The consequences of chromosome imbalance: using a trisomic human embryonic stem cell line to determine the primary defects in individuals trisomic for chromosome 13 (Patau syndrome)

Funding Type: 
SEED Grant
Grant Number: 
RS1-00308
ICOC Funds Committed: 
$0
Disease Focus: 
Diabetes
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Over 50% of all human conceptions have too much or little genetic material, often due to the gain or loss of entire chromosomes. The majority of these are spontaneously aborted, but a significant fraction develop to term. Perhaps the most well known is trisomy 21, Downs syndrome, in which the individual bears three rather than the normal two copies of chromosome 21. Aneuploidy results in severe developmental abnormalities including mental retardation and severe physiological abnormalities. In spite of its frequency and biomedical relevance, we known little about the primary causes of the developmental defects associated with aneuploidy. Unlike Mendelian inherited disease genes, aneuploidy is not due to disruptions in gene sequence rather it is due to changes in gene copy number. Until recently, we did not have the tools to determine the primary molecular defects in aneuplid individuals and studies necessary focused on secondary defects. However the development of human embryonic stem cells and more specifically the isolation of a human embryonic stem cell line containing three rather than the normal two copies of chromosome 13 now enables us to explore the primary defects associated with human aneuploidy. In this proposal we plan to use newly developed methods to explore global patterns of gene expression in normal and trisomic lines. In is our hope that these will provide a glimpse of the initial defects associated with human aneuploidy. If the primary defects are due to the misexpression of only one or two genes, this would offer the possibility of developing theurapetic drugs to compensate for the mis-expressed gene(s). In addition, we propose to explore strategies for rapidly establishing normal cell lines from the trisomic human embryonic stem cell line as a first step toward cell replacement therapy. .
Statement of Benefit to California: 
This grant focuses on the primary molecular and cellular defects associated with trisomy 13, also known as Patau syndrome. Approximately 1 in 8,000 live births are trisomic for chromosome 13. Trisomy 13 together with trisomy 18 and 21 account for about 2 of every 1,000 live births and therefore classify as rare diseases. Approximately 25 million Americans (and between 2 and 3 million Californians) suffer from over 6,000 rare diseases for which there is no financial incentive for companies to develop drugs. In response, congress enacted the very successful Orphan drug act that has resulted in an over tenfold increase in the number of drugs targeting rare diseases. More recently, congress enacted The Rare Diseases Act of 2002 in order to produce NIH-sponsored centers of excellence for research on rare diseases. Three of these centers are in California(UCSF, UCLA, UCSD). Due to the severe restrictions on federally funded research with human embryonic stem cells, to my knowledge there is no institutional backing for the application of hESC technologies toward the study and treatment of rare diseases. Funding the project proposed in this grant would be the first step towards laying the groundwork for a general strategy for CIRM to apply their resources toward helping the millions of Californians afflicted by rare diseases.
Progress Report: 
  • The goals of this proposal are to investigate endodermal differentiation and proliferation of human ES cells in culture. Endodermal differentiation is a necessary step towards making pancreatic beta cells, as well as other endodermal cells such as liver cells. Pancreatic beta cells generated from human ES cells could be used to treat type I diabetics. In the past two years, we have incorporated human ES cell culture technology into our laboratory and have been able to replicate data obtained by other research groups. While several other research groups and companies around the world are focused on making pancreatic beta cells as quickly as possible, we strongly believe that a more detailed understanding of the biology of human ES cell differentiation into endoderm will help the optimization of this protocol. Therefore, we have focused our efforts on testing a number of variables in the initial step of creating definitive endoderm. We have found that different human ES cell lines have very different capacity to differentiate into endoderm under the same culture conditions. In addition, we have recently focused our research effort on the post-translational modifications of key regulators of endoderm differentiation, and found a critical role for a poorly appreciated modification, namely a sugar modification called GlcNAcylation. In summary, developing a reproducible and efficient way to differentiate human ES cells into endoderm, as well as a thorough understanding of this key step, will allow us and others to elucidate the detailed set of molecular and biochemical events underlying this critical differentiation step, and will improve differentiation protocols.
  • The goals of this proposal are to investigate endodermal differentiation and proliferation of human ES cells in culture. Endodermal differentiation is a necessary step towards making pancreatic beta cells, as well as other endodermal cells, such as liver cells. Pancreatic beta cells generated from human ES cells could be used to treat type I diabetes. In the past two years, we have incorporated human ES cell culture technology into our laboratory and have been able to replicate data obtained by other research groups. While several other research groups and companies around the world are focused on making pancreatic beta cells as quickly as possible, we strongly believe that a more detailed understanding of the biology of human eS cell differentiation into endoderm will help the optimization of this protocol. Therefore, we have focused our efforts on testing a number of variables in the initial step of creating definitive endoderm. We have found that different human ES cell lines have very different capacity to differentiate into endoderm under the same culture conditions. IN addition, we have recently focused our research effort on the post-translational modifications of key regulators of endoderm differentiation, and found a critical role for a poorly appreciated modification—namely a sugar modification called GlcNAcylation. In summary, developing a reproducible and efficient way to differentiate human ES cells into endoderm, as well as thorough understanding of this key step, will allow us and others to elucidate the detailed set of molecular and biochemical events underlying this critical differentiation step, and will improve differentiation protocols.
  • We initiated a project on the role of post-translational modifications during hES cell differentiation into endodermal lineages, specifically on the GlcNAcylation sugar modification. We found that this modification appears to be important for endoderm formation in hES cell cultures. Identification of modified proteins is an important next step in understanding the mechanisms of this phenomenon and may ultimately provide a basis to develop assays for screening drugs that enhance endoderm/beta-cell formation.

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