Autologous cell engineered bladder augmentation

Some 3 million people in the UK experience incontinence and the cost of management is over £500 million per annum to the NHS. It can be caused by both congenital anomalies, such as a failure of the bladder in the foetus to form (bladder exstrophy), and acquired conditions such as a lost of the compliance of the bladder (neuropathic bladder) or bladder cancer. For end-stage bladder disease, the most commonly performed surgical procedure to augment or replace the bladder involves incorporating a vascularised, reconfigured segment of bowel (enterocystoplasty). Although this procedure can provide continence and enhance quality of life, it is commonly associated with serious complications, including infections, mucus production and stone formation, and long-term risk of cancer. These complications are primarily due to the fact that bowel mucosa is structurally and physiologically incompatible with the long-term exposure to urine. A number of bladder engineering strategies have been proposed and typically, these rely upon the incorporation of a natural or synthetic biomaterial into the bladder, which may or not be pre-cultured with cells derived from the patient (autologous). Despite some high profile reports, there is little evidence that bladder tissue engineering can yet achieve a functional outcome equivalent to current surgical techniques, and there remains an unmet clinical need.
The aim of this programme is to deliver functional bladder reconstruction to patients through an autologous cell engineering strategy. This project will bring together expertise in stem cells and urology (ICH, GOSH and UCLH) with urothelial cell biology and bladder tissue engineering (York) with the purpose of developing a novel technique for bladder reconstruction that will be applicable in the first instance to patients with bladder exstrophy, but eventually extended to all patients requiring bladder augmentation. The stages of the project will be firstly to develop methods to identify and isolate the most appropriate source of autologous epithelial cells in the case of bladder exstrophy. To meet this aim, cells derived from normal and diseased bladder and from biopsies of buccal mucosa will be isolated, and characterized. Expansion of the epithelial cells will be conducted using "Good Manufacturing Practices" techniques, a standard methodology used for pharmaceutical products, to provide a ready route for translation to the clinic. Human cells will be xenogeneic transplanted in 24 pigs for bladder augmentation and functional and histological data will be obtained at 3 and 6 months after transplantation. Principal outcome measures will be: safety as measured by survival; efficacy as measured by capacity and compliance of the neo-bladder. Secondary measures for this phase will include: surgical morbidity, post-mortem immunohistological analysis of both the augmented and native mucosa, and also 'reverse translational' outcome measures, where observations of cellular behaviour are used to develop hypotheses about stem cell/cell functional engraftment in these settings in vivo.

1. Sourcing of autologous cells. Bladder mucosa will be harvested from exstrophy babies undergoing primary repair according to ethical approval. Buccal mucosa epithelium will be obtained from discarded tissue during hypospadias repair. Cells will be isolated and expanded using established techniques and tested for their capacity to express differentiation-associated markers and for functional barrier properties by measuring trans-epithelial electrical resistance and permeability to urea and water. The harvest of differentiated sheets and suitable carrier for transport and delivery will be also refined.
2. Refine the procedures to allow completion of GMP standard operating. Appropriate standard operating procedures and batch manufacturing records will be based on cell culture optimization to define a robust and reproducible process able to be tested in a pre-GMP setting. All critical reagents will be identified, sourced and, if necessary, validated for GMP compliance. Human urothelial, exstrophy and buccal epithelial GMP-compliant cells will be produced/banked for transplantation into pigs.
3. Establish the porcine experimental surgical model of composite cystoplasty. A vascularised, de-epithelialized, seromuscular colonic segment will be developed in a total of 24 immunosuppressed pigs. After isolation, the patch will receive the tissue-engineered epithelial sheet in complex with the carrier (e.g. Vicryl mesh) to facilitate epithelial attachment. The pigs will be divided into 3 groups (8 animals each): one set will receive a bladder augment with a patch built with tissue derived from healthy urothelium; the second set from exstrophy-derived urothelium, and finally the third set will undergo augmentation using tissue from buccal mucosa epithelium. The animals will be sacrificed at 3 and 6 months post-augment. The assessment will include voiding behaviour records and post-mortem immunohistological analysis of both the augmented and native mucosa.

The aim of this programme is to deliver a functional bladder reconstruction, through a cell/tissue engineering strategy, to children affected by bladder augmentation, who require increased bladder capacity. Bladder exstrophy is a major congenital anomaly that affects the growth and function of the bladder. Despite improvement of the surgical techniques, the need for bladder augmentation in bladder exstrophy patients, in order to achieve an adequate bladder capacity is reported in up to 70% of children. In general, the incidence of neonates born with bladder exstrophy or other congenital malformations is increasing and the results of the operations currently used for their correction are often disappointing, leaving these children with profound disabilities and poor quality of life. This has significant deleterious effects on both the family and the community.
While we focus the work of this project on the bladder exstrophy, the use of a surrogate epithelium such as the buccal mucosa here proposed may have an impact also for other indications where bladder augmentation is required but autologous urothelium cannot be used, such as for bladder cancer patients. The evidence suggests an increasing trend in the incidence of this malignancy in recent years. In 2000, worldwide, 336,000 patients were diagnosed with bladder cancer. Bladder augmentation is part of the armoury of the urologist / paediatric urologist in the treatment of urinary incontinence. Urinary incontinence is associated with tremendous costs and with a significant decrement in health-related quality of life; it is proven that in addition to high economic costs, incontinence results in medical and psychological morbidity and adverse effects on quality of life. In general, urinary incontinence, secondary to congenital and acquired diseases, is experienced by some 3 million people in the UK and the cost of management is over £500 million per annum to the NHS.
At the moment, although bladder augmentation can provide continence and enhance quality of life, it is commonly associated with serious complications, such as mucus production, formation of stones, recurrent urinary infection, metabolic imbalance and for some, in the long term, malignant change. Management of these complications can require additional medication, bladder washouts and occasionally hospitalisation treatment.
The aim of this project is to provide a safe alternative to bowel segments for augment the bladder that will improve clinical outcomes and the quality of life for these patients and their families by reducing their risk of sequelae. This in turn will reduce the cost burden for the NHS of lifelong management of such complex patients and their complications. Once validated, the proposed surgical approach to composite cystoplasty will be easily and quickly translated into clinical application. We also envisage that this research may have, in the long term, a wider economic impact. This is based on the fact that our results could be of interest not only for the cure of congenital anomalies, but also for the treatment of acquired diseases in children as well as in adults of all ages. Moreover tissue engineering is linked to advances in technology and is likely to generate commercial interests. If our approach would prove to be correct, possible patents could be generated for commercially exploitable results. It is not our primary interest to develop a patent, but we believe that this could help acquiring future grants and fostering of future research.