Embryonic stem cells are unique in their ability to give rise to all mammalian tissues. Their potential application to human disease is enormous because they could be employed to repair or replaced damaged tissue. Although tremendous strides have been made in recent years in treating human disease, replacing damaged tissue remains almost completely beyond our grasp. Harnessing human embryonic stem cells for this purpose will open completely new areas of regenerative medicine. One shared characteristic of embryonic stem cells and adult stem cells that reside in many of our tissues is the ability to self-renew. Self-renewal is the ability of a stem cell to divide and give rise to a daughter cell that is undifferentiated and capable of giving rise to all the same lineages as the parent stem cell. Understanding how embryonic stem cells self-renew is critical for determining how to maintain these cells, how to differentiate them toward specific tissue lineages and how to expand more committed stem cells or progenitor cells in cell culture. A small number of genes that control embryonic stem cell self-renewal have been discovered, but our understanding of this process remains in its infancy. In this proposal, we investigate the molecular mechanism by which telomerase contributes to embryonic stem cell self-renewal. Telomerase is an enzyme complex expressed in embryonic stem cells, some tissue stem cells and in almost all human cancers. Most differentiated cells lack telomerase expression. Telomerase adds DNA repeats to structures at the ends of our chromosomes, termed telomeres. Telomeres are very important in protecting chromosome ends and in preventing chromosome ends from breaking down or sticking to other ends inappropriately. By maintaining telomeres, telomerase supports the ability of stem cells to divide a large number of times. In addition to its function in telomere synthesis, we recently discovered a second role for telomerase. We expressed the TERT protein component of telomerase in mouse skin and unexpectedly found that TERT activated resting tissue stem cells. Activation of skin stem cell by TERT triggered a regenerative program in skin leading to robust hair growth. We used a rigorous technique to show that this new activity does not involve TERT’s other role in telomere lengthening. It is absolutely essential to understand how telomerase carries out its functions in stem cells. Therefore, we have isolated proteins that strongly interact with TERT. In this proposal, we investigate the function of TERT in embryonic stem cells and determine the role of several TERT associated proteins in embryonic stem cell self-renewal.
Statement of Benefit to California:
This proposal will benefit California and its citizen in two general ways. First, I have recruited two new scientists to California from Texas to work on this proposal. These are new taxpayers and consumers, which will benefit local businesses. They would have been less likely to come to California in the absence of the CIRM grant program. Second, this novel grant will generate new intellectual property in the form of patents. These patents may in fact be licensed to California companies or be used to support the formation of new start-up companies. The growth of such companies has historically fueled much of the profound growth in California. The future of California is linked to new technologies in the stem cell, biotechnology and other technology sectors.
SYNOPSIS: The general goals of this proposal are to explore the functions of TERT, the catalytic protein subunit of telomerase, in embryonic stem cells. In Aim 1 the applicants will study the effect of TERT depletion and TERT overexpression on the self-renewal of murine and human ES cells. In Aim 2 the applicants will pursue the functional characterization of 4 proteins they have identified that associate with TERT in cells. They will use RNA interference to determine whether these proteins are necessary for the proliferation of human ES cells. SIGNIFICANCE AND INNOVATION: ES cells express telomerase, and a number of recent publications have demonstrated that telomerase can promote cell proliferation independent of its action at telomeres. The applicant, in particular, has found that this second function of telomerase can cause the expansion of hair follicle progenitor cells in vivo. This phenomenon was quite unexpected given that telomere length is not physiologically limiting for the proliferation of mammalian stem cells under the vast majority of circumstances. The mechanism underlying this pro-proliferative function of telomerase is not known, and the possible contribution of the pro-proliferative function of telomerase to ES cell self-renewal would be of interest. This applicant proposes to extend earlier work by studying the effect of TERT expression in mouse and human ES cell self-renewal and differentiation, and this proposal attempts to determine the mechanism by which TERT can influence the proliferation of at least some stem cells. If TERT influences the self-renewal of ES cells, and if the applicant is able to uncover the mechanism, then this work would be very significant for the stem cell field. As part of an effort to understand this new telomerase activity, the applicant has purified the telomerase multi-protein complex and used mass spectrometry to identify 4 new telomerase-associated proteins. Their roles in telomere maintenance versus cell proliferation are not known. STRENGTHS: The underlying phenomenon is quite unexpected and therefore quite novel, and the technical approaches the applicant takes toward trying to uncover the mechanism are reasonable, though of course it is uncertain whether these approaches will shed new light on the phenomenon. The applicant has developed important research tools that provide the foundation upon which this application is based. These include murine ES cells that conditionally induce or conditionally repress TERT expression, shRNA vectors that target human TERT, the cloning of 4 new TERT-associated proteins, and the preparation of antibodies against these proteins. The experiments are well designed, and the applicant clearly has the tools and expertise to perform them. The use of mouse ES cells in some of the experiments to benefit from mouse genetics is scientifically appropriate. WEAKNESSES: This proposal has a number of weaknesses: 1. Given that ES cells exhibit critical differences in their self-renewal potential and regulation relative to adult stem cells, it would not be surprising if they responded quite differently to TERT over-expression. From this point of view it might be inappropriate to expect to replicate the same phenomenon as observed in adult epithelial stem cells. Moreover, since it is not clear whether increased TERT expression will influence ES cell self-renewal, it is not clear whether this is a system that is capable of providing insight into the phenomenon described in the skin. Prior experiments conducted in hematopoietic stem cells did not detect the kinds of effects of increased TERT expression on stem cell function (Nature Med. 9:369) as the PI observed in epithelium, or which are contemplated in ES cells. The most critical scientific goal in light of this phenomenon reported by the PI is to uncover the underlying mechanism. Given that the work in hematopoietic stem cells suggests that this phenomenon does not occur in all stem cells upon TERT over-expression (though additional experiments clearly are required), the most scientifically sound approach would be to try to work out the mechanism in the epithelial stem cells that we know are affected by TERT. 2. Aim 1 is divided between studying the effects of telomerase over- and under- expression in murine and human ES cells. Murine cells have long telomeres, and it is already established that TERT null mice can be propagated for at least 4 generations before any significant pathology emerges. It is therefore not clear what the applicant would learn by decreasing TERT expression in murine ES cells. After many passages these cells would eventually lose sufficient telomeric DNA to undergo crisis due to chromosome instability. Similarly, what is to be learned by decreasing TERT expression in human ES cells that is not already evident from identical experiments in other human cell types? On the other hand, TERT overexpression has been shown to drive the proliferation of many cell types, including stem/progenitor cells, in vitro and in vivo. Perhaps the experiments proposed here could reproduce that result in ES cells. However, the important question is the mechanism underlying this effect and nothing is proposed here that would address that issue. Aim 2 proposes to study the functions of TERT-associated proteins. Reviewers feel that there is little or no reason to do these experiments in ES cells, as opposed to using much more easily propagated cell lines that would be much better suited to the initial biochemical and cell biological experiments that are essential at this early stage of analysis. 3. The applicant describes ES cells as "a highly physiologically relevant cell type in which to study TERT biochemistry". However, ES cells are not a physiological cell type in the sense that they change their properties relative to inner cell mass cells, while forming a cell line in vitro. Moreover, inner cell mass cells undergo many fewer divisions in vivo than adult stem cells like epithelial stem cells or hematopoietic stem cells. Thus, it is highly unlikely that TERT expression would have any effect on the inner cell mass in vivo. From this point of view, ES cells could be regarded as a highly unphysiological system for studying this phenomenon. DISCUSSION: This PI has a very strong track record, albeit limited history in stem cells. The basic phenomenon is interesting, and the approaches are reasonable, but are they applicable to ESCs? If the over-expression phenomenon applies to hESCs, this work would be valuable, yet the loss of function studies are not very illuminating. It is not clear what more will be learned by performing the proposed experiments in hESCs. Also, what will be learned in mouse cells? Why not start with a more tractable model?