The potential of human embryonic stem cells (hESC) to be used to treat devastating diseases has raised a significant level of expectation among the scientific community and patient groups as well. hESC are pluripotent cells, that is these cells have the potential to differentiate to any cell type and to self-renew. hESC cells are presently viewed as promising cell sources not only for disease treatment, but for regenerative medicine. Nonetheless, there are ethical and political questions about the use of human embryos in medical research that have made international news headlines and shifted some of the focus from the promise of the research to the legal battles that are presently being waged. As the field of hESC research matures, it is being realized that many areas of investigation need greater emphasis to develop and understand the true potential of hESC and their capacity to be used in disease treatment and regenerative medicine. Importantly, integrins are a class of functional proteins found on the cell surface of hESC, and many other cell as well, that are involved in cell-cell and cell-surrounding tissue (the extracellular matrix, ECM) communication that control important cellular process including cell division and cell shape determination. The integrin class of proteins has been greatly understudied in the hESC field. Integrins are closely linked to processes involved in cell migration and differentiation. hESC need to be characterized in terms of their integrin content and the role of integrins in hESC differentiation and pathway determination and this is the focus of the present application. We have been studying integrins on cancer cells for a number of years and now wish to apply our expertise to the study of hESC integrins and their interactions with the ECM. We have used proteins called disintegrins, which are integrin antagonists, to block integrin function. We will utilize the disintegrins and several other techniques designed to block integrin function or to block their intracellular production. These techniques will enable us to determine the importance of integrins to hESC proliferation and fate determination. Further, our studies will help to develop procedures that could control hESC cell fate determination. Thus, by appropriate blockage of integrin pathways, we could alter the fate of hESC by forcing the cells to differentiate, for example, into a muscle cell or a brain cell. As a specific illustration we will induce the generation of blood vessels by hESC using different ECM matrices and determine integrin involvement in this process by selective use of integrin antagonists. Previous work with mouse ESC showed an integrin-antagonist of a specific integrin known to be important to the process of vessel formation, inhibited this process. Thus, integrins apparently play important roles in cell fate determination and we seek to be able to selectively control integrin function, thereby controlling cell fate determination.
Statement of Benefit to California:
A significant number of Californians have family members suffering from serious medical conditions that can potentially be addressed by stem cell therapies. These conditions include cancer, diabetes, heart disease, Alzheimer’s, Parkinson’s, spinal cord injuries, along with nearly 100 other diseases and conditions. Recent advances in medicine have addressed the abilities of stem cells to potentially treat these conditions. Californians deserve the best possible medical care, and the federal government’s reluctance to fund the vital hESC research limits advances in this important area of medical science. The research proposed here would provide the medical community with mechanisms to control cell differentiation and force the hESC along the desired differentiation pathway. This would represent a significant breakthrough for cell based therapies for a specific disease or for regenerative medicine based procedures. The research proposed in this application is directed to identifying cell surface proteins that are involved in hESC propagation and differentiation; the studies we propose should lead to an understanding of the role of integrins in hESC proliferation and fate determination. Since this may ultimately lead to the ability to control cell fate by turning off or antagonizing a specific subset of integrins, we may be able to direct cell fate determination, that is control hESC differentiation. The integrin receptors on hESC surfaces provide adhesive ability and migration potential to the cells, as well as being involved in a number of important signal transduction pathways. When the ability of these surface proteins to bind to external factors is modulated, there is a dramatic effect on cell proliferation and differentiation potential. The ability to modulate these proteins and thereby control or alter the fate of hESC would be a powerful tool in using stem cells as therapeutics for people in the State of California with any of the diseases enumerated above. The ability to modulate integrin function would be most beneficial when advancing therapies for specific diseases or developing regenerative medicine procedures, and would provide physicians the capacity to specifically control the differentiation fate of hESC being employed for these procedures.
Integrins are known to be responsible for cell adhesion. However, over the last decade it has become clear that engagement of integrins also plays a role in biological processes (e.g. differentiation). Selective integrin antagonism, using either integrin antibodies, disintegrins, DDs or small inhibitory RNA (siRNA), will enable us to control or modulate hESC proliferation and differentiation pathways and to determine the importance of integrins in hESC proliferation and differentiation. Studies in mice suggest an important role for integrins in hESC proliferation and differentiation. Here the investigators will evaluate the role of integrins in hESC prolifertaion and differentiation, specifically targeting disintegrins (physiological integrin antagonists). SIGNIFICANCE AND INNOVATION: The interaction of stem cells with their environmental niche is pivotal in the control of their grafting, renewal, and differentiation. The focus of this proposal on integrins is timely and the approach is systematic and entirely feasible. The studies are likely to yield valuable information, and could result in the identification of specific integrins that drive the differentiation of human ES cells into specific differentiated progeny. Also, if cell type restricted integrins are uncovered, it may allow direct FACS sorting as these are surface determinants and antibodies exist for a subset of integrins that could be examined. Obtaining insights in signals that are important for ES proliferation and differentiation is of key importance. Aside from cytokines and morphogens, it is highly likely that signaling as a result of adhesion to specific extracelllular matrices will affect the fate of ESCs. Hence, systematic evaluation of the role of interaction with ECM may yield important new insights in how to manipulate the proliferation and differentiation of hESC. STRENGTHS: This strength of the proposal is that it combines investigators with extensive expertise in integrin and disintegrin moecules, the role of adhesion on endothelial differentiation, and investigators with knowledge of siRNA inhibition of gene expression. The hypothesis to be pursued is novel, and the methods to be used are relatively straigthtforward. Specific strengths include: 1) Experienced investigator with solid track record in the integrin field and access to a well-characerized set of reagents and protocols 2) Preliminary data with ES cells that document the roles of integrins in key steps of the differentiation process 3) Known role of integrins in the formation of the vasculature in general and endothelial lineages in particular 4) Interactive, strong environment for human ES cell work is being established and access to expertise on site appears well in hand. WEAKNESSES: Reviewer one: 1. A number of viral transduction steps are proposed, although the investigators do not acknowledge potential problems of silencing of viral vectors upon cell differentiation. 2. Due to non-specific nature of the differentiation process via EBs, it is not at all certain that inhibition of differentiation as a result of elimination of a given disintegrin will demonstrate that this molecule is important for endothelial differentiation, as it could eliminate an accessory cell that for instance secretes cytokines that allow differentiation of ESCs to endothelial cells. Hence, unless a more pure system for differentiation is employed wherein endothelial progenitors are highly enriched, this reviewer does not believe that data will be interpretable. Reviewer two: 1) The ability to purify specific subset of differentiated progeny from the human ES cells could enhance the proposal. 2) Not clear if cell autonomous versus non-cell autonomous roles can be dissected with the strategy being employed. Secondary effects are highly likely. DISCUSSION: This is a molecule-driven proposal in which the PI is seeking funding to study his molecule of interest (integrins). The work has value in that integrins are likely to play an important role in ESC biology and would have a potential practical application in that individual integrin 'signatures' would allow the development of methods of purifying cells. Although of potential interest, the proposal is a flawed one that is outside the fundable range. The issue of not being able to dissect cell autonomous versus non-autonomous effects is an important one that is not adequately addressed. A more pure system and more cell-specific manipulations may be required.