Basic Biology II
$1 447 956
A major challenge facing all stem cell therapies is regulating the behavior of stem cells – their survival, proliferation, self-renewal and differentiation – to regenerate the desired cell types and tissues. The success of these therapies depends on understanding how extracellular signals, such as growth factors, present in the stem cell niche regulate stem cell behavior through the activation of cell surface receptors. The receptor Kit is present on the surface of multiple human stem cell types, both pluripotent (embryonic and induced pluripotent) and adult (hematopoietic, cardiac and neural) as well as certain mature cell types. In addition, Kit is required for the maintenance of these cells. Our work will address the following question: how does a receptor that is expressed in stem and non-stem cells, function specifically in the context of stem cells to regulate self-renewal and survival? We hypothesize Kit achieves context-specific functioning by interacting with and activating other cell surface receptors, each of which is specific to a different stem cell type. Our previous work, combining bioinformatics predictions with experimental validation, has already identified IL-4R as a novel Kit-activated receptor in hematopoietic stem cells. Our proposed research will first extend this work to find similar Kit-activated receptors in the other stem cell types. To understand the significance of the above Kit-activated receptors, we will compare normal stem cells to those lacking one of these receptors and evaluate the contribution of Kit as well as each Kit-activated receptor to stem cell function. Our final objective is to study the role of microRNAs, a class of small RNA molecules, in the mechanisms by which Kit activation elicits changes in stem cell behavior. A rapidly growing body of evidence suggests that microRNAs play key roles in regulating cellular differentiation but their role in stem cell biology remains unclear. The research outlined in this proposal is aimed at uncovering a novel paradigm for context-specific activity of the pan-stem cell receptor Kit in multiple human stem cell types. In addition to investigating the mechanisms underlying Kit activity, our work may suggest new stem cell-targeted therapies using combinations of growth factors, that exploit the potential synergy between Kit and the Kit-activated receptors.
Statement of Benefit to California:
The goal of this proposal is to investigate novel mechanisms of action of the stem cell receptor Kit, which is present on embryonic and multiple tissue-specific adult stem cells and known to play a central role in regulating survival, proliferation and self-renewal. Our research is likely to both advance our knowledge of mechanisms underlying stem cell biology and motivate the development of novel stem cell-targeted therapies. The natural environment of stem cells within different tissues in the body provide a variety of cues in the form of growth factors to promote the survival and self-renewal of stem cells and, when needed, their proliferation and differentiation into various types of mature cells. The successful clinical use of human stem cells requires the ability to maintain and grow these cells, and to stimulate them to develop into the desired tissues. This requires a thorough understanding of the mechanisms by which stem cells respond to extracellular cues, which is the focus of our current and proposed work. We seek to uncover novel interactions between Kit and other stem cell receptors and determine the role of these interactions in regulating stem cell behavior. Our scientific method based on combining bioinformatics with experimental validation, has proven to be much faster than conventional approaches, having accelerated by several years our finding of a novel interaction between Kit and the IL-4 receptor in bone marrow stem cells. Our results are likely to inform novel stem cell-targeted therapies. Currently, administration of Kit ligand, also known as Stem Cell Factor, at doses necessary to stimulate bone marrow stem cells results in severe adverse effects due to the action of Kit on certain non-stem cells. Our findings on interactions between Kit and other stem cell receptors may provide a basis for combination therapies that utilize much lower doses of Kit ligand together with other growth factors to specifically target the desired stem cells while diminishing the adverse effects.
EXECUTIVE SUMMARY The applicant proposes to determine how Kit, a receptor expressed in several different stem cell types, provides cell-type specific signaling affecting stem cell function and fate. Kit signaling plays an important role in stem cell biology by controlling cell survival and proliferation. The proposed work pursues the hypothesis that cell type-specific signaling by Kit is achieved through interaction with and activation of cell type-specific co-receptors, named Kit-activated receptors. In Aim 1, the applicant will extend his/her current bioinformatics-based approach to computationally predict and then experimentally validate novel Kit-activated receptors and their signaling pathways in five human stem cell populations (embryonic, induced pluripotent, hematopoietic, cardiac, and neural). In Aim 2, the applicant proposes to determine the transcriptional outputs for specific responses to Kit and Kit-activated receptors. In Aim 3, the applicant will identify microRNAs (miRNAs) that regulate downstream Kit signaling. The reviewers found the proposed use of bioinformatics coupled with solid biological validation to be innovative and creative. They felt this proposal addressed an important problem, and studying it may lead to a better understanding of stem cell biology. The reviewers also stated the aims were logical and they appreciated the mechanistic focus. Despite these positive assessments, the reviewers raised several concerns about the overall scope of the proposal and its potential for impact. The review panel believed the applicant was overly ambitious in attempting to investigate too many cell types, raising concern that none of the identified pathways would be studied in enough depth to reach solid conclusions. Although the underlying hypothesis is based on convincing preliminary data that Kit employs the IL-4 receptor in hematopoietic progenitors to mediate Kit responses, the study’s potential impact was hard to gauge because limited evidence was presented to suggest this kind of mechanism would be generally relevant to Kit function in stem cells. The reviewers also thought Aim 3 was not needed and not well substantiated, and contributed to the proposal’s over-reaching nature. Several concerns were raised about the preliminary data and experimental design. Reviewers commented that some preliminary data were convincing, but other preliminary data as presented did not strongly support the claims. For instance, reviewers were unable to see how the provided clustering data supported the bioinformatics-based discovery of Kit and IL-4 receptor co-expression and by extension the proposed underlying discovery technology. Furthermore, results for the gene expression response to Kit ligand were described but not provided, and hence could not be evaluated. Questions regarding the purity of the stem cell populations to be studied constrained the reviewers’ enthusiasm for the quality of the gene expression profiles that may be obtained. Furthermore, the applicant does not address the possibility that Kit ligand may cross-react with the newly identified receptors but rather assumes a trans-activation mechanism, and a reviewer suggested several experiments to address this possibility. Other potential pitfalls were not adequately addressed in this proposal. The applicant is highly trained, productive, and well funded and commits an appropriate amount of time to this project. Reviewers also praised the proposed collaborations and research team, and they considered the facilities among the best. In summary, reviewers appreciated the proposal’s innovative bioinformatics-based predictive approach coupled with experimental biology, the applicant’s expertise, and the research team; however, the proposal’s over-reaching scope and concerns about the preliminary data dampened reviewers’ enthusiasm.