The stem cell microenvironment in the maintenance of pluripotency and reprogramming
Pluripotent stem cell research is just on the verge of beginning to fulfill its promise to revolutionize medicine. Whether they are derived from embryos, or from adult cells that have been reprogrammed, human pluripotent stem cells can be propagated indefinitely in the laboratory and can turn into a wide range of mature cell types, providing an renewable source of a wide range of types of human tissue for research or therapy. We are still learning about the best ways to grow and manipulate pluripotent stem cells, and how best to reprogram adult cells to the pluripotent state. In fact, our concept of what a pluripotent stem cell is and what it looks like is still emerging. Recent work has shown that pluripotent stem cells, in the embryo or in the laboratory, are not simply homogeneous monocultures. Rather, stem cell cultures are complex and highly dynamic ecosystems. They contain a spectrum of cell types, from the most primitive cells, to cells that are already well on the way to becoming particular specialized types of cell. Different subpopulations of cells within these ecosystems communicate with one another, and these interactions dictate cell behavior. Cells even produce a type of scaffold on which they grow, called the extracellular matrix, that helps guide its fate. Thus, the microenvironment of the stem cell-its neighbors, and the signaling molecules they produce, is a critical component to guiding the cells fate. Our understanding of this microenvironment is however still rudimentary. This project will study two key processes-stem cell renewal, or the means by which stem cells divide to produce more stem cells, and specification, or how stem cells chose to begin to specialize into more mature cell types. We will look at the regulation of these processes at very high resolution, to see how individual cells within the various subpopulations respond to signals around them, and to identify the critical signals that maintain stem cells in the primitive unspecialized state or help adult cells to become reprogrammed. By carefully dissecting the stem cell population, and identifying its various subcompartments, we will provide critical information for other scientists that will enable them to study stem cell regulation at a much more refined level. And, an enhanced knowledge of the signaling systems cells use to talk to one another will help us to propagate stem cells and to enhance the reprogramming of adult cells. All of these fundamental discoveries will facilitate work towards the application of pluripotent stem cells in medicine.
Over the past ten years there has been remarkable progress in human embryonic stem cell research, and much of this progress has been driven in recent times by the California Proposition 71 initiative. Advances in our understanding of stem cell growth and differentiation, the approach of the first clinical trials of human embryonic stem cell derived products, and the remarkable discovery of the reprogramming of adult cells to the pluripotent state, have raised the prospect that this research will soon begin to fulfill its promise to revolutionize medicine. California can take a leading role internationally in this process not only by accelerating the progress of basic discoveries to the clinic, but also by building the intellectual infrastructure for the next decade of discoveries in stem cell research. This proposal is based on a new framework to understand the structure of pluripotent stem cell hierarchies, to unlock the fundamental principles underlying the interaction of pluripotent stem cells with their immediate environment, and to discover how these interactions decide stem cell fate. The results will lead to concrete outcomes in terms of products and processes for stem cell manipulation in research and biotechnology, and they will provide enhanced means for the derivation and propagation of embryonic stem cells and pluripotent stem cells from adult tissues. The basic discoveries will also strengthen the intellectual basis of stem cell research in the State, and the project will provide outstanding training opportunities for California scientists.
Pluripotent human stem cell lines can develop into any cell type in the body. In order to exploit the power of these cells for use in research or therapy, we need to be able to grow them in the laboratory in pure form and efficiently turn them into specific types of mature cell. Current methods for growing pluripotent stem cells yield mixed populations of cells, some of which have limited capacity for growth and development, others that represent true pluripotent stem cells. Thus, the stem cell cultures are like an ecosystem, with many components. We are studying the culture microenvironment of the stem cell to learn how it regulates cell growth. In order to do this we have to understand the heterogeneity in stem cell cultures. To this end, we have developed techniques to analyze the molecular makeup of single stem cells, and to track their fates. These techniques enable us to identify the most primitive cells in the culture with the greatest potential for growth and differentiation. These cells tend to be spatially segregated from other more mature cells, in a specialized microenvironment that helps them to maintain their ability to grow and turn into specialized cells. We have identified some key components of the microenvironment that keep stem cells in the primitive pluripotent state. We will use these techniques to develop means to produce pure populations of specialized cells from stem cell cultures.
Our aim is to gain a detailed understanding of how human embryonic stem cells are regulated- how do embryonic stem cells decide whether to multiply to produce more stem cells, or to begin forming specialized cell types. We have found that human embryonic stem cultures are not homogeneous but are composed of different cellular subpopulations whose identities can be clearly defined at the molecular level. Only a minority of cells in the population has the capacity for self renewal, the ability to form new stem cells. This ability to divide to produce new stem cells depends on factors made by the stem cells themselves. Other cells in the culture have begun the process of specialization, with many on the way to becoming precursors of the central nervous system. Again, the choice to become a nerve cell depends on signals from surrounding cells in the culture. Understanding the conversations between subsets of stem cells is crucial to efforts to grow pure populations of stem cells or specialized cell types.
Human pluripotent stem cells are defined by their abilities to multiply indefinitely in the laboratory and to turn into any type of body cell. However, stem cell cultures are not composed of one cell type, but are heterogeneous. In this study, we took a close up look at human embryonic stem cell cultures by examining the properties of individual single cells, rather than studying the population as a whole. The result show that the population of cells in the culture dish comprises a complex hierarchy, from primitive cells with great capacity for multiplication through to cells just on the verge of becoming specialized cell types. Specific signals govern how cells at different stages of this hierarchy behave: whether they divide to form more stem cells, or begin to specialize into different cell types such as nerve cells. The cells themselves produce signals that govern how they move through this hierarchy. By understanding these internal dialogues between different populations in stem cell culture, we can learn how to control stem cell behaviour, and to understand why some stem cell lines respond differently to others under specific culture conditions. In turn, this knowledge will enhance our ability to propagate stem cells and to turn them into particular cell types useful in research or regenerative medicine.