The promise of human embryonic stem (ES) cells is that their use could revolutionize the treatment of many human diseases for which current treatments are ineffective. Many interventional therapies will rely on the manipulation of stem cell differentiation in order to selectively produce a particular cell or tissue type. To accomplish this, we will first need to increase our understanding of lineage commitment during early embryogenesis, and define how, once cells have differentiated into a particular cell type, organogenesis is regulated, i.e. how the movement and organization of cells is controlled. During embryogenesis, cells can move in ways that are very similar to the movement of cancer cells during metastasis. That is, cells have the ability to both migrate on and degrade a mixture of proteins called extracellular matrix (ECM). An example is heart formation, where the coordinated movement of the cells that make up the cardiac crescent is followed by reorganization and differentiation, including the invasion of cardiac progenitors into the cardiac jelly (ECM) to form the valves. Our hypothesis is that the same mechanisms are used to control invasive movements during early embryogenesis and metastasis. In support of this hypothesis, we have recently found that some of the cells present in embryoid bodies (cultures of differentiated cells derived from human ES cells) express genes and contain cell structures that are found in metastatic cancer cells. In particular, genes called Tks4 and Tks5 are selectively expressed in some cells. Furthermore, our preliminary experiments with zebrafish embryos have revealed that loss of Tks4 and Tks5 leads to very early severe developmental defects, consistent with loss of cell movements. Our goal here is to dissect the control and function of Tks4 and Tks5 during early development of human ES cells in culture. Understanding the mechanisms by which movement and ECM degradation is controlled during early human embryogenesis will set the stage for determining how to manipulate these properties in the future.
Statement of Benefit to California:
Each year large numbers of Californians are afflicted by diseases such as Parkinson's, Alzheimer's, and diabetes. Stem cell therapies may revolutionize how these diseases are treated in the future. For this revolution to occur, we will need to accomplish a number of goals. For example, for each disease to be treated, we will need to be able to produce the correct type of cell or progenitor (eg neuronal cell, pancreatic beta-cell), and deliver it to the patient so that it will function correctly. In some cases, we may also want to be able to control the movement of the cells, either promoting their migration or ensuring that they cannot migrate, depending on the disease to be treated, and the way in which the cells are to be delivered. The research proposed here is part of an overall effort to understand how the movement and development of human embryonic stem cells and the more differentiated progenitors that arise from ES cells are controlled. With this knowledge, we may in the future be better able to harness stem cell therapies.
SYNOPSIS: The overall goal of this proposal is to look for common signaling pathways that mediate migration between cancer cells, developing cells, and hESC, with a focus on the Tks4 and Tks5 genes and morphologically on the formation of podosomes. Tks4 and Tks5 were recently identified by the PI, and a knockout in Zebrafish shows (generalized?) developmental defects. The specific aims are (i) determine how expression of Tks4 and Tks5 is regulated (ii) determine which cells in embryoid bodies (EBs) express Tks4 or Tks5 and/or form podosomes and (iii) determine the phenotype of EBs lacking Tks4 and/or Tks5. SIGNIFICANCE AND INNOVATION: There are many similarities between cancer and stem cells, both phenotypically and at the level of expression. An important similarity is migration and the ability to break down matrix in order to migrate. The expression profiles mediating migration and loss of migration are important to characterize for safe translational therapies. This work will examine proteins known to be involved in motility, and their role in the motility of hESC. It is not clear how these studies would positively impact the hESC community, clinical applications, or major problems/questions faced in the field. The proposal is not innovative as no new technologies are being developed and no new capabilities for hESC researchers will be produced. STRENGTHS: The choice of general topic - the migration of stem cells - is a good one, and one that historically has been hard to study. The relationship between cancer and stem cells also deserves further exploration. The PI presents a clear and addressable hypothesis with straightforward molecular manipulations. Thus the proposal is feasible. WEAKNESSES: While nicely organized with a cogent and reasonable hypothesis, this proposal does not seem to be responsive to the call for funding only the most important, highest impact projects within CIRM. Taken together, all the aims except 1 are relatively high risk for the short time frame, and aim 1 can certainly be done independent of hESC. The problem is that the high risk is not justified by a high gain in the way the studies are designed. For example, the second aim should come first, because if the embryoid bodies of hESC do not express the gene, the rest of the proposal does not hang together. One reviewer performed a cursory search for the gene in one publicly accessible microarray database, which revealed that the gene may not be expressed in EBs, and the PI might have done their own, more general search in other databases to firm up the support. Ironically the PI cites the “Stem Cell Matrix” microarray data at her own facility, which should have been consulted in a similar manner. In addition, the studies to look at podosome formation in-vitro and the migration of hES cells on gelatin using video are likely to fail for a number of reasons. What will the cells migrate to? If the common feature of migration in cancer and hES cells is protease breakdown of the matrix, why is that specific matrix chosen for study? There are several questions about the experimental design. Aim 1 is supposed to “determine how expression of Tks4 and Tks5 is regulated in early embryogenesis” but is really an analysis of the regulatory elements of these genes, and independent of anything to do with embryogenesis. Lentiviral siRNA expression will be used to knock down Tks4 and Tks5 expression, and the ability to knock down both together might be needed for a phenotype. That may take some time. Also, the phenotype will be based on co-expression of various markers, some for relatively undifferentiated cells and some for more differentiated cell types, over the course of 3 weeks of culture. Is one of the markers GFP for liver? DISCUSSION: There was no further discussion following the reviewers' comments.