ESPU Meeting on Wednesday 8, June 2022, 15:50 - 16:40
Astgik PETROSYAN, Paola AGUIARI, Valentina VILLANI, Roger DE FILIPPO, Stefano DA SACCO and Laura PERIN
Children's Hospital Los Angeles, Urology, Los Angeles, USA
Wilms tumor (WT) accounts for 95% of renal pediatric malignancies and is characterized by uncontrolled proliferation of nephron progenitors (NP) without generation of functional nephrons. Due to the inability of isolating these human NP, little is known about WT initiation and growth. In this study, we isolated NP expressing SIX2 and CITED1 (the master genes regulating nephrogenesis) from WT samples and from human fetal kidneys (hFK) and performed in vivo and in vitro experiments to study the regulation of self-renewal vs differentiation of NP.
MATERIAL AND METHODS
WT and hFK samples were histologically analyzed, digested to single cell suspension, incubated with Smartflare-probe and SIX2+CITED1+ cells immediately sorted and processed for transcriptomics analysis. Xenografts were generated and tumor formation was assessed. Mechanisms regfulating self-renewal vs differentiation were studied. Digital Spatial Transcriptomics was performed to confirm the generated data.
Histology confirmed a different pattern of expression for SIX2 and CITED1 across WT with different prognosis and stages compared to hFK. When transplanted in vivo WT-NP demonstrated marked capacity of tumorigenesis, which in some instances induced metastasist. Single-cell RNA-seq after xenotransplantation of WT-NP defined precise cancer-associated cellular identities compared to WT-NP, confirming divergence from canonical nephrogenesis in WT-NP . In vitro experiments confirmed that modulation of integrin signaling leads to blockade of self-renewal in NP by decreasing CITED1 expression and inducing specification by stimulating the activation of LEF1. Digital Spatial Transcriptomics confirmed the generated data and pinpointed critical differences between favorable and unfavorable WT.
This work evidenced that SIX2+CITED+ cells in WT represent a population of cancer stem cells. Our characterization also highlights differences in self-renewal potential between favorable and unfavorable WT samples and confirms that integrin interplay exerts a key role in its regulation. These studies can help to increase our knowledge of human nephrogenesis and the development of new strategies aimed at halting tumor progression.
Nao IGUCHI 1, Sarah HECHT 2, Duncan WILCOX 3, Anna MALYKHINA 1 and Nicholas COST 4
1) University of Colorado Denver, Surgery, Aurora, USA - 2) Doernbecher Children's Hospital, Oregon Health & Science University, Pediatric Urology, Portland, USA - 3) Children's Hospital Colorado, Surgery, Aurora, USA - 4) Children's Hospital Colorado and University of Colorado Hospital, Surgery, Aurora, USA
Vincristine is the most common chemotherapy agents used in pediatric oncology. Despite well-known vincristine-induced peripheral neuropathy (VIPN), the potential impact of VIPN on lower urinary tract (LUT) function remains undefined. This study aims to investigate the effects of systemic vincristine exposure in childhood on LUT function by using a juvenile murine model.
MATERIAL AND METHODS
Using IACUC approved protocols, CD-1 mice (3.5-wk-old) received an intraperitoneal injection of 0.5 mg/kg of vincristine (experimental group) or saline (control group) twice per week for 4 weeks. Physiological recordings of LUT and detrusor function were conducted by cystometry in conscious and unrestrained mice and an in vitro organ bath at 5 weeks after the last treatment. Changes in gene expression in the bladders and the lumbosacral dorsal root ganglia (Ls-DRG) were examined.
Cystometry revealed that vincristine exposure induced increased functional bladder capacity, micturition volume and non-void contractions. Vincristine exposure also caused sexual dimorphic changes; in females, increased intravesical pressure at micturition and downregulations of a major player in bladder afferent firing, Htr3b, in the bladders, and Cav1.2 in the Ls-DRG, while male mice displayed detrusor overactivity, upregulations of IL-2, Trpa1 and Itga1 in the bladders and neuroinflammation-related genes in the Ls-DRG. These results suggest that that systemic vincristine exposure caused sensory neuropathy via sex-dimorphic mechanisms, leading to altered LUT function.
Systemic vincristine exposure in juvenile mice affected LUT function via sex-dimorphic mechanisms, which may clinically present as gender-specific signs of LUT dysfunction. A recent survey study of childhood cancer survivors from our group has implicated vincristine and/or doxorubicin exposure in LUT dysfunction with differences observed between male and female patients. Thus, follow-up urological assessment would benefit to pediatric cancer patients treated with these chemotherapeutic agents.
Kevin Xi CAO 1, Maria KOLATSI-JOANNOU 1, Navroop JOHAL 2, Peter CUCKOW 2, Paul WINYARD 1, Chris FRY 3 and David LONG 1
1) UCL Great Ormond Street Institute of Child Health, Nephro-Urology, London, UNITED KINGDOM - 2) Great Ormond Street Hospital for Children, Urology, London, UNITED KINGDOM - 3) University of Bristol, Physiology, Bristol, UNITED KINGDOM
Posterior Urethral Valves (PUV) remains one of the most severe paediatric conditions, responsible for most of the demand for childhood renal replacement therapy resources. We previously showed that the low-compliance bladder in PUV is strongly correlated with excess connective tissue or fibrosis. Finding an anti-fibrotic therapy is thus a promising therapeutic avenue for this disorder.
We performed a pre-clinical animal trial, demonstrating that a family of drugs, the soluble guanylate cyclase modulators (sGC) successfully delivered a dramatic anti-fibrotic effect.
MATERIAL AND METHODS
8-week old male C57 mice (n=36) were used to create a PUV-analogue surgical model, through partial-ligation of the urethra around a temporary 0.6mm metallic stent. After one week, SGC drugs were administered daily for two weeks and experiments performed on the 21st day.
Histology of the smooth muscle to connective tissue ratio (SM:CTr) was performed on paraffin-embedded bladder specimens utilising picosirius red staining to define smooth muscle and connective tissue elements. Biomechanical assessments of the stress-strain relationship of the dissected detrusor were performed on fresh bladder tissue mounted to an isometric force transducer, deriving Elastic Modulus, or stiffness from the steady-state tension after sequential muscle prep stretches (measured in kilopascals, kPa).
The partial bladder obstruction model generated a PUV-like morphology, reducing SM:CTr from 1.2 in sham surgery (no urethral ligation) to 0.5 and simultaneously increased elastic modulus or stiffness of detrusor from 50kPa to 170kPa. Treatment with either sGC activator, cinaciguat or sGC stimulator, BAY 41-2272 (both 10mg/kg) prevented these changes, maintaining SM:CTr and elastic modulus in the same range as sham.
SGC modulation in a preventative-equivalent dosing regimen described here demonstrates prognosis-altering potential for this on-market drug. Normalising bladder function in PUV and protecting kidney function gives hope for altering the natural history of this devastating condition.
Massimo GARRIBOLI 1, Jenny HINLEY 2, Rosalind DUKE 2, Paolo DE COPPI 3, Imran MUSHTAQ 4 and Jennifer SOUTHGATE 2
1) Evelina London Children's Hospital - Guy's and St Thomas NHS Foundation Trust, Paediatric Urology, London, UNITED KINGDOM - 2) University of York, Jack Birch Unit for Molecular Carcinogenesis Department of Biology, York, UNITED KINGDOM - 3) Great Ormond Street Institute of Child Health, London, United Kingdom, Stem Cells & Regenerative Medicine Section, Developmental Biology & Cancer Programme, London, UNITED KINGDOM - 4) Great Ormond Street Hospital, Paediatric Urology, London, UNITED KINGDOM
Xenotransplantation can represent intermediate step toward clinical translation (human-in-animal) or solution for shortage of donors (animal-in-human). There are controversies about survival of human cells in animal models.
We investigated whether human urothelial cells would survive in porcine bladder environment
MATERIAL AND METHODS
Bladder biopsies from children undergoing urological procedures (IRAS 118347) were isolated, cultured and differentiated before to be implanted onto a de-epithelialised, seromuscular intestinal segment of immunosuppressed pigs (PPL P963D67BC).
Animals were sacrificed at 4 weeks.
Augmented bladders' samples were stained (Haematoxylin&Eosin) and labelled with antibodies against CK7 and CK13 to examine the phenotype of the epithelium. Chromosome Y-painting looked for male human urothelium implanted in female pigs.
Serum samples were collected from animals pre- and post-immunosuppression and at termination and screened on unfixed urothelial cell sheets using secondary anti-IgM and anti-IgG fluorescent-conjugated antibodies to detect binding by indirect immunofluorescence microscopy.
Seven pigs underwent bladder augmentation.
Immunosuppression levels (Tacrolimus>10-15 µg/L)were achieved before surgery and maintained until termination without complications.
All surgeries proceeded well. 2 animals were sacrificed at 72 hours; the others after 4 weeks. Post-mortem no macroscopic abnormalities were found.
Histologically, the presence of bowel and bladder smooth muscle was confirmed.
In the two pigs sacrificed early, scant CK7-positive cells were identified and were heavily infiltrated with porcine CD45+ polymorphonuclear leucocytes.
Chromosome painting of epithelial cells from across the augmented tissue failed to identify male cells.
The presence of porcine natural IgM reaction to human cells was confirmed by the immunofluorescence results on pig’s serum
Despite successful immunosuppression, human cells didn't survive in porcine bladders. Innate host-reaction would have led to hyperacute rejection, particularly evident in the pigs sacrificed at early time point.
When designing human-in-pig model xenotransplantation, approaches to overcome hyperacute reaction are necessary
Massimo GARRIBOLI 1, Koichi DEGUICHI 2, Ellie PHYLACTOPOULOU 2, Paola BONFANTI 3 and Paolo DE COPPI 2
1) Evelina London Children's Hospital - Guy's and St Thomas NHS Foundation Trust, Paediatric Urology, London, UNITED KINGDOM - 2) Great Ormond Street Institute of Child Health, London, United Kingdom, Stem Cells & Regenerative Medicine Section, Developmental Biology & Cancer Programme, London, UNITED KINGDOM - 3) The Francis Crick Institute, London, United Kingdom, Epithelial Stem Cell Biology and Regenerative Medicine Laboratory, London, UNITED KINGDOM
Strategies for overcoming long-term complications of enterocystoplasty are needed and cell-seeded biological scaffolds could represent a solution. A functional urothelium is a stratified tissue that provide barrier function to urine. We previously presented a method for creating a porcine derived decellularised scaffold.
We now present a protocol for repopulating the bladder acellular matrix with functional human urothelium.
MATERIAL AND METHODS
Bladders from piglets were decellularised with a dynamic detergent-enzymatic treatment using peristaltic infusion. Primary human urothelial cells were isolated from biopsies of paediatric patients (IRAS 118347, REC reference number 14/LO/0226) and expanded in vitro.
Urothelial cells were then seeded (6 x 106 cells/cm2), cultured and differentiated on the scaffold. MTT assay was used to evaluate cell coverage. Engineered grafts were analysed by immunofluorescence to assess the expression of differentiation markers. Barrier function was tested using transepithelial electrical resistance (TER) and FITC-Dextran permeability test.
Histological evaluation of the scaffold confirmed absence of cellular elements with preservation of tissue matrix architecture (collagen and elastin). Following seeding, human urothelial cells proliferated up to 8.7-folds (8.70 x 106 cells/cm2) on the luminal surface of the scaffold after 7 days of culture. Histological and immunofluorescent staining confirmed the generation of multi-layered urothelium with the expression of markers specific for differentiated urothelium (Ki67, CK8, CK13, CK14, ZO-1). TER and Dextran permeability tests confirmed the formation of a tight barrier after recellularization (TER:1,569 vs 131 ohms.cm2, p<0.0001).
We produced a tissue-engineered bladder graft seeded with functional urothelium. Functional data suggest its use in the development of a tissue-engineered bladder for patients requiring bladder augmentation/substitution.
Yutao LU 1, Jens C DJURHUUS 1, Yazan F. H. RAWASHDEH 2, Rikke NØRREGAARD 1 and L. Henning OLSEN 1
1) Aarhus University, Department of Clinical Medicine, Aarhus, DENMARK - 2) Aarhus University Hospital, Department of Urology-Paediatric Urology, Aarhus, DENMARK
Progression of bladder fibrosis leads to changes in bladder storage and voiding functions and to damaged kidney function. In animal studies antifibrotic compounds, like pirfenidone or relaxin show promising effects in e.g., the lungs and kidneys. Precision Cut Slicing has been used for culturing solid organ slices in their natural structure with intact intercellular and cell-matrix interactions. The bladder walls' unstable architecture requires an advanced slicing technique to preserve the three main layers prior to culturing. Here, we describe a new viable protocol and application of precision cut human bladder slices (PCBS).
MATERIAL AND METHODS
Whole bladder wall samples of 1x1 cm from children undergoing ureteral re-implantation or other benign open bladder surgery and adult patients undergoing cystectomy due to urothelial tumours were used. Samples were stabilised by embedding in agarose for precision cut to preserve the original bladder wall structure. PCBS were exposed to TGF-beta to induce fibrosis. The viability of PCBS was assessed by ATP content. Gene expression of fibrosis markers were analysed.
PCBS were demonstrated to be viable for up to 96 hours. Incubation for 48 hours was however seen to be more optimal and consistent during TGF-beta stimulation. Exposure to TGF-beta significantly increased the gene expression of collagen 1 (Col-1) and connective tissue growth factor (CTGF). Fibronectin (FN) was not significantly changed.
Cultured whole bladder wall slices from one single patient can be exposed to different medications and thereby pointing at the most potential molecules in the treatment of bladder fibrosis. PCBS are expected to pave the way from the in vivo studies to the clinical trials.
Jessica PINOL 1, Rania TRIGUI 2, Mouloud ADEL 2, Haddad MIRNA 1, Chaumoitre KATIA 3, Di Bisceglie MATHIEU 3 and Alice FAURE 1
1) TIMONE ENFANT HOSPITAL, UNIVERSITY HOSPITAL CENTER, PAEDIATRIC SURGERY, Marseille, FRANCE - 2) AIX MARSEILLE UNIVERSITE, INSTITUT FRESNEL, Marseille, FRANCE - 3) UNIVERSITE HOSPITAL CENTER, NORTH HOSPITAL, MEDICAL IMAGING DEPARTMENT, Marseille, FRANCE
Spinal dysraphisms are the first cause of neurological bladder in children. Urodynamic remains the gold standard for renal risk analysis. Radiomics seems to be a promising alternative. Textural and geometric radiomic attributes calculated from the bladder wall in MRI could reflect microstructural changes. The objective is to estimate the diagnostic performance of radiomics on uroMRI to identify hostile bladders in children with congenital neurological bladder.
MATERIAL AND METHODS
In order to characterize the bladder state and functioning, it is necessary to succeed the segmentation of its wall in MR images. In this context, we propose a computer-aided diagnosis system based on segmentation and classification applied to the Bladder Wall (BW). The proposed automated system starts with the BW extraction using an improved levelSet based algorithm. After the segmentation step, we extract masks of ROIs containing only Bladder Walls, from which we compute different types of features that could help in the differentiation between severe and not-severe spina bifida cases.
Experimental results show that the association between textural features and other features describing wall's orientation, shape, thickness and boundaries similarities is interesting and leads, according to our proposed characterization method, to a good identification of high risk spina bifida patients.
Obtained results proves the efficiency of our proposed system, which can be significantly helpful for avoiding the fastidious manual segmentation and providing a precise idea about the spina bifida severity. Our ultimate plan is to increase our dataset so as to apply deep learning approaches and to correlate this results to urodynamics findings.
Biniam M. BEKELE, MD 1, Verena SCHÖWEL-WOLF 1, Janine KIESHAUER 1, Andreas MARG 1, Andreas BUSJAHN 2, Sarah DAVIS 3, Gayle NUGENT 3, Anne-Karoline EBERT 4 and Simone SPULER 1
1) Muscle Research Unit, Experimental and Clinical Research Center, a cooperation between the Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association and the Charité, Universitätsmedizin Berlin, Berlin, GERMANY - 2) HealthTwist GmbH, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association and the Charité, Universitätsmedizin Berlin, Berlin, GERMANY - 3) Charles River Laboratories, Michigan, USA - 4) University of Ulm, Department of Urology and Pediatric Urology, Ulm, GERMANY
Muscle stem cells (MuSCs) are a promising treatment option for urinary incontinence. Preclinical efficacy studies are required to demonstrate functional improvement in animal models before clinical trials are initiated. Bladder telemetry allows continuous recording of urodynamic parameters in freely moving rats. We aimed to demonstrate efficacy of MuSCs in restoration of urinary sphincter function using bladder telemetry.
MATERIAL AND METHODS
Human MuSCs were prepared from a male donor aged 14 without neuromuscular disorders. We surgically implanted telemetry sensors in the bladder of athymic male rats. Diurnal variation was assessed in standard 12-hour light/dark cycles. We injured the urethral sphincter by electrocauterization. Five days later, we injected 2 x105 MuSCs/animal (verum group, n=5) or placebo (cryopreservation medium; control group, n=5). Peak pressure (PeakP), base pressure (BaseP), Rise, Period, Intercontraction interval (ICI) and Peak Duration (PeakD) were recorded pre-injury and post-injection.
We observed higher PeakP, BaseP and shorter period, ICI during the dark (active) phase. Post-injection, PeakP, BaseP and Rise returned to pre-injury values in verum group while they were significantly decreased in control group (Delta PeakP; Control = -2.4±0.7, verum = 0.0±0.7, p = 0.007; Delta BaseP; Control = -1.7±0.5, verum = 0.2±0.7, p = 0.020; Delta Rise; Control = -0.69±0.53, verum = 0.1±0.24, p = 0.043). Injury-related tissue changes were only seen in the placebo group.
Bladder telemetry provides a longitudinally analyzable translational model for assessing efficacy in a rat model of sphincter injury. Injection of MuSCs resulted in functional restoration of the injured urethral sphincter.