endoscopic Combined Intrarenal Surgery in Korea: Matched Cohort Analyses versus Shock-Wave Lithotripsy.

PURPOSE: This study presents our initial experience with endoscopic combined intrarenal surgery (ECIRS) for large renal stones and compares the results of a propensity score-matched cohort of patients undergoing shock-wave lithotripsy (SWL). MATERIALS AND METHODS: A total of 100 adults underwent ECIRS for renal stones between August 2017 and January 2019. For comparison, 2172 patients who underwent a first session of SWL between January 2005 and May 2018 were included in the SWL cohort. Propensity score matching was performed using maximal stone length (MSL), mean stone density (MSD), and stone heterogeneity index (SHI) scores. Stone-free rate (SFR) and success rate were compared between ECIRS and SWL. RESULTS: In the ECIRS group, the mean MSL, mean MSD, and mean SHI were 28.7±15.2 mm, 1013.9±360.0 Hounsfield units (HU), 209.4±104.0 HU, respectively. The SFR was 70%, and the success rate was 82.0% in this group. Although the ECIRS group had larger, harder, and more homogeneous stones than the SWL group, ECIRS showed a higher SFR and success rate than SWL. After propensity-score matching, SFR and success rate remained higher with ECIRS than with SWL (both, p<0.001). In multivariate logistic regression, smaller stone size [odds ratio (OR): 0.947, 95% confidence interval (CI): 0.913-0.979, p=0.002] and lower Seoul National University Renal Stone Complexity score (OR: 0.759, 95% CI: 0.610-0.935, p=0.011) were independent predictors of successful ECIRS. CONCLUSION: ECIRS showed a higher SFR and success rate than SWL for large renal stones. Smaller stone size and lower complexity of stones were associated with a higher likelihood of successful ECIRS.

Effect of neoadjuvant chemotherapy on overall survival of patients with T2-4aN0M0 bladder cancer: A systematic review and meta-analysis according to EAU COVID-19 recommendation.

PURPOSE: In the context of the COVID-19 outbreak, the European Association of Urology (EAU) guidelines Rapid Reaction Group provided recommendations to manage muscle invasive bladder cancer (MIBC) based on priority levels: neoadjuvant chemotherapy (NAC) should be avoided for patients with T2-3N0M0 MIBC. This meta-analysis aims to evaluate the efficacy of NAC compared with radical cystectomy (RC) alone in improving the overall survival (OS) of patients with T2-4aN0M0 MIBC. MATERIALS AND METHODS: A systematic review was performed according to the PRISMA guidelines. The PubMed/Medline, EMBASE, and Cochrane Library databases were searched. The primary outcome was OS of patients with T2-4aN0M0 MIBC, and the secondary outcome was OS of patients with only T2N0M0 MIBC. RESULTS: Eight studies were included in this meta-analysis. Overall, the quality of all studies was relatively high, and little publication bias was demonstrated. The OS was significantly better in the NAC with RC group than in RC alone (HR, 0.79; 95% CI, 0.68-0.92; p = 0.002). A subgroup analysis was performed on only patients with T2N0M0 MIBC, and five studies were included. There was no difference in the OS between the NAC with RC and the RC alone groups (HR, 0.83; 95% CI, 0.69-1.01 p = 0.06). CONCLUSIONS: As recommended by the EAU guidelines Rapid Reaction Group, patients with T2N0M0 MIBC should strongly consider omitting NAC until the end of the COVID-19 pandemic. Whether to omit NAC in T3-4aN0M0 MIBC needs further discussion, and studies targeting only T2-3N0M0 MIBC are expected to proceed further.

Antibiotic prophylaxis for percutaneous nephrolithotomy: An updated systematic review and meta-analysis.

INTRODUCTION: A single dose of preventive antibiotics is known to be sufficient to reduce the rate of infection-related complications in percutaneous nephrolithotomy (PCNL). However, some studies reported that the extended dose showed lower complications for high-risk groups. Therefore, we performed a systematic review and meta-analysis comparing single- and extended-dose antibiotic prophylaxis for PCNL. MATERIALS AND METHODS: Relevant studies that compared single- and extended-dose antibiotic prophylactic therapies were identified. Articles were selected from PubMed, EMBASE, KoreaMed, and Google Scholar up to September 2021. Fever and systemic inflammatory response syndrome (SIRS) were compared by meta-analysis. A subgroup analysis was performed according to the degree of risk to the patient. RESULTS: A total of 10 articles were included in this study. There were no significant differences between single dose and extended dose in the rate of fever [p = 0.93, OR = 0.96, 95% confidence interval (CI) 0.44-2.13, I2 = 64%]. Extended dose showed lower rate of SIRS compared to single dose (p = 0.0005, OR = 1.81, 95% CI 1.30-2.53, I2 = 53%); in the subgroup analysis, extended dose also showed lower rates of SIRS compared to single dose in high-risk patients (p <0.0001, OR = 3.53, 95% CI 1.91-6.54, I2 = 36%). CONCLUSIONS: The results of our meta-analysis showed that single-dose antibiotic prophylaxis can be effective for PCNL, but extended-dose antibiotics can be required in high-risk patients to reduce post-PCNL infection-related complications.

Distribution of ureteral stones and factors affecting their location and expulsion in patients with renal colic.

PURPOSE: To evaluate the distribution of ureteral stones and to determine their characteristics and expulsion rate based on their location. MATERIALS AND METHODS: We retrospectively reviewed computed tomography (CT) findings of 246 patients who visited our Emergency Department (ED) for renal colic caused by unilateral ureteral stones between January 2013 and April 2014. Histograms were constructed to plot the distribution of stones based on initial CT findings. Data from 144 of the 246 patients who underwent medical expulsive therapy (MET) for 2 weeks were analyzed to evaluate the factors responsible for the stone distribution and expulsion. RESULTS: The upper ureter and ureterovesical junction (UVJ) were 2 peak locations at which stones initially lodged. Stones lodged at the upper ureter and ureteropelvic junction (group A) had a larger longitudinal diameter (4.21 mm vs. 3.56 mm, p=0.004) compared to those lodged at the lower ureter and UVJ (group B). The expulsion rate was 75.6% and 94.9% in groups A and B, respectively. There was no significant difference in the time interval from initiation of renal colic to arrival at the ED between groups A and B (p=0.422). Stone diameter was a significant predictor of MET failure (odds ratio [OR], 1.795; p=0.005) but the initial stone location was not (OR, 0.299; p=0.082). CONCLUSIONS: The upper ureter and UVJ are 2 peak sites at which stones lodge. For stone size 10 mm or less, initial stone lodge site is not a significant predictor of MET failure in patients who have no previous history of active stone treatment in the ureter.

Generation of murine hepatic lineage cells from induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells can be generated from somatic cells of individuals by retrodifferentiation using defined transcription factors. Similar to embryonic stem (ES) cells, iPS cells can be differentiated into a variety of specific cell types. However, to date, no detailed hepatic differentiation of mouse iPS cells has been reported. In this study, we successfully developed a stepwise protocol to induce hepatic differentiation of iPS cells reprogrammed from mouse tail tip fibroblasts. At day 25 of differentiation, the iPS cell-derived hepatocytes morphologically resemble mouse primary hepatocytes with a distinct polygonal shape. Immunostaining and reverse transcription-polymerase chain reaction analysis revealed expression of specific hepatic markers including alpha-fetoprotein, albumin and alpha-1-anti-trypsin. In addition, these iPS cell-derived hepatocytes successfully demonstrated mature liver cell functions in vitro. Furthermore, in vivo assays revealed that the mouse iPS cell-derived hepatocytes successfully engrafted into the recipient livers with typical hepatic morphology. Thus, iPS cell-derived hepatocytes may hold great promise as a unique system for basic liver research and liver regeneration in the near future.

TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116.

Trichostatin A (TSA), an inhibitor of histone deacetylase, is a well-known antitumor agent that effectively and selectively induces tumor growth arrest and apoptosis. Recently, it was reported that hTERT is one of the primary targets for TSA-induced apoptosis in cancer cells but the mechanism of which has not yet been elucidated. In the present study, to better understand the epigenetic regulation mechanism responsible for the repression of hTERT by TSA, we examined expression of hTERT in the HCT116 colon cancer cell line after treatment with TSA and performed site-specific CpG methylation analysis of the hTERT promoter. We found that TSA-induced the demethylation of site-specific CpGs on the promoter of hTERT, which was caused by down-regulation of DNA methyltransferase 1 (DNMT1). Among the demethylated region, the 31st-33rd CpGs contained a binding site for CTCF, an inhibitor of hTERT transcription. ChIP analysis revealed that TSA-induced demethylation of the 31st-33rd CpGs promoted CTCF binding on hTERT promoter, leading to repression of hTERT. Taken together, down-regulation of DNMT1 by TSA caused demethylation of a CTCF binding site on the hTERT promoter, the result of which was repression of hTERT via recruitment of CTCF to the promoter.

Hepatic differentiation of cord blood-derived multipotent progenitor cells (MPCs) in vitro.

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells that possesses practical and ethical advantages. We previously reported a novel UCB-derived adult stem cells which we termed umbilical cord blood-derived multipotent progenitor cells' (MPCs). MPCs were capable of differentiating into functional neuronal cells. Under appropriate conditions lasting several days or weeks, we now show that the MPCs differentiate into hepatocyte-like cells in vitro; their properties were verified using reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, periodic acid-Schiff (PAS) staining of accumulated glycogen and an enzyme-linked immunosorbent assay (ELISA). We also found that hepatic differentiated cells expressed hepatocyte specific markers, such as albumin, hepatocyte nuclear factor (HNF)-1alpha, HNF4, cytokeratin (CK)-8, CK-18, tyrosine amino transferase (TAT), and CYP2B6. Moreover, albumin was secreted, which suggests that MPCs from UCB possess multi-differentiation potential and have the capacity to differentiate into functional cells of hepatic lineage in vitro.

Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood.

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types, including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.

Detection of heterozygous nonsense mutations in genes of interest using an Escherichia coli-based stop codon assay.

Since nonsense mutations are closely associated with severe conditions of genetic disorders, including familial cancers, rapid and precise detection of those mutations is very important for research purposes and molecular diagnosis. Currently, screening methods such as the FASAY (functional analysis of separated alleles in yeast) and the Y-SC (stop codon assay in yeast) are used for functional detection of nonsense mutations in genes of interest. But these yeast-based approaches are time-consuming, expensive and complicated. In order to circumvent these problems, we, in the present study, devised a novel Escherichia coli-based screening method, the E-SC (E. coli stop codon assay) for the detection of heterozygous nonsense mutations in genes of interest. Our strategy was based on the fact that the plasmid replicating with a low copy number in E. coli allows an effective separation of normal and mutant alleles. Moreover, it relies on the expression vector, resulting in the formation of white and blue colonies for mutant and normal alleles through the expression of PCR-amplified fragment/lacZ fusion protein respectively. To evaluate the applicability of the E-SC method for the detection of the heterozygous truncating mutation, PCR-amplified exon 7 of the StAR [steroidogenic acute regulatory protein; causative gene of the CAH (congenital lipoid adrenal hyperplasia)] and RT (reverse transcription)-PCR-amplified full-length cDNA of MeCP2 (methyl-CpG-binding protein 2; causative gene of Rett syndrome) were used. The E-SC showed an almost 1:1 ratio of blue/white colonies in all patients examined, whereas the control samples produced blue colonies only. These results demonstrate that the E-SC system is useful for rapid and precise detection of known and unknown heterozygous truncation mutations in genes that cause genetic disorders and familial cancers.