The goal of this proposal is to develop cell-based therapies that lead to the better healing of traumatic head injuries. Our first strategy will be to use genetics and embryology in zebrafish to identify factors that can convert human embryonic stem cells into replacement skeleton for the head and face. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. As zebrafish develop rapidly and can be grown in large numbers, a growing number of researchers are using zebrafish to study how and when cells decide to make a specific type of tissue – such as muscle, neurons, and skeleton - in the vertebrate embryo. Recently, we have isolated two new zebrafish mutants that completely lack the head skeleton. By studying these mutants, we hope to identify the cellular origins and genes that make head skeletal precursors in the embryo. These genes will then be tested for their ability to drive human embryonic stem cells along a head skeletal lineage. Our second strategy will be to test whether a population of cells, similar to the one that makes the head skeleton in the embryo, exists in the adult face. We have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation. In this proposal, we use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can give rise to new head skeleton in response to injury. Traumatic injuries to the face are common, and treatment typically involves grafting skeleton from other parts of the patient to the injury site. Unfortunately, the amount of skeleton available for grafts is in short supply, and surgeries often result in facial disfigurement that causes psychological suffering for the patient for years to come. Here we propose two better treatments that would lead to more efficient healing and less scarring. The first treatment would be to differentiate human embryonic stem cells, a potentially limitless resource, into skeletal precursors that can be grafted into the head injury site. By understanding the common pathways by which head skeletal cells are specified in the zebrafish embryo and human embryonic stem cells, we will be able to generate skeletal replacement cells in large quantities in cell culture. The second treatment would be to stimulate adult stem cells already in the face to regenerate the injured head skeleton. If successful, our experiments on zebrafish jaw regeneration will allow us to devise strategies to augment the natural skeletal repair mechanisms of humans.
Traumatic injuries to the head, such as those caused by car accidents and gunshot wounds, are commonly seen in emergency rooms throughout California. Current treatments for severe injuries of the head skeleton involve either grafting skeleton from another part of the body to the injury site or, in cases where there is not sufficient skeleton available for grafts, implanting metal plates. Although these operations save lives, they often result in facial disfigurement that causes psychological suffering for the patient for years to come. For this reason, there is enormous interest in cell-based skeletal replacement therapies that will heal the face without leaving disfiguring scars. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. Thus, we are using the zebrafish embryo to rapidly identify factors that can make head skeletal precursors, and then asking if these same factors can instruct human embryonic stem cells to form skeletal replacement cells. In addition, we have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation, and we will use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can regenerate the face. The successful completion of these experiments would allow us to both generate unlimited amounts of head skeletal precursors for facial repair and stimulate latent skeletal repair mechanisms. The combination of these approaches will lead to therapies that promote a more natural healing of the face, thus allowing Californians to eventually resume normal lives after catastrophic accidents.
Injuries to the craniofacial structures are an important clinical problem, as are birth defects that affect these structures, and regenerative medicine approaches for these problems are under-studied. Relatively little is known about the fate choice mechanisms that lead to development (and potentially regeneration) of the skeletal structures of the face from neural crest stem cells. The goal of the work is to identify genes involved in neural crest-derived generation of skeletal structures. The approach is to take zebrafish genes which have been identified to be important in this pathway and overexpress them in human embryonic stem cells (hESC). These genes will then be used to generate preskeletal neural crest cells from hESC. The applicant will take advantage of two zebrafish mutants that were identified during the applicant’s training. In both of these mutants, early neural crest markers are lost. Combined with other fate-specific genes for neural crest cells, experiments will be performed to determine if the same gene repertoire will lead to neural crest and craniofacial skeletal fate determination by hESC. The final aim will probe the role of adult skeletal progenitors and neural crest stem cells in adult zebrafish jaw regeneration toward the long-term clinical goal of stimulating regeneration of damaged craniofacial structures in adult human. The reviewers felt that the grant was very ambitious and exciting.
The proposal was described in the reviews as innovative and bold, not the least derivative, and also of considerable practical importance. The potential impact on regenerative medicine was thought to be very high. Reviewers also appreciated the care with which the proposal was written, and the scientific issues were well-articulated. Thoughtful consideration was given to potential experimental pitfalls and alternative strategies. The impressive body of useful preliminary data was also considered a strength. The proposal was unusual in planning a relevant use of human embryonic stem cells as a compliment to the work in a genetically accessible model organism (the zebrafish). One reviewer was particularly struck by the last part of this proposal based on data showing that the principal investigator (PI) has identified a putative adult stem cell population in the adult jaw that contributes to the remarkable ability of zebrafish to regenerate mandibular structures after surgical excision. Aim 3 includes studies to determine if these cells are essential for adult jaw regeneration, and expansion of this part of the grant into a larger part of the research plan would have strengthened the grant.
The major concern raised by reviewers was that neither of the genes to be used for these studies has been cloned, and though the applicant is the one who identified the mutations in fish, the lack of the cloned genes (as the crucial reagent) may be a problem in getting the work started. One candidate gene has been narrowed down to a 6-gene region and one of the genes in this region has no human homolog. If one of the operative ‘genes’ that was identified in fish is actually an enhancer sequence, it could be completely unrelated to neural crest development, and the identification of the elements could also be a long process, delaying the application of the work to hES cells. Also one gene is dominant making the cloning potentially more difficult. The applicant also does not present convincing evidence that expression of only the two genes of interest will be sufficient to drive hESCs to a neural crest cell fate. In addition, if the operative gene products in these mutations turn out to be secreted factors that are not even expressed in crest, this would greatly alter the strategy that the PI would need to use in the hESC system. Enthusiasm was reduced because so much of the grant was based on contingency, and reviewers were concerned that this represented a general problem with the thought processes underlying the research proposal. The study plan was not, then, impeccably crafted, with pitfalls and alternatives adequately addressed. Nonetheless, because the applicant and institutional support were so strong, and there are many ways to solve the cloning problem, these concerns were balanced to generate considerable support for the application. Furthermore, a reasonable Plan B is to continue more intensive work on jaw development in the zebrafish model.
The applicant brings a great educational pedigree to the work, and has a superb recent publication record including papers in Development and Neuron. The applicant’s graduate and post-doctoral education were all in first-rate programs and labs. The applicant’s focus has been systems development, most recently craniofacial, and so this application is also made in the context of current NIH, March of Dimes, and CIRM Seed grant funding. The applicant is an Assistant Professor in a new stem cell center. The thoughtful and detailed career development plan focuses on the applicant’s interest in developing an academic career in hESC biology. The applicant has carefully selected several strong mentors. It was the strength of the applicant that was the major factor in determining the enthusiasm for this proposal, given the high risk of developing the entire proposal based on the risky plan to clone the relevant zebrafish genes in a short period of time.
The stem cell biology group at the applicant’s institution and the intellectual environment surrounding the applicant is similarly strong, with the newly set-up core lab being an especially important feature. The mentoring plan was thoughtful, and included a detailed career development plan taking advantage of the strengths of the institution. The institution has provided a substantial start-up package for the applicant, and the letters from institutional officials made it clear that the institution is invested in the career development of the applicant.