Funding opportunities

Regulation of Adult Stem Cell Proliferation and Number

Funding Type: 
New Faculty I
Grant Number: 
RN1-00581
Funds requested: 
$3 019 782
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Our research focuses on developing new tools and models for the next generation of doctors and scientists in all specialties of regenerative medicine. The major obstacles in regenerative medicine are the limited number of pre-existing stem cells and the lack of human models to study organ repair. Our first aim is to identify the mechanisms that regulate adult stem cell proliferation. We propose to use this knowledge to produce cell-permeable proteins to reactivate proliferation in dormant stem cells. Our second aim is to identify how the adult stem cell fate is specified. This knowledge will be used to increase the number of pre-existing stem cells in an organ. Our third aim is to develop a model of human organ development, where we can test the approaches of promoting organ-stem cell populations to form, activating stem cell populations to divide, and activating signaling centers to sustain patterned growth. As a model to study adult stem cell quiescence and activation, we use the stem cells of the hair follicle to identify proteins that regulate cell cycle. We think that regulate the cell cycle of the hair stem cells will also function in other adult stem cells. The products of this research will be cell-permeable proteins that mimic the activation of hair stem cells and could be applied to other organ systems to induce regeneration. As a model to study stem cell identity, we are using both embryonic and adult models to determine whether the signals that induce embryonic stem cell identity are the same as in the adult. Our third model is to study epidermal organ-like structures produced by human ES cells. By developing reporters for early development of epidermal organs, we will be able to study how stem cell compartments form during human organ development. We hope that the wide community of scientists at UCSD will benefit from our epidermal organ studies so that these advances can be applied to other organ-specific stem cell compartments.
Statement of Benefit to California: 
A major goal of regenerative medicine is to replace organs and tissues lost from disease or injury using our own body’s cells. Our research focuses on approaches to induce pre-existing stem cells to divide and to develop models of human organ development to study regeneration. This research will greatly benefit the next generation of regenerative doctors and scientists and benefit the California economy now through the development of new tools and jobs. The major obstacles in regenerative medicine are the limited number of pre-existing stem cells and the lack of human models to study organ repair. Our aims are to identify the mechanisms that regulate stem cell proliferation and that induce stem cell formation, using the hair follicle as a model. The hair follicle regenerated itself 10-20 times during a lifetime and its stem cells are readily accessible. We hope to translate our findings in the hair follicle into developing cell-permeable proteins to induce stem cells in other organs to divide. The products of this research will aid California by helping to speed recovery and to provide therapies for diseases once thought to cause permanent damage. These tools could reduce the suffering and long-term health consequences following organ damage, which should benefit all Californians. We also propose to generate a human model of organ development that should provide the next generation of California scientists and doctors with the lead in developing innovative treatments of diseases in all organ types. This approach should also benefit the health, biotechnology, and pharmaceutical industries of California.
Review Summary: 
SYNOPSIS: The applicant will study the role of Ras pathway signaling in stem cell proliferation. The primary model system will be the hair stem cell compartment, also known as the “bulge”. The focus will be on murine hair stem cells, with the approaches primarily being genetic. In Aim 1 the applicant will use mouse genetics and protein transduction to determine the effects of specific Ras pathway lesions on hair stem cell proliferation in vivo. In Aim 2 the applicant will ask whether genes in the Sprouty family, which are Ras pathway inhibitors, affect embryonic stem cell numbers in the blastocyst and adult stem cells in the skin and hair follicle. Aim 3 will develop human teratomas as models for adult stem cell generation and organogenesis. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The research proposal uses skin as a model system to address more basic questions of how the number of stem cells is determined and whether activation of quiescent stem cells would foster organ regeneration. The 3 aims seem disparate but all focus on this basic question. Skin has the advantage that the stem cell compartments and timing of activation/quiescence of the stem cells is known. The first two aims use designer mice to explore the mechanisms and molecules that control bulge (skin stem cells) to proliferate in aim 1 and the mechanisms and molecules that regulate the stem cell compartment during organogenesis in aim 2. The third aim is a long term, possibly high risk/high gain development of a human ESC teratoma model to study stem cell fates and potential “organizing centers” and to generate reporter cell lines for human adult stem cells and organizing centers. Aim 1A is to determine the effect of activated Raf in the bulge stem cell population. It is well-known that hyperactivation of the Ras pathway will inhibit the proliferation of normal cells via an Arf and p53-dependent pathway. One reviewer commented that this has been done in bulge cells already by others. Repeating those experiments in the skin with activated BRAF would not constitute a signficant advance. This sub aim could probably be incorporated as a possible follow-up to Aim1B, depending on the outcome of those experiments. Reviewers found Aim 1B to be more interesting. Activation of Ras has been shown to have disparate effects on subpopulations of skin cells; it activates basal keratinocytes and sebocytes, but inhibits nail and hair progenitors. The question to be addressed here is which downstream pathways mediate these different biological outcomes. To do this the applicant will create 4 new mouse strains conditionally expressing different alleles of Ras which selectively activate different downstream pathways. One reviewer was concerned that validation of these strains would require substantial in vivo analyses to demonstrate that the mutations have had the anticipated molecular effects on downstream pathways. Another drawback is that the proposal does not discuss how the strains will be analyzed to verify their molecular and biological properties, but is limited to a simple descriptive analysis of whether the various cell types are increased or reduced in numbers. In Aim 1C the applicant will ask whether the anti-proliferative effect of Ras in bulge cells is mediated by C/EBP alpha through the construction of doubly mutant mice. Rather than take a broad approach, the applicant has decided to test just this very specific model and no follow-up to examine other pathways is proposed. A reviewer commented that if this hypothesis is not correct little will be learned, and that perhaps this approach is premature. It has already been shown that specific deletion of C/EBP alpha does not have a hair cell phenotype, probably because of compensation by other C/EBP family members. The applicant provides no reason why this will also not be the case in these experiments. It would seem only prudent to investigate the possibility of such compensation in cell culture systems before going through the time and expense of constructing and analyzing multiple mutant mouse strains. Aim 1D is interesting because it asks whether transient expression of specific proteins can activate bulge stem cell proliferation. The problem is that it lacks any preliminary data showing that TAT-transduction of these stem cells in vivo is even possible. Control experiments are also lacking (e.g. WT Akt, WT C/EBP). Aim 2A will be to test genetically whether loss of Sprouty, a Ras/Raf inhibitor, affects stem cell numbers in the mouse blastocyst. The affects of activated Ras alleles will be addressed in the same way. The problem here is that single/double and triple Sprouty KO mice have already been made, with no reported effect on embryogenesis. The applicant argues that a partial effect on blastocyst cell number may have been overlooked, but he has no evidence for this. It therefore seems a tenuous argument on which to base a large series of experiments involving multiple mutant mice. Moreover, the direction from here is not clear and not discussed in the proposal. Descriptive results, limited to partially-penetrant phenotypes at best, do not make a compelling Aim. Aim 2B is to generate mice that conditionally express Sprouty 4. This is said to be “gain-of-function” but it is not clear why it would be, since the normal protein will be expressed at only a modest level. As above, no analysis of the mice is proposed to demonstrate its molecular properties (is it really gain-of-function?). No follow-up to understand the molecular basis of any phenotype is proposed. Aim 2C is to investigate the effects of specific C/EBP phospho site mutants on a bulge cell line in vitro. The assays to be used are not described, nor is it clear why TAT transduction technology is necessary, instead of stable gene transduction. No control using WT C/EBP is indicated. No follow-up beyond a descriptive analysis is proposed. In Aim 3A the applicant will transduce human ES cell lines with a keratin-luciferease fusion gene, and create teratomas from these cell lines. He will specifically screen for teratomas that express a keratin-luciferase fusion gene, to identify those undergoing epidermal development. One reviewer commented that the reason for doing these experiments is very poorly explained (limited to saying that these will be a “tool to study organ development”), and the subsequent analysis is also very poorly explained: “Label-retaining studies will help identify quiescent stem cell compartments and generate cell fate maps. In situ hybridization and immunostaining will help identify organizing centers and adult stem cells…”. Aim 3B is to isolate teratoma-derived hair stem cells. The Aim contains insufficient justification for why cells expressing Keratin 15 and C/EBP alpha are necessarily stem cells, and no information about how their stem cell characteristics would be validated. The proposal to do gene expression profiling on these cells versus hair stem cells isolated from animals is unjustified, and little more than a shot in the dark. In sum, this is an ambitious proposal to study multiple aspects of the Ras signaling pathway in stem cell biology. A very large number of transgenic mice will be generated, with insufficient justification and inadequate analysis. Each sub-aim is quite superficial. Aim 3 is poorly explained, and significantly detracts from the focus of the other experiments. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant has a good publication record and excellent training. He obtained his M.D. and Ph.D. degrees from Washington University in St. Louis in 1998, where he did graduate work with Stanley Korsmeyer and a research fellowship with Steve Dowdy. He then joined the UCSF Department of Dermatology, where he was promoted to Assistant Professor in Residence in 2004, and simultaneously completed a postdoctoral fellowship with Gail Martin. In 2006, he was appointed Assistant Professor (Step II) in the Department of Dermatology. His publication record has been of good quality (first author publications in Nature and PNAS during his PhD, a first author publication with Dowdy in PNAS during his residency, and one co-first author publication with Martin as a post-doc), but one reviewer pointed out that his productivity has been low, calling into question the applicant’s ability to complete this extremely ambitious proposal. Moreover, the applicant has no prior experience in stem cell biology. The applicant presents his vision as well as a career development plan. He has a KO8, which ends in 2009. Overall, reviewers felt that this was a well-qualified physician investigator with a good track record. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Reviewers agreed that the research environment at UCSD is strong, and their track record is excellent. However, there was disagreement as to the strength of the institutional commitment to this investigator. Some reviewers noted that the institutional letter is exceptionally strong in its support of Dr. Yu and of their stem cell biology program. Dr. Yu was appointed Assistant Professor in 2006 and has been provided additional funds, technical assistance, and post doctoral trainees to supplement his K08 and maintain “an active and productive laboratory for 3 years”. He has protected time with minimal clinical and teaching responsibilities. The institution has a formal mentoring program, which is supplemented by informal interactions and collaborations with several members of the mentoring committee. Another reviewer expressed concerns that the overall level of institutional commitment is more limited than this letter conveys. Although there is some mention of departmental funds for technical help and postdoctoral support, the extent of those funds is not stated, nor is it stated whether these will be provided going forward. The letter states that the applicant has a bay to do experiments in, but it is unclear how much laboratory space has been assigned to his lab and whether this bay is in someone else’s lab. There is also no statement that funds will be available to support the research itself. No mention of salary support is made, nor is there any indication of independent lab space. DISCUSSION: Response to this application was mixed. Reviewers commented that the use of skin as a model system to reactivate organ formation is interesting and creative, and the study concept of organizing centers is very intriguing. The applicant is well-trained, and the institution believes that he is a rising star in cutaneous biology and is thus poised to become a successful physician-scientist. The major weakness cited was that this is an overly ambitious proposal, particularly in Aims 2 and 3 - the teratoma system as a model for organ formation could be its own grant. The applicant's publication record, while satisfactory, is too weak to justify this ambitious plan. In addition, the proposal is not well thought-out and is poorly organized, the genetics are complicated, and the aims are broad and superficially addressed
Conflicts: 

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