Derivation of hES Cells in Novel Key Regulators of Cell Survival and Growth
Human ES (hES) cells have the dual abilities to self-renew and differentiate making them powerful tools to study and treat diseases. Normal hES cells are a renewable source of differentiated cells to use in cell-based therapies. Disease-specific hES cells lines are powerful tools to study disease etiology and test disease treatments. Less than 30% of embryos give rise to hES cell lines. Given that embryos, particularly disease-specific embryos, are scarce and cells grown in the absence of key growth factors often accumulate genetic changes, improvements in the method used to make hES cell lines would be beneficial. This project will fill vital gaps in our knowledge about key signaling factors in the growth of hES cells and improve the method used to isolate and maintain hES cell lines. We previously demonstrated that neurotrophins (NTs) are potent survival factors for hES cells. Our preliminary data suggest that NT receptors are expressed in the inner cell mass (ICM) cells of the human blastocyst-stage embryo, the source of hES cells. We propose to test the ability of NTs to improve the survival of ICM cells and increase the efficiency of making new hES cell lines. The hypothesis is that NTs are important for embryo growth and will improve the efficiency of deriving new hES cell lines. The growth of hES cells depends on a balance of signals acting through multiple signaling pathways. We propose to fill the gaps in our knowledge of the pathways that control hES cell survival, proliferation, and differentiation. We established a system to perform a high-throughtput screen in hES cells to identify factors that affect hES cell survival, proliferation and differentiation. We propose to screen a high content siRNA library to identify kinases and phosphatases that affect hES cell growth. The hypothesis is that high-throughput screening can identify key regulatory pathways controlling hES cell survival, proliferation, or differentiation. The target molecules identified in Aim 2 are druggable. We propose to use those drugs (agonists or antagonists) to determine their effect on hES cell growth and stability. Genetic stability will be assayed by comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) analysis. Drugs that improve the growth and stability of hES cells will then be used to isolate new hES cell lines and the efficiency compared to that of current methods. The hypothesis is that drugs that target kinases and phosphatases identified in Aim 2 as key regulators of hES cell growth will improve the efficiency of making new hES cell lines and ensure their genetic stability. The results of this study will improve the ability to establish hES cell models of human diseases, even those for which the disease-specific embryos are scarce. Further, the improved culture conditions will reduce the probability of accumulating genetic changes that can occur when cells grow in adverse conditions.
Stem cell biology holds tremendous promise to decipher and treat human disease. Human ES (hES) cells have the dual abilities to self-renew and differentiate making them powerful tools to study and treat diseases. Normal hES cells are a renewable source of differentiated cells to use in cell-based therapies or drug screens. Disease-specific hES cells lines are powerful tools to study disease etiology and test disease treatments. Several roadblocks remain before the full potential of hES cells can be realized. These include inefficiency in establishing new hES cell lines and their poor growth. Only about 30% of embryos give rise to hES cell lines. Given that embryos, particularly disease-specific embryos, are scarce and growth in the absence of key growth factors can contribute to genetic instability, improvements in the method used to make hES cell lines would be beneficial. The studies proposed here will fill vital gaps in our knowledge of the key factors for optimal growth of hES cells and improve our ability to isolate genetically stable hES cell lines. Improvements in the efficiency of establishing hES cell lines and their quality will allow us to establish models of a variety of human diseases, even those for which the disease-specific embryos are scarce. These models of human disease can be used to study the disease pathology and will eventually yield effective treatments. Human diseases have a detrimental effect on personal freedom and earning power of the patients and create a financial burden for their families and California. Knowledge gained from these studies could aid in developing therapies that might benefit many people.