Telomeres are the ends of our linear chromosomes and protect the genetic material against enzymatic attack. They are essential for long-term survival of cells and prevent genome instability. Telomeres shorten with each cell division, and when they become critically short they cause a cessation of cell growth, or cell death. This limits the number of times cells can divide and therefore represents a powerful tumor suppressive mechanism. Stem cells however, possess unlimited proliferation potential, which can only be achieved by counteracting replication-associated telomere loss. They do so by activating telomerase, which is an enzyme that specifically elongates telomeres. Similarly, cancer cells activate telomerase to gain immortality, pointing out a parallel between stem cells and cancer cells. Telomerase activation has other side effects besides providing immortality, which are deregulation of the packaging dynamics of the genetic material, as well as altering gene expression, both of which represent undesired side effects that could interfere with the use of stem cells for therapy. We have discovered the mechanism for an alternative pathway for telomere lengthening, called ALT. This proposal is designed to investigate the effects of ALT on stem cell generation, long-term culture, genome stability and differentiation. The goal of this proposal is to develop an alternative method to maintain telomeres, potentially eliminating telomerase expression-associated side effects.
Statement of Benefit to California:
The use of stem cells for therapy is critically dependent on the capabilities to prevent the cells from transformation and the accumulation of other deleterious alterations during their handling and growth. Telomeres are the natural ends of linear chromosomes. They shorten with each cell division, until they cause a cessation of cell growth, or cell death. This limits the number of times cells can divide and represents a powerful tumor suppressive mechanism. Telomere shortening also limits the efficiency of reprogramming of aged cells and cells from older individuals. Telomerase is an enzyme that lengthens telomeres, and is activated during the reprogramming of cells, but also in cancer cells and renders them immortal. Telomerase activation has other side effects besides providing immortality, which are deregulation of the packaging dynamics of the genetic material, as well as altering gene expression, both of which represent undesired side effects that could interfere with the use of stem cells for therapy. We discovered the mechanism for an alternative pathway to telomerase for telomere length maintenance (ALT), and this proposal is designed to investigate the effects of ALT on stem cell generation, long-term culture, genome stability and differentiation. This project will benefit California and its citizens by providing the means for telomere lengthening in aged cells, stem cells and reprogrammed cells, while avoiding the deleterious side effects of telomerase.
Maintenance of the ends of chromosomes, or telomeres, is essential for prolonged survival of stem cells and other self-renewing cell types, including many cancers. This maintenance is typically accomplished through the activity of telomerase, an enzyme that counteracts progressive telomere shortening upon cell division by catalyzing the addition of deoxynucleotides to chromosomal ends. A second mechanism of telomere maintenance termed Alternative Lengthening of Telomeres (ALT) has also been described in certain cell types, but its role in stem cell biology is unknown. In this proposal, the applicant seeks to explore the effects of ALT activation on human induced pluripotent stem cells (iPSC) and to determine whether ALT might enable avoidance of potentially deleterious side effects that can be associated with telomerase expression in conventional iPSC.
Significance and Innovation
- Reviewers did not support the central hypothesis that telomerase is a risk factor for oncogenic transformation in iPSC, as telomerase alone is not a driver of carcinogenesis. It is unclear how ALT would be any different in this respect.
- Reviewers were not convinced that iPSC produced with ALT would have superior properties to those produced conventionally.
- Telomerase activity is not considered to be a major unsolved problem in human embryonic stem cells, nor by extension in hiPSC. Thus, while the proposal might provide new insights into telomere protection during and after reprogramming, the findings are not likely to be of high impact.
Feasibility and Experimental Design
- It is not clear that the proposed approaches would enable experimental separation of telomerase and ALT components with sufficient rigor to test the central hypothesis.
- The experimental design is contingent on the success of the first subaim to successfully reprogram cells with ALT. If this is not accomplished, the remainder of the proposal becomes superfluous.
- The number of cell lines to be tested per donor group is not sufficient to yield meaningful results.
- The proposal exhibits a general underestimation of the time and labor intensiveness of iPSC generation and maintenance.
- Preliminary data on ALT assays is excellent but not clearly relevant to reprogramming.
Principal Investigator (PI) and Research Team
- The PI has excellent qualifications and a proven track record of publications in investigations of telomere dynamics during aging and cancer development, but limited experience with human stem cells.
- The research staff has extensive experience with telomere biology but lack proficiency with human stem cells. The team will need to rely heavily on core facilities for stem cell expertise, and thus a letter of support should have been included.
Responsiveness to the RFA
No relevant concerns were highlighted by reviewers under this review criterion.