Investigation of the molecular pathways that integrate self-renewal and cell structure in embryonic stem cells.
New Faculty I
Human embryonic stem cells (hESCs) can give rise to virtually all types of specialized adult cells in human body. Thus they are thought to be the potential source of the cell-transplantation therapy for the treatment of diseases such as Parkinson's disease, myocardial infarction, and diabetes mellitus. While hESC research has made a considerable progress in developing methods to generate specialized cell types including neurons, cardiomyocytes, and insulin-secreting cells, studies to identify the key mechanisms that allow hESCs to maintain such an enormous differentiation capacity (pluripotency) have just begun. Because obtaining the pure undifferentiated cell population establishes the basis of hESCs-based therapeutic approach, these studies are essential to find the way to grow hESCs without losing their pluripotency. The goal of the proposed study is to determine the core mechanism to maintain pluripotency in ESCs. Our preliminary data have indicated that a specific signaling pathway (like a hormone that mediates various biological information) may be responsible for supporting the differentiation capacity in hESCs. Based on this finding, we will focus on investigating the role of the signaling pathway in hESCs through a series of molecular experiments, and developing novel technologies which enable us to grow hESCs as a pure population of undifferentiated cells that would further standardize the hESCs-based therapeutic strategy.
Statement of Benefit to California:
Our research will focus on identifying the mechanism that regulates the multiple differentiation capacity (pluripotency) in hESCs. With the knowledge obtained through the project, we will concentrate on developing novel technologies that will allow us to culture uniformly pluripotent hESCs which is not possible under the current culture protocols. The establishment of such a new method would impact virtually all hESCs-based application programs as it involves a common basic process to expand hESCs before turning into any type of adult cells for the therapeutic purposes. It is therefore predictable that the new methodology will be promptly translated as an intellectual property to be commercialized, and would substantially activate the biotechnology field in the State of California. More importantly, the new methodology will be provided to the Institutes in California at the highest priority where the method will accelerate the process to apply the hESCs-based transplantation approach for the clinical settings that would further substantiate the enhancement of the medical environment for California citizens.
SYNOPSIS: The general goal of this research proposal is to test the role of AMP activated kinase (AMPK) in stem cell self renewal and stem cell cytoskeletal structure, using murine and human embryonic stem cell (mESC, hESC) systems. Previous work by the PI has shown that inhibition of Glycogen Synthase Kinase-3 (GSK3), and consequent activation of Wnt signaling, can help sustain ES cell self renewal. Since GSK3 also plays a role in energy metabolism, the PI considered whether there is a general link between metabolism and developmental processes such as regulation of stem cell fate. He proposes that like GSK3, other regulators of energy metabolism, specifically AMPK, might have additional roles in cell fate specification. He focuses on AMPK since it serves as a cellular sensor for glucose deprivation or hypoxia, and because studies in flies have implicated AMPK in maintenance of epithelial cell integrity. It is therefore proposed that an effect of AMPK on stem cell self renewal will be mediated via regulation of epithelial cell-cell interactions. In Aim 1, the PI will test whether up- or down-regulation of AMPK alters ES cell self renewal and affects other signaling pathways known to be involved in that process. He will further test whether low oxygen environments promote ES cell self renewal via activation of AMPK. Studies in flies have implicated myosin regulatory light chain (MRLC) as a key target for AMPK, in a signaling pathway that affects cell shape and epithelial integrity. Therefore, in Aim 2, the PI will ask whether AMPK influences epithelial cell morphology by controlling the phosphorylation of MRLC, and whether this affects the assembly of cell junctions. He will further test whether non-muscle myosins, which associate with MRLC, are involved in stem cell self renewal, and whether other cell shape regulatory pathways such as those mediated by RHO and ROCK are downstream mediators of AMPK’s effects in this process. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The PI makes the case that getting the data sufficient for the AMPK work to be considered for an RO1 would take two years, hence the need for CIRM funding. On the other hand he proposes (at least at first) to use three presidential ‘approved’ lines for the research, which should be eminently NIH-fundable. So the question is whether the novel mechanism of AMPK regulation of self-renewal is a hot enough question for CIRM to devote a large grant to the work. The PI presents an interesting hypothesis, proposing a link between energy metabolism and self renewal, based on an understanding of the developmental biology that brings model systems’ work to bear on hESC. However, the proposed experiments for the most part are either excessively speculative, or relatively derivative. For instance in Aim 1, it is unclear what the basis is for the hypothesis that regulation of energy metabolism in general, and AMPK in particular, is a key to understanding stem cell self renewal. This idea is apparently based on the essential role of GSK3 in Wnt signaling. This has led the PI to propose that other regulators of energy metabolism, like AMPK, will also be important in stem cell differentiation. The logic behind this leap remains elusive. The role of GSK3 in the Wnt pathway has nothing to do with its role in glycogen biosynthesis. GSK3 is a kinase that in different cells phosphorylates different substrates acting in many pathways that are largely if not entirely unrelated and independent. To use this as a basis for studying AMPK is tenuous. Moreover, much of Aim 2 is largely derivative of recently published work from others showing that AMPK regulates epithelial cell polarity in Drosophila by phosphorylating MRLC. The applicant proposes to do the same experiments in ES cells. The rest of Aim 2 proposes unjustified leaps to test the roles of myosin and RHO in stem cell self renewal. Myosin is targeted because it interacts with MRLC. Not only does this assume that AMPK will be important for stem cell renewal, but it also assumes that this particular pathway downstream of AMPK is the relevant one. The connection of all this to RHO seems entirely hypothetical and indirect. No specific links are cited by the PI to justify these experiments. Although one reviewer expressed that (s)he believes in the leap of faith that the AMPK and ROCK signaling pathways will be interacting with each other, there are lots of self renewal mechanisms that could have been the focus of attention and the PI does not make a strong case for his favorite signaling system. The PI was involved in identifying RHO-ROCK signaling as an important factor in ES cell self renewal as a post-doc, and now has some small but compelling preliminary data suggesting that AMPK signaling may also play a role in hESC self-renewal. He has shown that cells treated with an AMP analogue and AMPK agonist (AlCAr) maintain colony formation in the absence of LIF, and maintain OCT4. However, a major weakness is that the proposal reads much like a compilation of a large number of descriptive assays, and it is not clear that upon completion of the proposed projects the field will have been moved much beyond what is already shown in the preliminary data. If addition of AMPK agonists relieves the reliance of mESC cultures for LIF, it is expected that the cells will reflect this activity in other stem cell assays. Likewise, if antagonism of AMPK disrupts cell epithelial integrity, this will likely disrupt cell shape and lead to decreased self renewal. If AMPK is a major player in linking cell shape to self renewal, it seems unlikely that MLRC will be the sole target of this, and its not clear how direct or indirect the linkage will be. The overall hypothesis is interesting, but it will be important to clarify whether the observed effects are quite direct, or reflect late downstream phenotypes that are only correlative with the disruption of the self renewal phenotype. There was consensus among the reviewers that the experimental plans amount to a huge list of experiments to be performed, that design strategies are hard to discern, and that there is no prioritization of experimental approaches. It is suggested to zero in on self renewal ,keeping the myriad differentiation experiments to the minimum needed to confirm pluripotency. Although many of the experiments listed are likely to yield important information on the control of hESC fate, the experimental design looks very shotgun and could benefit from some outline of the PI’s impression of the most important studies to be done. Specific suggestions to the research plan were as follows. 1) When experiments are listed with their sub-hypothesis side-by-side, they often lack in form. For instance, the last experiment listed in Aim 1a is to grow hESC on a stromal layer known to induce neuronal differentiation and then look at “neural differentiation capacity:” But if the real purpose of these experiments is an exhaustive look at stem cell fate vis-à-vis AMPK signaling and energy homeostasis, the optimal design would be to have a look at both neural and non-neuronal fate. 2) The consideration of using adenoviral vs. lentiviral vectors is a non-decision that runs through the grant. Why not choose lentiviral vectors with reporters? There are lots of reasons to do that rather than the transient infections. 3) The PI needs to provide end-points for FACS analysis. 4) Since microarray work on H1 cells has been done by the investigator (Table 1), he could check the microarrays to see which myosins are expressed at the message level. 5) What are the details of the energy experiments? How will hypoxia be monitored? Is the lab equipped to have steady low oxygen levels over long periods of culture without introduction of reperfusion injuries when the cells are passaged, which would lead to enormous variability in message-level readouts. Similarly, commercial media are very high in glucose, and ES cells reportedly have to be taken down gradually to truly hypoglycemic levels so as to accommodate the aphysiologic, oxidative stress caused by high glucose media typically used in cell culture. Since the question here is energetics, why not look at basic mitochondrial function of the cells, since they also are a theoretical control point distal to AMPK signaling? The naive discussion of energetics contrasts with the sophisticated discussion of AMPK in other contexts. 6) The PI would find the work in mesenchymal stem cell differentiation fate choice based largely on energetic considerations to be informative in the proposed ESC work, and it is somewhat surprising that the work is not mentioned, especially since fate choice based on constraining cell shape is an important part of this literature. 7) In terms of grantsmanship the vague plans for years 3, 4 and 5 are weaknesses of the proposal. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI received his MD from the Oita Medical University in 1987 and his PhD from the Juntendo University School of Medicine in 1996. He was a research fellow in pulmonary medicine at Cornell from 1996-2000 and then held a variety of short research associate or staff scientist positions at various places until being appointed an Assistant Professor in the Department of Biochemistry at UC Riverside in 2006. During his time as a research associate in the Brivanlou lab he published two papers in good journals on hESC and the role of the Wnt pathway in their self renewal. Since 2004 he has published only two methods papers. There is no current outside support. The PI is very well qualified for this project. He is fully capable of mESC and hESC cultures and most of the assays will not be particularly challenging for him. The PI is clearly committed to the study of stem cell fate and describes a plan to focus on how this relates to cell shape. This does provide him with a potential niche to develop within the field. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: UC Riverside has benefited considerably from CIRM funding and now has (according to the support letter) 30 investigators involved in stem cell research. The institution is also looking forward to the establishment of a new medical school campus, and is apparently looking to Dr. Sato to be a leader in this new endeavor. So they have an investment in his success. The description of facilities and interactions suggests that the investigator is in an appropriately supportive environment to carry out the planned experiments. He occupies 1500 square feet of independent lab space. His start up package included new equipment and access to core facilities, but no mention is made of research and salary support. It is also said that he can spend a minimum of 50% time in research, but the actual teaching burden is not indicated. The PI is also a member of the UCR stem cell research center, which was recently funded by CIRM. A designated mentor would be a good idea, even though the PI has independence, to help with grantsmanship. DISCUSSION: During a brief discussion, a reviewer stated that the PI pulled together disparate literature to come up with the interesting hypothesis that AMPK, a kinase involved in energy regulation, plays a role in stem cell self renewal. However, there was consensus among the reviewers that the proposal simply consists of a list of experiments with no endpoints of analysis proposed, and that overall it is not competitive.