BACKGROUND: Interstitial cystitis (IC) is a chronic and intractable disease that can severely deteriorate patients’ quality of life. Recently, stem cell therapy has been introduced as a promising alternative treatment for IC in animal models. We aimed to verify the efficacy and safety of the human perirenal adipose tissue-derived stromal vascular fraction (SVF) in an IC rat model. METHODS: From eight-week-old female rats, an IC rat model was established by subcutaneous injection of 200 lg of uroplakin3A. The SVF was injected into the bladder submucosal layer of IC rats, and pain scale analysis, awakening cytometry, and histological and gene analyses of the bladder were performed. For the in vivo safety analysis, genomic DNA purification and histological analysis were also performed to check tumorigenicity and thrombus formation. RESULTS: The mean pain scores in the SVF 20 ll group were significantly lower on days 7 and 14 than those in the control group, and bladder intercontraction intervals were significantly improved in the SVF groups in a dose-dependent manner. Regeneration of the bladder epithelium, basement membrane, and lamina propria was observed in the SVF group.In the SVF groups, however, bladder fibrosis and the expression of inflammatory markers were not significantly improved compared to those in the control group. CONCLUSION This study demonstrated that a perirenal adipose tissue-derived SVF is a promising alternative for the management of IC in terms of improving bladder pain and overactivity.

Background: Tissue engineering can be used for bladder augmentation. However, conventional scaffolds result in fibrosis and graft shrinkage. This study applied an alternative polycaprolactone (PCL)-based scaffold (diameter = 5 mm) with a noble gradient structure and growth factors (GFs) (epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) to enhance bladder tissue regeneration in a rat model. Methods: Partially excised urinary bladders of 5-week-old male Slc:SD rats were reconstructed with the scaffold (scaffold group) or the scaffold combined with GFs (GF group) and compared with sham-operated (control group) and untreated rats (partial cystectomy group). Evaluations of bladder volume, histology, immunohistochemistry (IHC), and molecular markers were performed at 4, 8, and 12 weeks after operation. Results: The bladder volumes of the scaffold and GF group recovered to the normal range, and those of the GF group showed more enhanced augmentation. Histological evaluations revealed that the GF group showed more organized urothelial lining, dense extracellular matrix, frequent angiogenesis, and enhanced smooth muscle bundle regeneration than the scaffold group. IHC for α-smooth muscle actin, pan-cytokeratin, α-bungarotoxin, and CD8 revealed that the GF group showed high formation of smooth muscle, blood vessel, urothelium, neuromuscular junction and low immunogenicity. Concordantly, real-time polymerase chain reaction experiments revealed that the GF group showed a higher expression of transcripts associated with smooth muscle and urothelial differentiation. In a 6-month in vivo safety analysis, the GF group showed normal histology. Conclusion This study showed that a PCL scaffold with a gradient structure incorporating GFs improved bladder regeneration functionally and histologically.

BACKGROUND: Despite major progress in stem cell therapy, our knowledge of the characteristics and tissue regeneration potency of long-term transported cells is insufficient. In a previous in vitro study, we established the optimal cell transport conditions for amniotic fluid stem cells (AFSCs). In the present study, the target tissue regeneration of long-term transported cells was validated in vivo. METHODS: For renal regeneration, transported AFSCs were seeded on a poly(lactide-co-glycolide) scaffold and implanted in a partially resected kidney. The target tissue regeneration of the transported cells was compared with that of freshly harvested cells in terms of morphological reconstruction, histological microstructure reformation, immune cell infiltration, presence of induced cells, migration into remote organs, expression of inflammation/fibrosis/renal differentiation-related factors, and functional recovery. RESULTS: The kidney implanted with transported cells showed recovery of total kidney volume, regeneration of glomerular/renal tubules, low CD4/CD8 infiltration, and no occurrence of cancer during 40 weeks of observation. The AFSCs gradually disappeared and did not migrate into the liver, lung, or spleen. We observed low expression levels of proinflammatory cytokines and fibrotic factors; enhanced expression of the genes Wnt4, Pax2, Wt1, and Emx2; and significantly reduced blood urea nitrogen and creatinine values. There were no statistical differences between the performance of freshly harvested cells and that of the transported cells. CONCLUSION: This study demonstrates that long-term transported cells under optimized conditions can be used for cell therapy without adverse effects on stem cell characteristics, in vivo safety, and tissue regeneration potency.
Amniotic Fluid ; Blood Urea Nitrogen ; Cell- and Tissue-Based Therapy ; Creatinine ; Cytokines ; Female ; In Vitro Techniques ; Kidney ; Liver ; Lung ; Polyglactin 910 ; Regeneration ; Spleen ; Stem Cells

BACKGROUND: Kidney ischemia-reperfusion (IR) via laparotomy is a conventional method for kidney surgery in a mouse model. However, IR, an invasive procedure, can cause serious acute and chronic complications through apoptotic and inflammatory pathways. To avoid these adverse responses, a Non-IR and dorsal slit approach was designed for kidney surgery. METHODS: Animals were divided into three groups, 1) sham-operated control; 2) IR, Kidney IR via laparotomy; and 3) Non-IR, Non-IR and dorsal slit. The effects of Non-IR method on renal surgery outcomes were verified with respect to animal viability, renal function, apoptosis, inflammation, fibrosis, renal regeneration, and systemic response using histology, immunohistochemistry, real-time polymerase chain reaction, serum chemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and Masson's trichrome staining. RESULTS: The Non-IR group showed 100% viability with mild elevation of serum blood urea nitrogen and creatinine values at day 1 after surgery, whereas the IR group showed 20% viability and lethal functional abnormality. Histologically, renal tubule epithelial cell injury was evident on day 1 in the IR group, and cellular apoptosis enhanced TUNEL-positive cell number and Fas/caspase-3 and KIM-1/NGAL expression. Inflammation and fibrosis were high in the IR group, with enhanced CD4/CD8-positive T cell infiltration, inflammatory cytokine secretion, and Masson's trichrome stain-positive cell numbers. The Non-IR group showed a suitable microenvironment for renal regeneration with enhanced host cell migration, reduced immune cell influx, and increased expression of renal differentiation-related genes and anti-inflammatory cytokines. The local renal IR influenced distal organ apoptosis and inflammation by releasing circulating pro-inflammatory cytokines. CONCLUSION: The Non-IR and dorsal slit method for kidney surgery in a mouse model can be an alternative surgical approach for researchers without adverse reactions such as apoptosis, inflammation, fibrosis, functional impairment, and systemic reactions.
Animals ; Apoptosis ; Blood Urea Nitrogen ; Cell Count ; Cell Movement ; Chemistry ; Creatinine ; Cytokines ; DNA Nucleotidylexotransferase ; Epithelial Cells ; Fibrosis ; Immunohistochemistry ; Inflammation ; Kidney ; Laparotomy ; Methods* ; Mice* ; Nephrectomy* ; Real-Time Polymerase Chain Reaction ; Regeneration*

BACKGROUND: We fabricated anti-inflammatory scaffold using Mg(OH)2-incorporated polylactic acid-polyglycolic acid copolymer (MH-PLGA). To demonstrate the anti-inflammatory effects of the MH-PLGA scaffold, an animal model should be sensitive to inflammatory responses. The interleukin-10 knockout (IL-10 KO) mouse is a widely used bowel disease model for evaluating inflammatory responses, however, few studies have evaluated this mouse for the anti-inflammatory scaffold. METHODS: To compare the sensitivity of the inflammatory reaction, the PLGA scaffold was implanted into IL-10 KO and C57BL/6 mouse kidneys. Morphology, histology, immunohistochemistry, and gene expression analyses were carried out at weeks 1, 4, 8, and 12. The anti-inflammatory effect and renal regeneration potency of the MH-PLGA scaffold was also compared to those of PLGA in IL-10 KO mice. RESULTS: The PLGA scaffold-implanted IL-10 KO mice showed kidneys relatively shrunken by fibrosis, significantly increased inflammatory cell infiltration, high levels of acidic debris residue, more frequent CD8-, C-reactive protein-, and ectodysplasin A-positive cells, and higher expression of pro-inflammatory and fibrotic factors compared to the control group. The MH-PLGA scaffold group showed lower expression of pro-inflammatory and fibrotic factors, low immune cell infiltration, and significantly higher expression of anti-inflammatory factors and renal differentiation related genes compared to the PLGA scaffold group. CONCLUSION: These results indicate that the MH-PLGA scaffold had anti-inflammatory effects and high renal regeneration potency. Therefore, IL-10 KO mice are a suitable animal model for in vivo validation of novel anti-inflammatory scaffolds.
Animals ; Ectodysplasins ; Fibrosis ; Gene Expression ; Immunohistochemistry ; Interleukin-10* ; Kidney ; Mice ; Mice, Knockout* ; Models, Animal ; Regeneration

BACKGROUND: The preservation of stem cell viability and characteristics during cell transport from the bench to patients can significantly affect the success of cell therapy. Factors such as suspending medium, time, temperature, cell density, and container type could be considered for transport conditions. METHODS: To establish optimal conditions, human amniotic fluid stem cells' (AFSCs) viabilities were analyzed under different media {DMEM(H), DMEM/F-12, K-SFM, RPMI 1640, α-MEM, DMEM(L), PBS or saline}, temperature (4, 22 or 37 ℃), cell density (1 × 10⁷ cells were suspended in 0.1, 0.5, 1.0 or 2.0 mL of medium) and container type (plastic syringe or glass bottle). After establishing the transport conditions, stem cell characteristics of AFSCs were compared to freshly prepared cells. RESULTS: Cells transported in DMEM(H) showed relatively higher viability than other media. The optimized transport temperature was 4 ℃, and available transport time was within 12 h. A lower cell density was associated with a better survival rate, and a syringe was selected as a transport container because of its clinical convenience. In compare of stem cell characteristics, the transported cells with established conditions showed similar potency as the freshly prepared cells. CONCLUSION: Our findings can provide a foundation to optimization of conditions for stem cell transport.
Amniotic Fluid ; Cell Count ; Cell Survival* ; Cell- and Tissue-Based Therapy ; Female ; Glass ; Humans ; Stem Cells* ; Survival Rate ; Syringes

Urine-derived stem cells (USCs) are considered as a promising cell source capable of neuronal differentiation. In addition, specific growth factors and extracellular matrix are essential for enhancing their neuronal differentiation efficiency. In this study, we investigated the possibility of neuronal differentiation of USCs and the role of laminin and platelet-derived growth factor BB (PDGF-BB) as promoting factors. USCs were isolated from fresh urine of healthy donors. Cultured USCs were adherent to the plate and their morphology was similar to the cobblestone. In addition, they showed chromosome stability, rapid proliferation rate, colony forming capacity, and mesenchymal stem cell characteristics. For inducing the neuronal differentiation, USCs were cultured for 14 days in neuronal differentiation media supplemented with/without laminin and/or PDGF-BB. To identify the expression of neuronal markers, RT-PCR, flow cytometry analysis and immunocytochemistry were used. After neuronal induction, the cells showed neuron-like morphological change and high expression level of neuronal markers. In addition, laminin and PDGF-BB respectively promoted the neuronal differentiation of USCs and the combination of laminin and PDGF-BB showed a synergistic effect for the neuronal differentiation of USCs. In conclusion, USCs are noteworthy cell source in the field of neuronal regeneration and laminin and PDGF-BB promote their neuronal differentiation efficiency.
Chromosomal Instability ; Extracellular Matrix ; Flow Cytometry ; Humans* ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; Laminin* ; Mesenchymal Stromal Cells ; Neurons* ; Platelet-Derived Growth Factor ; Regeneration ; Stem Cells* ; Tissue Donors

BACKGROUND: To evaluate mid-term oncological and functional outcomes in patients with prostate cancer treated by robot-assisted laparoscopic radical prostatectomy (RALP) at our institution.METHODS: We retrospectively reviewed the medical records of 128 patients with prostate cancer who underwent RALP at our institution between February 2008 and April 2010. All patients enrolled in this study were followed up for at least 5 years. We analyzed biochemical recurrence (BCR)-free survival using a Kaplan-Meier survival curve analysis and predictive factors for BCR using multivariate Cox regression analysis. Continence recovery rate, defined as no use of urinary pads, was also evaluated.RESULTS: Based on the D'Amico risk classification, there were 30 low-risk patients (23.4%), 47 intermediate-risk patients (38.8%), and 51 high-risk patients (39.8%), preoperatively. Based on pathological findings, 50.0% of patients (64/128) showed non-organ confined disease (≥T3a) and 26.6% (34/128) had high grade disease (Gleason score ≥8). During a median follow-up period of 71 months (range, 66–78 months), the frequency of BCR was 33.6% (43/128) and the median BCR-free survival was 65.9 (0.4–88.0) months. Multivariate Cox regression analysis revealed that high grade disease (Gleason score ≥8) was an independent predictor for BCR (hazard ratio=4.180, 95% confidence interval=1.02–17.12, p=0.047). In addition, a majority of patients remained continent following the RALP procedure, without the need for additional intervention for post-prostatectomy incontinence.CONCLUSION: Our study demonstrated acceptable outcomes following an initial RALP procedure, despite 50% of the patients investigated demonstrating high-risk features associated with non-organ confined disease.
Classification ; Follow-Up Studies ; Humans ; Medical Records ; Prostatectomy ; Prostatic Neoplasms ; Recurrence ; Retrospective Studies ; Urinary Incontinence

BACKGROUND: To evaluate mid-term oncological and functional outcomes in patients with prostate cancer treated by robot-assisted laparoscopic radical prostatectomy (RALP) at our institution. METHODS: We retrospectively reviewed the medical records of 128 patients with prostate cancer who underwent RALP at our institution between February 2008 and April 2010. All patients enrolled in this study were followed up for at least 5 years. We analyzed biochemical recurrence (BCR)-free survival using a Kaplan-Meier survival curve analysis and predictive factors for BCR using multivariate Cox regression analysis. Continence recovery rate, defined as no use of urinary pads, was also evaluated. RESULTS: Based on the D'Amico risk classification, there were 30 low-risk patients (23.4%), 47 intermediate-risk patients (38.8%), and 51 high-risk patients (39.8%), preoperatively. Based on pathological findings, 50.0% of patients (64/128) showed non-organ confined disease (≥T3a) and 26.6% (34/128) had high grade disease (Gleason score ≥8). During a median follow-up period of 71 months (range, 66–78 months), the frequency of BCR was 33.6% (43/128) and the median BCR-free survival was 65.9 (0.4–88.0) months. Multivariate Cox regression analysis revealed that high grade disease (Gleason score ≥8) was an independent predictor for BCR (hazard ratio=4.180, 95% confidence interval=1.02–17.12, p=0.047). In addition, a majority of patients remained continent following the RALP procedure, without the need for additional intervention for post-prostatectomy incontinence. CONCLUSION Our study demonstrated acceptable outcomes following an initial RALP procedure, despite 50% of the patients investigated demonstrating high-risk features associated with non-organ confined disease.

PURPOSE: We evaluated 5 different rat models using different agents in order to establish a standard animal model for interstitial cystitis (IC) in terms of the functional and pathologic characteristics of the bladder. METHODS: Five IC models were generated in 8-week-old female Sprague-Dawley rats via transurethral instillation of 0.1M hydrogen chloride (HCl) or 3% acetic acid (AA), intraperitoneal injection of cyclophosphamide (CYP) or lipopolysaccharide (LPS), or subcutaneous injection of uroplakin II (UPK2). After generating the IC models, conscious cystometry was performed on days 3, 7, and 14. All rats were euthanized on day 14 and their bladders were obtained for histological and pro-inflammatory-related gene expression analysis. RESULTS: In the cystometric analysis, all experimental groups showed significantly decreased intercontraction intervals compared with the control group on day 3, but only the LPS and UPK groups maintained significantly shorter intercontraction intervals than the control group on day 14. The histological analysis revealed that areas with severe urothelial erosion (HCl, AA, and UPK) and hyperplasia (CYP and LPS), particularly in the UPK-treated bladders, showed a markedly increased infiltration of toluidine blue-stained mast cells and increased tissue fibrosis. In addition, significantly elevated expression of interleukin-1b, interleukin-6, myeloperoxidase, monocyte chemotactic protein 1, and Toll-like receptors 2 and 4 was observed in the UPK group compared to the other groups. CONCLUSIONS: Among the 5 different agents, the injection of UPK generated the most effective IC animal model, showing consequent urothelial barrier loss, inflammatory reaction, tissue fibrosis stimulation, and persistent hyperactive bladder.
Acetic Acid ; Animals ; Animals* ; Chemokine CCL2 ; Cyclophosphamide ; Cystitis, Interstitial* ; Female ; Fibrosis ; Gene Expression ; Humans ; Hydrochloric Acid ; Hyperplasia ; Immunization ; Injections, Intraperitoneal ; Injections, Subcutaneous ; Interleukin-6 ; Mast Cells ; Models, Animal* ; Peroxidase ; Rats* ; Rats, Sprague-Dawley ; Toll-Like Receptors ; Urinary Bladder ; Uroplakin II