Basic Biology II
The discovery of induced pluripotent stem (iPS) cell technology promises to revolutionize our understanding of human disease and to allow the development of new cellular therapies for regenerative medicine applications. The ability to reprogram a patient's fibroblasts to iPS cells creates the opportunity to expand human cells with a specific genetic defect and to study that defect in a defined cell population, either to understand the basic biology of the disease or to study potential therapeutics. Furthermore, the genetic defects in iPS cells can be repaired and the iPS cells used as a source for cellular therapies after differentiation to specific cell lineages. Although tremendous strides have been made in recent years in treating human disease, replacing damaged tissue remains almost completely beyond our grasp. Harnessing human iPS stem cells for this purpose will open completely new areas of regenerative medicine. However, a limited understanding of iPS cell self-renewal and differentiation is a major roadblock in realizing this long-term goal. One shared characteristic of iPS 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. Senescence pathways – pathways that cause dividing cells to permanently stop dividing – represents a significant barrier in the reprogramming process to engineer new iPS cells. Understanding how iPS 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. In this proposal, we investigate the molecular mechanism of self-renewal and senescence in human iPS cells using skin cells isolated from patients with a defect in the enzyme telomerase. 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. People with telomerase mutations develop a stem cell disease – dykeratosis congenita. In this disease, patients have defects in skin, blood and lung – tissues that depend on tissue stem cell function to maintain these organs during life. We will reprogram skin cells from dyskeratosis patients to understand how senescence responses limit iPS cell self-renewal and differentiation to specific cell lineages.
Statement of Benefit to California:
This proposal will benefit California and its citizen in two general ways. First, I have recruited new scientists to California from Texas and from Brazil 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 program and its strong emphasis on human stem cell biology. 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.
EXECUTIVE SUMMARY In this proposal, the Principal Investigator (PI) probes human induced pluripotent stem cell (iPSC) senescence and self-renewal mechanisms by using skin cells isolated from Dyskeratosis Congenital (DC) patients. In DC patients, telomerase mutations lead to tissue stem cell defects in skin, blood, and lung. Telomerase is an enzyme complex that protects chromosomal structural integrity and is expressed in human embryonic stem cells (hESCs), some tissue stem cells, and almost all human cancers. Since DC is well characterized on a genetic level, the applicant proposes to utilize DC fibroblasts to gain a better understanding of telomerase’s function in cellular senescence and study the mechanisms of self-renewal and senescence in human somatic cell reprogramming. In the first Aim, the applicant will generate DC-iPSCs and compare the senescence, self-renewal, and the dynamics of telomerase regulation during reprogramming of normal and diseased fibroblasts. The second aim will examine the differentiation of iPSCs from DC patients towards specific cell fates. Reviewers agreed that the proposal described significant and innovative reprogramming research. The applicant’s focus on basic molecular and cellular reprogramming mechanisms was well developed and appreciated by reviewers. In particular, one reviewer praised the proposal’s creativity in using the derivation of iPSCs from patient material to test for the cellular defect leading to DC. One reviewer questioned the impact of the proposed research, since another group had recently published highly similar studies. However, collectively, the reviewers concluded that this proposal offered a unique insight and that the field would benefit from having two groups pursuing this line of work. Another reviewer felt the proposal was limiting its impact by its singular focus on differentiation to the hematopoietic stem cell fate. Despite these minor concerns, the review panel considered the project to be one of high potential, likely to provide new insights regarding telomerase and senescence responses in iPSCs, and broadly applicable to aging studies and regenerative medicine. Reviewers were highly enthusiastic about the proposal’s feasibility and experimental design. The panel found the preliminary data to be very strong and demonstrated the research team’s ability to generate reprogrammed DC fibroblasts. Reviewers stated that the applicant described experiments clearly, designed well-organized specific aims, and devised the experiments to give meaningful results. One reviewer particularly appreciated the strength of the in vitro model system to test the effects of telomerase component mutations on the senescence of progenitor cells. While one reviewer felt the chimeric model proposed in Aim 2 might be out of the proposal’s scope, another reviewer praised the direct in vivo assessment of DC-iPSC behavior by generating human-mouse chimeras that would facilitate an examination of the relevance of the in vitro observations. Although reviewers had minor concerns about some aspects of the experimental plan, these few criticisms were outweighed by enthusiasm for the likely success of the project in revealing novel insights into the role telomerase in stem cell senescence. Reviewers unanimously extolled the applicant and the research team. They considered the applicant to be a highly trained and well-published scientist. The facilities are outstanding and the proposed collaborations are strong. One reviewer remarked that the applicant’s level of commitment towards this project is good and enhances the proposal’s chances for success. In summary, despite a few minor criticisms, the reviewers were highly enthusiastic about this proposal’s innovative approach and high impact potential and considered the well-qualified applicant and team to heighten the probability of the project’s success.