Embryonic stem (ES) cells are primal undifferentiated cells that retain the ability to produce an identical copy of themselves when they divide and differentiate into other cell types. ES cells can act as a repair system by replacing other cells, tissues and organs in a diseased human body. The nature of the regulatory processes which control ES cell differentiation, however, are not well understood. Cell-microenvironment interactions are essential for ES cell functionality. Proteases, including matrix metalloproteases (MMPs), have the ability to alter the microenvironment in response to physiological stimuli. Twenty-five, soluble and membrane-attached, MMPs are known to exist in humans. Membrane-type (MT)-MMPs are anchored to the plasma membrane and, in contrast to the soluble MMPs, are well-suited for cleaving neighbor proteins. The available knowledge about ES cell MMPs is extremely limited. This lack of knowledge is responsible for our inability to modulate differentiation pathways of human ES cells and to control the MMPs in a clinically advantageous manner. We have developed a well-controlled and reproducible method by which we can control the directional differentiation of human ES cells into neuronal cells. When we tested if MMPs are affected during this differentiation process, we identified the presence of a proteinase called MT6-MMP which was significantly affected. Based on our preliminary data coupled with our knowledge of MMPs, we hypothesize that MT6-MMP contributes to the neoplastic transformation of human ES cells into cancer cells resulting in brain tumors in vivo. To gain a fundamental knowledge about stem cell MMPs and to validate this hypothesis, we have designed the following specific aims: (1) Determine the roles of MT6-MMP and MT1-MMP (control) in an in vitro model of human ES cell differentiation into the uniform neural precursor, (2) Identify the intracellular proteases involved in the activation of MT6-MMP and MT1-MMP in human ES cells and the uniform neural precursors, and (3) Determine the roles of MT6-MMP and MT1-MMP (control) in the malignant transformation of the ES cell-derived neural precursors into malignant cells which are capable of generating tumors in mice. The most advanced analytical methods, including the use of genetic engineering, genomics and proteomic techniques, high resolution microscopy, lentiviral gene expression of the wild-type and mutant MMPs and related cutting-edge methods will be used to achieve our aims. All of these methods are well established and are used in the day-to-day operations of our laboratories. Our extensive and diversified experience with both MMPs and human ES cells bolsters the confidence of the PI and the Collaborator that they will determine the importance of pericellular proteolysis in the normal and malignant differentiation of human ES cells.
Statement of Benefit to California:
The term “stem cells” identifies the population of cells as the source of other, more specialized cells. The very earliest cell which is an immediate descendant of the fertilized egg is a totipotent embryonic stem cell. We have only very recently begun to appreciate and gain an understanding of the specific individual steps from the very many molecular events involved in the ES cell differentiation. To date, our means to control and to modulate these differentiation processes are limited. In all known cell types proteases are powerful regulators of cell functions. Proteases regulate the cell microenvironment and the cell surface adhesion signaling receptors and, therefore, have a long-lasting effect on cell behavior. Unfortunately, our knowledge of stem cell proteinases is non-existent. This lack of knowledge contributes to our inability to modulate differentiation pathways of human ES cells and to control stem cell MMPs in a clinically advantageous manner. Because synthetic, highly potent, specific inhibitors of MMPs have been developed and tested in clinical trials in cancer, an opportunity exists to use these inhibitors to specifically modulate the activity of stem cell MMPs and through the control of the individual MMPs to modulate stem cell proliferation, fate decision, differentiation into specific cell types and the efficiency of preferential stem cell migration towards the site of a lesion. We believe that our proposed research will lead us to a far better and very necessary understanding of the role of membrane proteinases in ES cell differentiation. We believe our results will lead us to develop novel and effective means to control and to specifically modulate the differentiation processes of ES cells. The means to control stem cell differentiation will ultimately lead to novel, stem cell-based, therapies the development of which will benefit the state of California. We also believe that a broadly understandable way to describe the benefits to the State of California that will flow from the stem cell research we propose to conduct is to couch it in the familiar business concept of “Return on Investment”. The novel therapies and reconstructions that will be developed and accomplished as a result of our research program and the many related programs that will follow will provide direct benefits to the health of California citizens. This program and its many complementary programs will generate potentially large, tangible monetary benefits. These financial benefits will derive directly from two sources. The first source will be the sale and licensing of the intellectual property rights that will accrue to the state and its citizens from this and the many other stem cell research programs that will be financed by the CIRM. The second source will be the many different kinds of tax revenues that will be generated from the increased bio-science and bio-manufacturing businesses that will be attracted to California by the success of the CIRM.
This proposal is based on the observation that when the human ES cell line (H9) differentiates into neural precursors, there is down regulation of MT6-MMP. Given the level of transcripts encoding other MMPs (matrix metalloproteases), and the reports of elevation of MT6-MMP in human brain tumors, the hypothesis to be tested is that MT6-MMP promotes neoplastic transformation of neural stem/progenitor cells into cancer stem cells which ultimately produce brain tumors in vivo. There are three specific aims. First, the applicants will determine the roles of MT6-MMP in vitro when human ES cells differentiate into neural precursors. In this regard, the parental H9 cells and transfectants expressing wild type, mutant, and chimeric constructs will be used. Aim 2 will identify the furin-like protein convertases involved in the activation of MT6-MMP in human ES cells while aim 3 will determine the roles of MT6-MMP in malignant transformation by implanting MT6-MMP ES trasfectants into the brain of nude mice. SIGNIFICANCE AND INNOVATION: There is no doubt that understanding the normal and malignant differentiation of hES cells is important, and the present proposal incorporates well-in-hand technologies of genetic engineering, genomic and proteomic techniques, and high resolution microscopy to study matrix metalloproteinase roles in these processes. Pericellular proteolysis and membrane-tethered MMPs are definitely under-studied in stem cell and human embryonic stem cell biology. The PI is an expert is this field, and brings a fair amount of technology and experience to this work that has potential significance toward understanding stem cell transformation that contributes to neoplasia. STRENGTHS: This is an innovative proposal since there is very little known about MMPs in ES cell biology in general. Thus, the application will break new ground with respect to findings that are expected to be novel. There is also an increasing belief that brain tumors that arise within the CNS have their origins from neural stem cells so the results of this proposal will have clinical relevance. It is helpful the preliminary studies have shown that MT6-MMP, that is associated with brain tumorigenesis, is downregulated during in vitro differentiation of the H9 hES cell line into neural precursor cells. The overall quality of the research is high, the timelines are reasonable and achievable, most aims are feasible, and there are appropriate collaborations set up amongst the investigators. Other strengths of this proposal include the high standing of the primary investigator in the field of MMP biology and his continued high level of productivity. Several unique reagents will also be generated as a result of this proposal in order to probe the role of MT6-MMP in ES cells in general. WEAKNESSES: Although MT6-MMP is altered following differentiation, it certainly is unclear if MT6-MMP alteration drives the differentiation or if MT6-MMP is merely one of the many changes that occurs as a result of cellular differentiation. If the former, the clinical significance of this proposal will be tremendous. If the latter, then the relevance of the proposal is questionable. Nonetheless, this is a seed grant to probe unproven and highly risky projects so the current proposal as laid out could be what CIRM is looking for. It is stated simply that other MMPs are not altered but no data are shown except for MT1-MMP. This is of some significance since other MMPs have been implicated in the origin of brain tumors as well. The focused attention on MT6-MMP at the exclusion of other MMPs may thus result in the wrong MMP member being examined. Certainly, the inhibitors proposed for this project (GM6001, AG3340 and SB3CT) are not specific or selective for MT6-MMP. One is concerned that about whether a single mutation of MT6 in ES cells will be sufficient to produce malignant transformation. Brain tumor cells generally have acquired several mutations and it has been hard to transform neural cells into brain tumors based on a single alteration. Thus, whether the experiments of aim 3 (generating transfectants with mutant MT6-MMP) are sufficient to yield interpretable results can be questioned. If the transfectants do not result in brain tumors, they may simply indicate that other mutations are required in addition to a pivotal role for MT6-MMP. However, the experiments as they are designed currently may simply lead to the wrong conclusion of MT6-MMP being unimportant when additional co-mutations are necessary. Finally, the experiments on furin-like proprotein convertase is not related to the rest of the work. DISCUSSION. The PI has experience and a number of grants, and the preliminary MT6-MMP data are helpful. The weakest point in this proposal is the assumption that hESCs engineered with the MMPs will generate tumors in grafted mice. There are very few cases where a single mutation can cause tumorigenesis, therefore there is no reason to believe that tumors will form. Reviewers were also concerned about whether the MT-MMP6 expression changes were a cause or consequence of differentiation. Another issue is how the PI will separate solely neural cell effects from the MMPs' vasculogenic effects.