The functional role of protein O-GlcNAcylation in hESC pluripotency and differentiation

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: 
Human embryonic stem cells can be changed into virtually any cell type in the adult body. Because of this unique capability, stem cells have the potential to cure a vast majority of existing human disorders. However, the mechanisms that govern the definition and function of stem cells has not been completely elucidated, and several hurdles exist and need to be overcome before stem cells can be used in the clinic. For example, the factors which govern the conversion of stem cells into a variety of tissue types such as liver, heart, and brain tissue - are not well understood. Our research employs a unique multidisciplinary approach to bridge this information gap. Proteins govern the daily life of cells by controlling when genes are activated, how cells communicate with one another, and several other critical processes. The action of proteins inside cells is commonly turned on and off by the appending to, or removal of, sugars from proteins. Though this control mechanism is well established in other areas of health and human disease, it has not been widely studied in the context of stem cell biology. The proposed research will examine how the sugars found on proteins impact processes such as the differentiation of stem cells into neurons, the generation of pluripotent stem cells, and how the genetic reprogramming of stem cells is actually carried out by cellular proteins. The results of these studies may lead to a greatly increased understanding of how stem cells retain their ability to be changed into other cell types, and also how the fate of stem cells is decided upon differentiation. Both are critical areas that need to be explored to enable modern regenerative medicine to realize its full potential as a tool for the treatment of human diseases.
Statement of Benefit to California: 
Programs funded by CIRM and other state granting agencies will allow California to continue to be at the frontier of stem cell research for the development of new treatments to cure human diseases. Research such as ours will hopefully enable modern medicine to access exciting new areas such as spinal regeneration, and finding treatments for neurodegenerative disorders for which there is currently little hope for curing. Some illnesses which could be potentially impacted include multiple sclerosis, Alzheimer’s, Parkinson, and Batten diseases. Several hurdles exist, however, which need to be overcome before results from the exciting field of stem cell research can be used in the clinic. For example, the factors which govern conversion of stem cells into a variety of tissue types that may find uses in regenerative medicine such as the liver, heart, and brain, are not well understood. Our research employs a unique multidisciplinary approach to bridge this information gap. In particular, our research will examine how the sugars which are attached to proteins control processes such as the vast genetic reprogramming that accompanies the conversion of stem cells into mature tissues. Through initiatives like CIRM, California will continue to lead the nation in the discoveries resulting from multidisciplinary scientific research which will fuel tomorrow’s medical advances.
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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