Dissecting Pathways of p53-Mediated Inhibition of Cellular Reprogramming

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01497
ICOC Funds Committed: 
$0
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 
The possibility of generating patient-specific pluripotent stem cells has tremendous utility for treating patients with a wide variety of diseases. Recently, it has become possible to derive cells from patients and to subject them to a process known as cellular reprogramming to form induced pluripotent (iPS) cells specific to that patient. This technology, however, currently has some limitations. First, the reprogramming process is not very efficient. Second, the process required to reprogram cells entails introduction of genes that can promote cancer. Therefore, it is a critical goal to define the mechanisms that control the ability of cells to undergo reprogramming, to provide alternate strategies to generate iPS cells more efficiently and more safely. One approach recently demonstrated to enhance the efficiency of reprogramming is through inactivation of a gene known as p53 in the cells to be reprogrammed. Inactivation of p53 also allows iPS cells to be generated without the introduction of cancer-causing genes. Despite increasing efficiency of iPS cell formation, p53 inactivation also has some limitations, as it can cause chromosomal instability in the cell, contributing to cancer development itself. P53 affects a vast network of proteins in the cell, and therefore, if select components of that network could be targeted instead of p53 itself, it might provide a means to increase the efficiency of reprogramming without causing genetic instability and promoting cancer. Here, we propose to identify mediators of p53 function in limiting reprogramming to determine if it is possible to uncouple this function from its ability to maintain chromosomal stability. In this way, we could devise strategies to achieve an enhancement of reprogramming efficiency without the deleterious consequences of compromising chromosomal stability and promoting cancer. Inactivation of such key p53 reprogramming mediators may also allow generation of iPS cells without introducing cancer-causing genes, as does p53-deficiency. Thus, these experiments may lead to both improved efficiency and safety of reprogramming.
Statement of Benefit to California: 
The use of stem cells to regenerate and restore tissues is an important strategy for treating a variety of diseases. However, a limitation of this strategy is the possibility of immune rejection of foreign cells by an individual’s body. Therefore, generation of stem cells specific to a given patient, which will not be rejected by that individual’s body, holds great promise for the treatment of a variety of human diseases. Recent technology called cellular reprogramming allows the conversion of cells derived from an individual into stem cells that can be used for therapeutic purposes. This technology still needs further development, in the sense that it is inefficient and not adequately safe. In this grant we explore the ways in which this process is controlled, with the aim of enhancing the efficiency and safety of this process, to facilitate its therapeutic applications. This technology should benefit Californians of a variety of ages, with a broad spectrum of diseases ranging from neurodegeneration to diabetes.
Progress Report: 
  • Over the past year, we have analyzed five induced pluripotent stem (iPS) cell lines engineered from different individuals with a genetic stem cell disease. Dyskeratosis congenita is a rare disease affecting stem cells in multiple tissues. Patients with dyskeratosis congenita develop life-threatening bone marrow failure and pulmonary fibrosis, and are highly prone to cancers. In addition, they develop defects in skin, nails and many other organs. Dyskeratosis congenita is caused by mutations in an enzyme - telomerase - that is particularly important in stem cells. Telomerase elongates telomeres, caps that protect chromosome ends. If telomerase is defective, telomeres shorten and loss of the protective cap at telomeres can cause serious problems in stem cells. It has been very difficult to study this disease because isolating stem cells from dyskeratosis congenita patients is challenging. To overcome this problem, we engineered iPS cells from five patients. This is a way to change skin cells into cells that closely resemble embryonic stem cells - stem cells that can give rise to all tissues within the body. We studied these iPS cells from dyskeratosis congenita patients and found that the type of effects on telomerase were very specific and depended on the specific gene that is mutated in the patient. For example, mutations in TERT, the catalytic protein in the telomerase complex, resulted in a 50% reduction in telomerase activity in the patient's iPS cells. In contrast, mutations in the protein dyskerin, seen in the X-linked form of the disease, reduced telomerase activity by a much greater amount - 90% compared to controls. Mutations in another telomerase protein, TCAB1, left telomerase activity unaffected, but made the enzyme mislocalize within the nucleus. We studied how telomeres elongated with reprogramming of skin cells to iPSCs for each patient. Normal cells from healthy people show significant elongation of telomeres during the making of iPSCs, because telomerase is reactivated during this process. For TERT-mutant patients, elongation still happened, but elongation was significantly blunted. For dyskerin-mutant iPS cells and TCAB1-mutant iPS cells, elongation was completely blocked by the mutations and instead, telomeres shortened during this process and with passage in culture. Importantly, the much more severe telomere defect in dyskerin-mutant and TCAB1-mutant cells corresponds closely with the severity of the disease in the patients themselves. Our data show that iPS cells are a very accurate system for studying dyskeratosis congenita and revealed for the first time that the severity of the disease correlates with the severity of the telomerase defect in stem cells. These findings create new opportunities to study stem cell diseases in cell culture and to develop therapies that could specifically reverse the disease defect.
  • Over the past year, we have generated and analyzed new induced pluripotent stem (iPS) cell lines engineered from different individuals with a genetic stem cell disease. Dyskeratosis congenita is a rare disease affecting stem cells in multiple tissues. Patients with dyskeratosis congenita develop life-threatening bone marrow failure and pulmonary fibrosis, and are highly prone to cancers. In addition, they develop defects in skin, nails and many other organs. Dyskeratosis congenita is caused by mutations in an enzyme - telomerase - that is particularly important in stem cells. Telomerase elongates telomeres, caps that protect chromosome ends. If telomerase is defective, telomeres shorten and loss of the protective cap at telomeres can cause serious problems in stem cells. It has been very difficult to study this disease because isolating stem cells from dyskeratosis congenita patients is challenging. To overcome this problem, we engineered iPS cells from dyskeratosis congenita patients. This is a way to change skin cells into cells that closely resemble embryonic stem cells - stem cells that can give rise to all tissues within the body. We studied these iPS cells from dyskeratosis congenita patients and found that the type of effects on telomerase were very specific and depended on the specific gene that is mutated in the patient. Normal cells from healthy people show significant elongation of telomeres during the making of iPSCs, because telomerase is reactivated during this process. In iPS cells from patients with dyskeratosis congenita by contrast, telomere elongation during reprogramming is compromised. These findings create new opportunities to study stem cell diseases in cell culture and to develop therapies that could specifically reverse the disease defect.
  • Over the past year, we have generated and analyzed new induced pluripotent stem (iPS) cell lines engineered from individuals with a genetic stem cell disease. Dyskeratosis congenita is a rare disease affecting stem cells in multiple tissues. Patients with dyskeratosis congenita develop life-threatening bone marrow failure and pulmonary fibrosis, and are highly prone to cancers. In addition, they develop defects in skin, nails and many other organs. Dyskeratosis congenita is caused by mutations in an enzyme - telomerase - that is particularly important in stem cells. Telomerase elongates telomeres, caps that protect chromosome ends. If telomerase is defective, telomeres shorten and loss of the protective cap at telomeres can cause serious problems in stem cells. It has been very difficult to study this disease because isolating stem cells from dyskeratosis congenita patients is challenging. To overcome this problem, we engineered iPS cells from dyskeratosis congenita patients. This is a way to change skin cells into cells that closely resemble embryonic stem cells - stem cells that can give rise to all tissues within the body. We studied these iPS cells from dyskeratosis congenita patients and found that the type of effects on telomerase were very specific and depended on the specific gene that is mutated in the patient. Normal cells from healthy people show significant elongation of telomeres during the making of iPSCs, because telomerase is reactivated during this process. In iPS cells from patients with dyskeratosis congenita by contrast, telomere elongation during reprogramming is compromised. These findings create new opportunities to study stem cell diseases in cell culture and to develop therapies that could specifically reverse the disease defect.a

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