New Faculty I
$3 065 520
To fix a broken car, the mechanic either repairs or replaces the defective part. Similarly, one of the most promising approaches physicians foresee for treating human disease and ameliorating the aging process is regenerative medicine. One of the major aims of this field is to restore function by repairing or replacing damaged organs. Scientists envision a day when people with heart failure can be cured with hearts grown from their own cells, and a future in which dialysis machines are not needed because patients with damaged kidneys can be furnished with new ones. However, organ engineering is highly complex, and the field of regenerative biology is still in its early stages. Therefore, it is important to provide proof of principle and lay the foundation for creation of new organs by first using as simple a system as possible, and teeth provide an excellent model system for organ replacement. Their physiology is less complex than many other organs, but their development has much in common with that of other organs. This means that much of the information obtained from studying tooth regeneration will be generally applicable for building other organs. Teeth are a relatively safe prototype for organ regeneration, and there is a significant need for replacement teeth, as those born without teeth due to genetic defects, as well as elderly patients, patients with caries and periodontal disease, and victims of physical trauma all need new teeth. Our ultimate goal is to take advantage of basic biological principles in order to develop new therapeutic approaches. As such, we seek to help lay the groundwork for the formation of new teeth that can replace missing teeth, in the same way that permanent teeth replace the primary teeth formed in early childhood. This ambitious goal must be built on the proper foundation if it is to succeed. Therefore, we will first concentrate on a more readily achievable objective, which is to understand a naturally occurring version of regeneration by studying the continuous growth of the mouse incisor. This unusual tooth depends on the presence of adult stem cells to constantly produce all the cell types of the mature organ. Toward this end, we propose in this application to first analyze the biological processes that regulate the stem cells in this remarkable tooth. We will develop tools to grow the mouse stem cells outside of the animal in order to better understand them. Subsequently, we propose to translate what we have learned from the mouse model into human cells by learning how to induce human embryonic stem cells, fetal cells, or adult cells to become tooth progenitor cells. This last step will help lay the necessary groundwork for growing human teeth and blaze the trail for regeneration of larger organs.
Statement of Benefit to California:
The promise of stem cell biology for regenerative medicine lies in the ability of these remarkable cells to give rise to more differentiated cell types that, individually or as part of a bioengineered organ, can replace structures that have been damaged by disease or aging. We propose to help lay the groundwork for organ regeneration by focusing on the tooth as a prototype organ. In addition to addressing the health issues posed by dental decay and tooth loss, which require prosthetic replacements that are functionally inferior to natural teeth, our project will help to pave the way for safe clinical applications of human embryonic stem cells in regeneration of larger organs, such as hearts or lungs. We anticipate that our research will be a significant step towards making the promise of regenerative medicine from adult stem cells and human embryonic stem cells a reality. Our studies will provide a much-needed model system that will allow us to study the basic mechanisms underlying guided development of organs from stem cells, which will be central to fulfilling the therapeutic potential of stem cell-based organ regeneration. Eventually, stem cell-based therapies will reduce health care costs for Californians by improving treatment for diseases for which we currently do not have effective therapies. Our work could provide economic benefits to the state by helping to lay the groundwork for commercial efforts to regenerate teeth as well as other solid organs, such as the heart and pancreas. Such developments would be of great benefit to California by making the state a leader in a field that is poised to become economically important in the future. The State of California will also stand to benefit from the intellectual property generated by this research, as generalizable principles regarding the use of stem cells, in vitro differentiation of cells, scaffolding materials, and organ bioengineering may be patentable.
SYNOPSIS: This proposal concerns the very interesting model of tooth regeneration as a model for organ regeneration in adults. There are 3 aims. The first aim uses genetic mouse models to determine the mechanisms that enable self-renewal and differentiated progeny from murine adult dental epithelial stem cells. Using the Sprouty mouse, the PI will investigate how SPRY2 and SPRY4 function to repress epithelial stem cells, and determine whether reduced Spry2/4 function affects the kinetics of ameloblast and odontoblast formation from progenitor stem cells. The applicant has localized the cells and will further define them by lineage tracing, gene expression, and cell surface markers. Additionally, he proposes analysis of these cells in mouse models modulating the two pathways (FGFs and sonic hedgehog) known to regulate tooth morphogenesis. The second aim is to analyze the growth and differentiation of these murine dental epithelial stem cells in vitro using growth factors, media and, importantly, substrata. A follow-up subaim is to then test these cultured cells for their ability to form new teeth when transplanted under the kidney capsule with embryonic mesenchyme or mesenchymal stem cells, or subcutaneously, or into the dental niche. The third aim is translating these findings to the human. It is unclear if there are human adult dental epithelial stem cells normally, but patients with cleidocranial dyplasia (CCD) continually generate new teeth. Here two major approaches will be used: 1) culturing ameloblast precursors from fetal teeth, extracted molars, or from CCD patients and 2) from human ESCs. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This application is well-constructed and is a pleasure to read. The proposal, which deals with a highly significant biological issue with future clinical applications, presents the tooth as an organ with less complex physiology that can be studied as a model for general organ regeneration. One of the major strengths of the proposal is the use of the tooth as a prototype for organ replacement, and this work could enable the development of techniques at an early stage which could be perfected for later application in humans. The proposed studies will characterize, functionally analyze, and culture human dental epithelial stem cells, and will derive dental epithelium from human ESCs. These cells are relatively uncharacterized as compared to dental mesenchymal stem cells, so the proposed studies are important. The ability to use hESCs to regenerate dental epithelium, and the enamel tissue secreted by dental epithelial-derived ameloblasts, would be significant. Another strength is the proposed effort to identify human adult epithelial dental SCs, and/or to differentiate them from progenitor fetal or embryonic SCs. The PI states that these studies will “launch the mouse incisor as a new system for the study of epithelial cells”, when in fact this model has been studied for decades. Nevertheless, these studies are innovative in that they use novel mouse mutants exhibiting distinct tooth phenotypes. These mice will be used to identify signaling pathways regulating replacement tooth formation, and will be tested for their ability to direct human DSCs, and hESCs, in tooth regeneration efforts. A key risk of the proposal is whether the cells will grow in culture, but this is matched by an alternative line of work with hES cells which also utilizes cutting-edge work on isolation of the best hES cells for the purpose. The timeframe does seem realistic as the cell culture starts from day one which will be important and will be progressively informed by early work on fundamental cell biology. The third aim is also risky since each of the approaches relies on learning from the collaborators, who are expert in ameloblast or hESCs. Human post-natal and embryonic teeth obtained from aborted fetal tissues and human ESCs will be obtained in collaboration with colleagues at UCSF and used for tooth regeneration studies. The proposed use of human ESC lines (4 UCSF stem cell lines, and 2 Valencia Stem Cell Bank lines) is considered a strength, but apparently many of the proposed lines, which will be obtained from collaborator Dr. Susan Fisher, have not yet been generated. One reviewer feels that as currently proposed, the experimental design is overambitious and lacks focus. For example, in Aim 2B it is not clear what will be done, and how the results will be analyzed and quantified – in vitro filter assays, kidney capsule implants, and subcutaneous implants are all proposed, on implants grown from 4 days to 4 weeks, but experimental details are not provided. Similarly, the expression of various fluorescently-tagged FGFs and Spry gene constructs in cultured human dental epithelial SCs will be used to “observe, measure, and localize the distribution of members of the pathway under conditions that lead to self renewal vs. differentiation." It is not clear what this means, and detailed experimental procedures need to be described here. This reviewer recommended that the proposal should be revised to consist of a more focused study of a few of the signaling pathways included in this study, perhaps focusing on the very interesting Fgf/Spry interactions in dental epithelial SC proliferation/differentiation. The proposed study of CCD dental epithelium is also quite interesting, and would be another area on which to focus, by providing detailed studies whose goal is to elucidate the molecular nature of disrupted signaling pathways resulting in supernumerary tooth regeneration in these patients. In general, a more detailed description of the proposed experimental studies would have been helpful, along with strong preliminary data demonstrating the ability to perform each of the experimental techniques in the proposed studies. Evidence of which of the proposed reagents/transgenic animals/cell lines have been established should have been presented more clearly. Finally, detailed methods for quantitating the results in a statistically significant manner, including descriptions of the numbers of samples required, should be incorporated into the study plan. The three aims are ambitious, but reviewers pointed out that a team of collaborators will provide needed expertise and reagents. The clear experimental plan would provide valuable data on the characteristics and functions of dental epithelial progenitor cells, and the proposal is based on solid preliminary data which provide strong support for both the team and the concept. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Klein is an outstanding candidate for a Physician-Scientist New Faculty Award. He obtained his MD-PhD from Yale where he completed his pediatric residency. He did a clinical genetics fellowship at UCSF and did his research in the lab of Gail Martin. Upon finishing his fellowship in 2007, he was hired as Assistant Professor in the Departments of Orofacial Sciences and Pediatrics and the Institutes for Human Genetics and Regeneration. He has received numerous honors and awards for his research, including the Chancellor’s Scholarship, UC Berkeley, NIH Medical Scientist Training Program, and Young Investigator Research Grant from the Society for Pediatric Research Annual Meeting, AA Pediatrics, and has already been invited to speak at four international conferences. He has 11 publications since 1995, both clinical and basic studies, in top level peer-reviewed journals including American Journal of Medical Genetics, Clinical Genetics, and Human Reproduction. The preliminary data for this research was published in 2006 in Developmental Cell. He is currently supported by a K08 Award from the NIDCR (07/01/07-06/30/12). He has very strong collaborators with the necessary expertise for his proposed research. The career development plan is another of the numerous and substantial strengths of this proposal, with well-defined goals for the science, a long-term vision for the PI’s involvement in regenerative medicine, and clinical applications for the work. The PI clearly sees clinical responsibilities developing as a closely integrated aspect of the project and in stem cell science. There are excellent plans for mentoring with a formal advisory committee to give direct feedback. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: There is a very strong institutional commitment letter detailing the start-up package (salary for the 5 years) and protected research time (90%). The PI has been provided with a 12-bench laboratory, office, and support space in the UCSF Parnassus Heights research campus. The environment is excellent. The Human Embryonic Stem Cell Center at the Parnassus campus will be used to conduct the proposed hESC research. This laboratory is supported only by private funds, and it has an embryo bank and equipment for hESC work. The UCSF CIRM application for a shared research and teaching facility was recently chosen for funding as well. The Department of Obstetrics and Gynecology and the Center for Reproductive Sciences, in conjunction with the Institute for Regenerative Medicine, is available for use by all investigators as a core facility. The start up package has provided all of the necessary equipment, including microscopy requirements, although funds for an additional fluorescent dissecting microscope are requested in this application. UCSF has had an excellent track record for supporting the development of junior faculty, and the institute has local plans for further associate professors and additional investment in hES programs. DISCUSSION: This interesting proposal is from a very strong candidate who has good mouse models of murine epithelial-derived stem cells. This is an outstanding candidate for MD/PhD award, and one reviewer commented that this was the best application that he/she reviewed. The proposal is ambitious with many different approaches, and although one reviewer commented that the studies are over-ambitious and lack focus, other reviewers noted that in each case there are close collaborators from whom the applicant can learn. The studies are bound to provide information that will move the field forward. One minor comment was that the proposal lacked a detailed description of the experiments; for example, the reagents (transgenic animals, cell lines, inducible lines etc.) weren't fully described, the design lacked the number of animals needed for experiments, and there was no description of how data would be analyzed. However, panelists commented that the PI already has many of the animals and reagents needed and will generate the rest with the strong collaborators in place under this 5-year award.