Basic Biology V
$1 111 200
Recommended if funds allow
Human embryonic stem cells (hESCs) can be maintained in culture indefinitely while retaining the capacity to generate any cell type of the body, therefore offering a potentially renewable source of cells for cell replacement therapy applications . hESCs also represent a platform for addressing some fundamental questions in basic biology, such as how stem cells retain the ability to produce more of themselves and how they give rise to more specialized cells. If the full potential of hESCs in both research and clinical application is to be realized, a greater understanding of the regulation of their fate is critical. Recently, we developed a new culture medium that allows us to efficiently propagate hESCs without loss of their potential to produce specialized cells. The key components in this culture medium are two small molecules that can modulate the function of β-catenin, a protein important for many cell functions. The main goal of this project is to understand how modulation of β-catenin’s function can control the fate of hESCs. Our study will be important not only for optimizing culture conditions for efficient and unlimited expansion of hESCs but also for manipulating and controlling the generation of specific cell lineages for use in regenerative medicine. Given that β-catenin is also a key player in the pathogenesis of diverse human cancers, our research is likely to identify novel β-catenin targets that could provide leads for anticancer drug development.
Statement of Benefit to California:
Currently, there is no cure or effective treatment for Parkinson’s disease, spinal cord injury, diabetes, cancers, and other pathological conditions. Many patients in California suffering from these afflictions could benefit from therapies using cells derived from human embryonic stem cells, which can give rise to any type of endogenous cell in the body. To realize clinical application of human embryonic stem cells or their derivatives, we must learn how to control the expansion of human embryonic stem cells, and more importantly, how to control the differentiation of human embryonic stem cells into specific cell types. Currently, human embryonic stem cells are routinely propagated on feeder cell layers, in medium containing serum or serum replacements whose components are incompletely defined. Consequently, the mechanism underlying human embryonic stem cell propagation and differentiation is still poorly understood. We recently developed new conditions that allow us to efficiently control the expansion and differentiation of human embryonic stem cells. In this study, we will further investigate how human embryonic stem cell fate is controlled under these conditions. Our study will be important not only for optimizing culture conditions for efficient and unlimited expansion of human embryonic stem cells but also for manipulating and controlling the generation of specific cell lineages for use in regenerative medicine.
This Fundamental Mechanisms application reports a preliminary discovery of a new culture medium enabling efficient propagation of human embryonic stem cells (hESCs) without loss of their potential to differentiate into specialized cell types. A key component of this medium is a cocktail of small molecules that modulates the function of beta-catenin, a protein with important roles in many cellular processes. Based on these observations, the applicant proposes three aims to investigate the molecular mechanisms through which modulation of beta-catenin function controls the fate of hESCs. In the first two aims, the applicant will explore the biology of beta-catenin in hESCs and how its subcellular localization impacts the self-renewal properties of these cells. In the third aim, the applicant will focus on molecular pathways through which beta-catenin promotes hESC differentiation. Significance and Innovation -Reviewers generally agreed that improving hESC growth performance is an important goal for the field. Development of a small molecule cocktail that increases cloning efficiency of undifferentiated hESCs would be significant. -Details of the pathways to be studied have been extensively described in other tissues and cell types, and thus new insights to be obtained about beta-catenin mechanisms may prove incremental rather than transformative. - Investigating the proposed mechanisms in human cells adds value, as much of the prior work has been conducted in the murine system. Feasibility and Experimental Design - Reviewers acknowledged promising preliminary data in support of the hypotheses to be tested, although their confidence for supporting a project of this scale would have been greater if the data had been peer-reviewed and published. -Many of the proposed experiments are open-ended in nature, with few details provided for how potentially large numbers of newly identified genes would be prioritized and validated. - Some reviewers believed that instead of proposing large-scale screens within Aims 2 and 3, the applicant should focus more of the investigation around numerous factors that have previously been identified as components or targets of beta-catenin and its associated pathways, and for which a wealth of information is available in the literature. - The application does not consider investigating additional, potentially beneficial effects of the proposed culture conditions, such as improved karyotype stability. Principal Investigator (PI) and Research Team - The PI has made key contributions to field of stem cell biology and has published extensively in the field. A strong letter of support from an institutional Chair has been included. -The team assembled can perform all the experiments in this proposal. Responsiveness to the RFA - The work proposed will make extensive use of hESCs and is appropriate for the RFA.