Augmentation or replacement of the bladder is often necessary for the treatment of adults with bladder cancer and children with spinal cord injury or spina bifida. Current surgical techniques utilize segments of intestine or stomach as a substitute for bladder wall. Use of intestinal segments is associated with many complications including infection, stones, salt imbalance, and most concerning, cancer. An ideal substitute for bladder wall would be bioengineered bladder tissue. Ideally, a bioengineered graft would consist of cells that are genetically normal and free of cancerous mutations, promote blood vessel growth, survive long-term and regenerate. Stem cells appear to be the ideal solution for bioengineering tissue.
Preliminary clinical trials have demonstrated the feasibility of using bioengineered tissue for bladder augmentation. The bladder is lined by a very unique cell type called “urothelium”. The ability to induce human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC) into urothelium would provide a major advancement in the tissue engineering field, scientifically and clinically. In addition, deciphering the mechanisms of hESC to urothelial differentiation would facilitate investigation of deviated differentiation into urothelial cancer stem cells; the “seeds” of bladder cancer.
Bladder cancer is the fourth most common type of cancer and caused 15,000 deaths last year. Treatment often requires removal of the bladder. Like other tumors, bladder cancer is believed to originate from the transformation of stem cells into cancer stem cells (CSCs). Potential markers of urothelial CSCs have been identified. Surprisingly, the scientific community has not yet addressed the study of normal human urothelial stem cells and differentiation of hESC to urothelium. The investigation of mechanisms and markers involved in the differentiation of hESC into urothelium will yield important facts about normal and abnormal differentiation and will ultimatley help predict the malignancy of bladder cancers and improve treatments.
Our specific aims are to induce the differentiation of hESC into urothelium via cell signaling. We will also investigate the genes involved in this process. And, we will test the feasibility of transplanting hESC-derived urothelium into a bladder.
This investigation will lead to advances in stem cell biology in an important area not addressed by other scientists. The successful completion of this project will improve human health, indirectly through increased knowledge of differentiation pathways relevant to normal development and neoplasia, and directly through development of novel methodologies for bioengineering tissue for adults and children with urologic disorders and cancer. We are working in a very novel field, which has a high potential to save lives and to vastly improve the quality of life for many patients who need their bladder removed or enlarged.
The scientific community has not yet addressed the study of urothelial stem cells and differentiation of human embryonic stem cells (hESC) to urothelium. Our investigation of mechanisms and markers involved in the differentiation of hESC into urothelium will yield important facts about normal and abnormal differentiation and will help predict the malignancy of bladder cancers and improve treatments. This project will also advance the field of regenerative medicine. Adults with bladder cancer and children with spina bifida often need bladder reconstruction. Current surgical techniques use segments of intestine as a substitute for bladder wall. Use of intestinal segments is associated with many complications including cancer. Preliminary clinical trials have demonstrated the feasibility of using bioengineered tissue. The ability to induce hESC or induced pluripotent stem cells (iPSC) into urothelium would provide a major advancement in the regenerative medicine field, both scientifically and clinically.
Due to its high rate of recurrence, bladder cancer carries the highest lifetime cost to treat of all cancers. The successful completion of this project will improve human health, indirectly through increased knowledge of differentiation pathways relevant to normal bladder development and bladder cancer, and directly through development of novel methodologies for bioengineering tissue for adults and children with urologic disorders and cancer. These benefits will come to the citizens of California first. In addition to healthcare, this research will benefit the California economy by developing new protocols and technologies that could be adapted for other organs and tissues. Any health benefits, patents, new biotechnology or clinical trials would start in California. This research exemplifies the intent of CIRM bringing together clinical scientists with basic and translational scientists to develop stem cell treatments for the California public while at the same time advancing stem cell biology.
The goal of the proposed studies is to develop a protocol for the differentiation of human embryonic stem cells (hESC) into urothelial cells, a unique cell type lining the bladder that might prove useful for treating patients with bladder defects or cancer. In Aim 1, the principal investigator (PI) intends to improve the efficiency of hESC differentiation into definitive endoderm (DE). Next, the PI proposes to enhance the efficiency of hESC-derived DE differentiation into urothelium via inductive signaling from bladder epithelium and mesenchyme. For the third Aim, the PI will employ overexpression and knockdown approaches in order to verify the function of candidate genes in this process. Finally, in Aim 4, the PI will test the feasibility of transplanting hESC-derived urothelium into bladder.
The reviewers acknowledged that this proposal addresses an understudied and important problem that could lead to advances in the development of regenerative therapy for bladder repair. In general, this is an innovative project in that very few labs are addressing this topic, and reviewers found that some of the experimental approaches, i.e. the co-culture system and bladder transplantation models, were interesting. On the other hand, other aspects of the proposal lacked novelty, especially Aim 1, since others have already published data and protocols on DE generation from hESC. As a result, reviewers were uncertain what advantages the proposed protocol would offer over alternative methodologies and therefore questioned the extent to which this work would have impact.
While the proposal was well written, the reviewers identified a number of weaknesses in the research plan that led them to question its overall feasibility. First, several lines of investigation, such as those in Aim 3, were not adequately supported by the preliminary data, leaving the impression that this effort might be somewhat premature. A similar opinion was expressed for Aim 4, which reviewers praised for its translational aspirations but cautioned that the more basic aspects of urothelial differentiation have yet to be worked out. Reviewers found the experimental design to be overly ambitious, noting that the extensive gain and loss of function experiments proposed for Aim 3 lacked a clear rationale for candidate gene prioritization and were missing critical details regarding the stages of differentiation to be analyzed. Considering the ultimate goal is to derive clinically relevant urothelium from hESC, reviewers were surprised that no attempts to systematically analyze and identify the active factors released from the co-cultured bladder cells were proposed. Reviewers also noted that the applicant mistakenly listed 2 different DE markers to be analyzed although they represent the same gene. As much of the proposal hinges on the ability to efficiently achieve DE differentiation, the reviewers worried that this technical error might be indicative of the applicant’s inexperience with this pathway. To ensure interpretable results, a reviewer emphasized that it would be critical to quantify the percentage of DE or urothelial cells prior to polymerase chain reaction-based marker analyses.
The PI is an associate professor and holds a leading clinical position; based on his/her urological background, the reviewers were convinced s/he would be very committed to this specialized research program. The PI has been productive and has assembled a very capable group of collaborators who have been already working together on this project for several years. The co-investigator has considerable experience in stem cell differentiation and has published widely. Reviewers did note that insufficient lab personnel were dedicated to this complex project.
In summary, reviewers found this proposal to be innovative and significant. However, an overly ambitious scope and a premature research plan led them to question its feasibility and potential for impact.