Pericellular proteolysis in human ES cell differentiation and tumorigenesis
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.
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.