| Given appropriate conditions, stem cells can self-renew for long periods of time while maintaining the ability to differentiate into various functional cell types in the body. It is these characteristics that not only make stem cells a useful system in which to study tissue and organ development, but also give them great potential for regenerative medicine. Given the success of well-practiced cell-based therapies (e.g., hematopoietic stem cell transplantation for treating hematological diseases and pancreatic islet cell transplantation for type I diabetes), it is conceivable that this approach could be applied to many other serious medical conditions where cells are lost because of disease, injury, or aging. Whether they are cell-based or stimulation of the body's own repair, it is clear that a better understanding of stem cell biology is required in order for these approaches to be realized.;Herein, several strategies that aim to uncover regulators of cardiac development are reported. In particular, the first protocol for monolayer, serum-free differentiation of ESCs into cardiomyocytes is described. Using this protocol, several signaling pathways are found to be of significance to cardiac development. Our results indicate that BMP and Wnt signaling sit high in the order of mesodermal and subsequent cardiac specification and that inhibition of Wnt signaling is important in later stages of differentiation. Previously, BIO and BMP-4 have been reported to stimulate self-renewal of mESC's. In this study, a novel cardiac mesoderm induction effect of BIO on ESC's was identified, and a strong positive role for BMP-4 in cardiac mesoderm formation was observed. Additionally, by conducting a small scale screen using our defined cardiac differentiation protocol, we demonstrate a positive role for calcium/cAMP signaling in the specification of cardiogenic mesoderm, and a positive role for inhibition of TGF-beta signaling in the specification of true cardiac cells from mesodermal cell types. While calcium/cAMP signaling is important in various differentiation programs, its particular importance in the specification of cardiogenic mesoderm was previously unknown. And while TGF-beta has previously been reported to positively impact mesodermal fate specification, its inhibition aiding in specification of cardiac cells from mesoderm was unknown.;While monolayer conditions for the differentiation of neural1 and endoderm2 derivatives have been described, to our knowledge, similar success has yet to be achieved in cardiac differentiation. Use of a similar basal differentiation condition should facilitate comparisons of different ESC derived populations.;A high-throughput screen of primary adult human beta cells for molecules that can induce proliferation is also described. A new serum-free culture condition that maintains beta cells better than previous conditions was formulated and used for screening. Several unknown molecules are described that induce expression of proliferative markers, as well as several known molecules. In particular, a positive role for inhibition of P38MAPK signaling and the importance of MEK/ERK signaling in beta-cell cycle regulation are demonstrated.;Small molecules have long been associated with biological discoveries. Our understanding of biological processes often develops from discovering or designing ways to perturb a given process and observing the effects of the perturbation. Although genetic approaches have been widely used for this purpose, the small-molecule approach clearly offers some distinct advantages. Small molecules provide a high degree of temporal control over protein function, which generally acts within minutes or even seconds, and their effects are often reversible, which facilitates both rapid inhibition and activation. Their effect can also be finely tuned by varying concentrations of the compound of interest. Moreover, because of the inherent difficulty of genetic manipulation for many types of stem cells (e.g., low transfection efficiency or poor clonal expansion), small-molecule tools are especially useful for the stem cell field.;Lastly, a method for the direct reprogramming of fibroblasts to a cardiac fate is described. Using specific combinations of virally overexpressed transgenes as well as growth factors and small molecules uncovered in our differentiation studies, efficient cardiac conversion can be achieved. Though the overexpressed genes are often associated with their role in pluripotency, we demonstrate that the conversion does not progress through a pluripotent intermediate. |
