The bladder serves as a reservoir for the storage and emptying of urine. Spina bifida and bladder cancer are conditions that adversely affect the bladder, and many patients must undergo bladder replacement. Unfortunately, current bladder replacement techniques require the use of intestine, which is associated with significant perioperative morbidity and long-term complications. Recently, tissue engineered bladder segments derived from autologous biopsy have been identified as an alternative to the use of intestine for bladder replacement in both animal studies and in clinical trials.
Unfortunately, suitable unaffected donor cells may not exist in patients with spina bifida or bladder cancer. In these situations, hESCs are envisioned as a viable source of cells from which the desired tissue can be derived. Combining the techniques learned in tissue engineering over the past few decades with this potentially endless source of versatile cells could lead to novel sources of replacement organs such as the bladder.
This proposal will build upon principles from both tissue engineering and developmental biology with the intent of bioengineering bladder tissue derived from hESCs to avoid the complications and morbidity associated with the use of intestine for bladder replacement.
In addition to NIH-approved hESC lines, this proposal will involve the use of non-NIH approved hESC lines that were developed in xenogen-free and pathogen-free conditions, which would make them eligible for future clinical applications. This is in contrast to the approved hESC lines on the NIH registry, which were contaminated with mouse products.
In summary, this proposal will investigate the targeting of lineage-specific differentiation of hESCs toward the bladder cell lineages for bladder tissue engineering applications. Due to ongoing clinical studies using constructs derived from autologous bladder cells, the transition to clinical applications should be greatly shortened. The findings will greatly improve the lives of patients with spina bifida and bladder cancer, and will help to solidify California’s leadership in these fields.
Statement of Benefit to California:
This proposal will utilize novel hESC lines that were developed outside of the United States in political and social environments that have allowed rapid advancements in human embryonic stem cell research over the past 10 years. These cell lines include those that were derived in xenogen-free and pathogen-free conditions that render them eligible for future clinical applications. As these cell lines have not been used in the United States thus far, this proposal will allow the importation of these novel stem cell lines, which eventually can be made available to all interested scientists in California and the rest of the United States.
In addition, this proposal will investigate the targeting of lineage-specific differentiation of hESCs toward the bladder cell lineages for bladder tissue engineering applications. Due to ongoing FDA-approved clinical studies using tissue engineered constructs derived from autologous bladder cells, the transition to clinical applications should be greatly shortened. The findings will greatly improve the lives of patients with spina bifida and bladder cancer by avoiding the use of intestine, and will help to solidify California’s leadership in these fields.
SYNOPSIS: The PI is a young physician-scientist who proposes a study directed at differentiating bladder cells from hESC for use in tissue engineered bladder grafts. The specific aims of the proposal are (i) to determine if hESC lines (various new and approved) maintain their undifferentiated state in a proprietary medium (from a colleague in Korea) without contact with xenogeneic proteins or cells and (ii) to determine whether hESC treated with growth factors and/or genetically modified with transcription factors can be used to induce hESC to differentiate into bladder cell lineages.
INNOVATION AND SIGNIFICANCE: Bladder replacement would have a major impact on healthcare. The ability to differentiate hESCs reliably to bladder-related cell types would be a useful demonstration for the hESC community. However, innovation is limited by the fact that the PI picked an application area that may not represent the greatest need. The proposal is a hard sell because the investigator is using hES cells for an application that has been one of the great successes of adult tissue stem cell engineering. Thus, it is not clear why one would choose hESC-derived bladders versus adult (stem) cell-derived equivalents.
STRENGTHS: The background of the PI is a strength of the proposal, given the experience with bladder-related cell biology. The link to clinically and translationally relevant collaborators and experience amongst this group makes it likely that successful hESC differentiation of cells could be quickly directed toward clinical endpoints. The interface with a Phase II trial of engineered bladder constructs from adult stem cells is a plus.
WEAKNESSES: The first aim is laudable but is a bit disconnected from the second aim. There are a few problems with this aim. It is unclear whether the proprietary medium formulation would be available to other investigators. Although the PI states that all the cell lines he explores will be grown on STO (which is mouse) and also in xenogeneic-free conditions, he doesn’t clearly state that he is going to compare the cells grown under the two conditions. The comparison (and statistical considerations) are just implied. The length of culture isn’t stated. Cells will be analyzed every 10 passages for markers that characterize the undifferentiated state and also for karyotypic abnormalities — but how many passages will be performed? There also is no discussion of what markers will be used to characterize the EB’s.
The second aim is not likely to be successful. The investigator provides no details that would suggest that they have any insight on how to differentiate the hESCs to bladder cells. They have some experience showing differentiation of a related stem cell into bladder cells with minimal stimuli, but why would one think that the same cues that drive the final steps of differentiation of a late-stage precursor could also be used to drive differentiation from hESCs which have so many steps to get to that precursor? There is no discussion of the specific growth factors that will be used (some are mentioned), and there is no novelty to the idea of trying cocktails to differentiate hESCs. If the investigator fails with one cocktail, how does that inform them to prioritize which next cocktail to try? How many combinations (of different factors, different concentrations) will be tried before declaring victory or failure?
If the FACS will be used to provide analysis of the early stages of differentiation, then why not use FACS to pull out intermediate progenitor cells to enrich the population for cells likely to be conducive to the growth factor and transcription factor manipulations? There is no plan to examine the contractile phenotype of the cells. PDGF and VEGF are involved in differentiation of many different cell types, and so unless the population they are applied to is somehow restricted, they are likely to get a mixed bag of cells and inefficient generation of the cells of interest.
The use of a virally-introduced transcription factor is potentially interesting if it has already been shown to be involved in bladder differentiation, but doesn't seem to fit the goals of generating a clinically useful cell (having been infected with a virus). The investigators provide very little detail with regard to how they will interpret or investigate this aspect of the work.
The use of co-cultures is extremely vague.
Given the above, probably a better approach for starting is to default to the conditioned medium strategy just to see if the hESC lines are easily pushed to bladder cell lineages. The PI states that he will use conditioned medium if his primary methods don’t work, but it would be a good idea to go in reverse. There are lots of options for conditioned medium that are EASY to get and have proven useful in pushing other stem cells to a bladder cell lineage fate (bladder carcinoma lines, primary mouse uroepithelial cells, urinary bladder smooth muscle cells, and others).
DISCUSSION: There was no further discussion following reviewers' comments.