A major challenge in the development of regenerative medicine strategies involving human embryonic stem cells (hESCs) remains the routine and reproducible culturing of hESCs in the laboratory without loss of their developmental potential. Outside their natural environment, cell culture conditions in the laboratory represent artificial cellular environments that cause cellular stress and may harm the cell physiology. Most human cells respond to cellular stress conditions by activating mechanisms that are capable of reducing the damage to the cell. However, activating these cellular stress response mechanisms may often also have consequences for the proliferation and developmental capacity of the cells. To response DNA damage, most cells for example inhibit the progression of the cell cycle until the DNA has been repaired. In previous work, our laboratory has characterized the regulatory proteins and mechanism that control stress responses in mammalian immune cells. We are able use computational simulations of these complex regulatory mechanisms to gain insights and direct specific experiments. Here we propose to determine how stress responses are regulated in hESCs. We will make quantitative measurements regarding the presence of the key regulatory proteins and construct a computational model of the regulatory networks as it pertains to hESCs. This allows us to compare different hES cell lines, provide important insights on their physiological regulation, and predict the effects of pharmacological treatments. As the very first molecular and computational description of important regulatory mechanisms, the results will also form the basis for future studies that monitor the molecular changes during tissue development and the role these molecular components play in such tissue development. In addition, we propose to engineer molecular tools that allow us to monitor the molecular stress level in the cell. Engineered proteins inserted into hESCs will be used as sensors of cellular stress. By monitoring their activity we will optimize laboratory methods and techniques related to growing stem cells in the laboratory. These studies may have result in improving current technologies and make a critical step in developing regenerative medicine more reliable and routine.
Statement of Benefit to California:
A major challenge in the development of regenerative medicine strategies involving human embryonic stem cells (hESCs) remains the routine and reproducible culturing of hESCs in the laboratory without loss of their developmental potential. Outside their natural environment, cell culture conditions in the laboratory represent artificial cellular environments that cause cellular stress and may harm the cell physiology. The proposed research is focused on characterizing the molecular stress responses in hESCs. The research will significantly contribute to an understanding of stem cell biology and the molecular mechanism that regulate the capacity for self-renewal and for tissue development. The molecular characterization will contribute to an understanding how different hESC lines differ and what their respective advantages may be. And finally the proposed research will result in improved laboratory methodologies for the handling of hESCs. The research will benefit the State of California and its citizens by (1) enhancing the scientific knowledge base of the molecular mechanisms that control hESC physiology and developmental capacity (2) improving the methodologies for culturing and manipulating hESCs thereby increasing the likelihood of successful of regenerative medicine strategies (3) making California a leader in stem cell systems biology by constructing a computational model that allows for virtual cell studies by simulations (4) training a junior PI and two talented postdoctoral fellows with proven scientific track records in stem cell biology, enabling them to contribute to Regenerative Medicine in their own future laboratories.
SYNOPSIS: The PI proposes a look at NFkB and p53 signaling in HES cells with a goal of improving the reproducibility of culturing them. The specific aims are to (1) (Broadly) characterize NFkB signaling in several HES lines, in response to inflammatory, cytokine and mechanical manipulation, and to characterize the role of NFkB in HES senescence and proliferation (2) Characterize the signaling components related to p53 in response to metabolic and genotoxic stress, and study the role of p53 in self-renewal and (3)Construct lines that have reporters for monitoring RelA and p53 activity in HES, and use these lines to examine how cell culture procedures and feeder layers trigger stress responses. Then these lines will also be used in a small molecule screen to find products that reduce the HES cell stress in culture. SIGNIFICANCE AND INNOVATION: NFkB biology has been relatively unstudied in HES cells and is certainly going to be important, and the PI has terrific background and potential for this study. Bringing a mathematician or modeler into the stem cell field would have made the grant much more significant. In this proposal, the investigator argues that outside of their natural environment hESCs have cellular stressed. As there is a link between cellular stress responses and transcription factors, the control, at least in part, is by the transcription factors p53 and NF-kB. The author suggests investigation of behavior of these factors in hESCs. He proposes three specific aims. One is the characterization of the NF-kB signaling system in hESCs. The second is to characterize the p53 signaling system in hESCs. I’m not sure due to a lack of a verb in the first sentence of the aim (on pages 2, 7, 9, and the abstract page), but I think that the third specific aim involves improving the condition of cultures of hESCs based on the knowledge gained from their state of stress. All of these aims could have been done using registry lines, and therefore could be funded by the NIH. There are no innovative approaches in this proposal. The significance is based on a possible connection between the level of stress and differentiation. STRENGTHS: The major strength of the proposal is the background of the PI in NFkB signaling. This is a leader in the field who has done great work establishing himself in the NF-kB pathway. The experiments are well designed and controlled. And clearly, the knowledge of the status of activation of NF-kB and p53 are important information necessary to our in-depth understanding of hESCs. Aim 3 has the potential to develop lines that would be broadly useful to HES cell biologists, since NFkB is at the nexus of so many cellular stress responses. WEAKNESSES: The grant is too unfocused, even though NFkB is at its center. The first aims are really a transfer of ongoing work in the lab, going on in other cell lines. The success of the NFkB work done by the PI to date may well depend in part on the homogeneity of the cells used for the studies, and there is no acknowledgement that the highly heterogeneous nature of cells (within a single HES line) may make the kind of data generation done in other cells more difficult. In aim 3, it is clear that culture conditions are stressful to cells, part of why they senesce in culture as they do. Here there would be a great opportunity to lower stress by practical means as well as doing a small molecule screen. Which library is going to be used? Aside from the fact that it is technically challenging to stably transfect hescs the way he proposes, it is important to remember that p53 and NF-kB might have additional or independent functions other than a readout of stress for hESCs. It is not clear that the reporters will behave appropriately, or what the information would be in the context of hESCs. Progress in model building in these computational models of biologic functions requires an ongoing dialogue between the mathematicians and the biologists. Bringing a mathematician from California into the stem cell fold would have been a big boon, especially in the spirit of the seed grant RFP. Instead, the models will be built up by biologists (presumably in consultation with the mathematician at Hopkins, Levchenko but this is not stated in the application). The whole point of the computational models, acknowledged by the PI, is that they should be used to help guide experiments, and there is absolutely no indication how the models will be used to guide experiments in the proposal. Based on the explanation provided in specific aim 1, there is little to indicate that the computational modeling of NF-kB signaling will have value in our understanding of the pathway in hESCs outside of theoretical possibilities. There is the assumption by the PI that the kinds of stresses imposed on other cells (such as some of the immune cells he has worked on) will be operative in HES cells. Are toll-like receptors present on undifferentiated stem cells to interact with LPS? Are TNF receptors present on HES cells to interact with TNF as a stressor. The answers are certainly in the literature, and some effort to find them to support the work in HES cells, would have shown more interest in HES cell biology, rather than a simple transfer of experiments from one cell type to another. NFkB biology has been relatively unstudied in HES cells and is certainly going to be important, and the PI has terrific background and potential for this study. However, the grant is unfocused, some critical research designs are too vague, and there is some considerable overlap with other work (just differing the cell type). DISCUSSION: Whereas the one reviewer recommended removing aim 3, another reviewer felt that the application would have been much stronger if it had been focused only on aim 3.