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.
The goal of this effort is to elucidate the role of extrinsic factors in the human embryonic stem cell (hESC) microenvironment in the regulation of pluripotency. Specifically, the proposal is based on the fundamental observation that hESC cultures are heterogeneous as well as the applicant’s own research suggesting that only a subset of the hESC population, referred to as primordial embryonic stem cells (pESCs), fully expresses a pluripotent phenotype. In Aim 1, the applicant proposes to characterize this pESC population, as well as “less pluripotent” hESC populations, by gene and protein expression profiling. For the second Aim, the applicant will examine his/her hypothesis that the hESC microenvironment is crucial in determining cell fate by disrupting the pESC niche and attempting to reconstitute it with various combinations of hESCs as defined by the cell surface markers that were identified in Aim 1. Finally, the applicant proposes to identify key factors secreted by the stem cell niche to maintain pluripotency. In all three aims, induced pluripotent stem cells (iPSCs) will be studied in parallel to address similar questions about the role of the microenvironment in reprogramming and iPSC pluripotency.
Reviewers agreed that this proposal addresses a major unsolved problem and could have a significant impact on the field of stem cell biology. The nature of the microenvironment and the identity of the component molecules that contribute to the extrinsic regulation of pluripotency are poorly understood. Greater knowledge of these extrinsic cues would lead to improved methodologies to maintain pluripotent stem cell lines and may also improve the efficiency of iPSC reprogramming. Reviewers found the proposal to be highly innovative, both in its experimental approaches and the problem addressed.
Reviewers praised the research plan and its sound rationale, based on logical hypotheses explored through well-formulated specific aims. They appreciated the strong preliminary data, noting that it supports the proposal’s central hypothesis about hESC culture heterogeneity and the existence of pESCs. An important aspect of the preliminary data is that it demonstrates successful application of real-time PCR analysis to single ESCs defined by their spatial location or phenotype, as this technology is crucial to the success of the proposal. However, one reviewer would have been more confident if the applicant had included data to define the colony-forming efficiency of each population. Reviewers were particularly enthusiastic about Aim 1, focused on defining pESCs that are proposed to sit atop the ESC hierarchy. Powerful technologies for interrogating gene and protein expression will be used and these experiments should generate important data. One reviewer also appreciated that these techniques would also be applied to iPSCs, although another was uncertain as to how these studies would be designed. One reviewer raised concerns about the feasibility of Aim 3, noting that it represents a considerable amount of work given the broad range of factors and pathways that will be examined. In addition, there was some concern that changes in cell surface protein expression may be a consequence of perturbed signaling pathways and may not be coupled to changes in the pluripotency of the cell. Overall, reviewers considered the feasibility of the project to be high.
Reviewers praised the applicant as a leader in the stem cell field with a long track record studying the biology of hESCs. They appreciated the enlistment of collaborators, both in academia and industry, with expertise in cutting edge technologies for single-cell gene profiling and live cell imaging. The reviewers judged the research team and resources to be excellent.
Overall, reviewers were extremely enthusiastic about this proposal. They emphasized that it addresses a significant but underappreciated scientific problem using innovative technologies and that the logical research plan is supported by strong preliminary data.