Use of small organic molecules to enhance, control, and understand survival and self renewal of human embryonic stem cells in vitro

Funding Type: 
SEED Grant
Grant Number: 
RS1-00308
ICOC Funds Committed: 
$0
Disease Focus: 
Diabetes
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Overview of proposed research: Our long-term goals are to develop better defined media for in vitro culture of human embryonic stem cells (hESC) and to understand mechanisms regulating survival and self renewal of stem cells in vitro. Achievement of these goals is necessary to facilitate work with hESC and for future therapeutic applications of stem cells to human disease. Our specific strategy is to identify small organic molecules that promote survival and self renewal in hESC when added to culture media. We have already created a focused chemical library containing biologically active molecules that are effective at very low doses. Some of these molecules have growth factor activity when tested with human cells. The first molecule that we tested in this library acts as a potent growth factor for mouse ESC (mESC) cultured without fibroblast feeder cells or a protein substrate for cell attachment. Our goals in the proposed project are to screen our focused chemical library to identify additional chemicals that enhance survival and self renewal of hESC and to characterize the effect of each “hit” on attachment, growth, chromosomal stability, and pluripotency. We will meet these goals by first purchasing about 150 chemicals to augment our already existing focused chemical library. These chemicals will be structurally similar to those we already know to be highly biologically active. We will then screen our augmented focused library using hESC. Cells will be cultured in various doses of each chemical, and the effect on growth will be monitored after 24 hours by comparing cell number in control and treated groups. Chemicals that significantly increase cell number during 24 hours will be considered “hits”, and assays will then be performed on each “hit” to determine their effects on cell attachment, survival and growth, chromosomal stability, and pluripotency. In addition to our own focused library, we also have access to five diverse chemical libraries at our campus that we can screen in the unlikely event that no additional “hits” are found in our focused library. Potential contribution to therapies: There is a great need for developing better defined culture media for the growth and maintenance of hESC. Advances in this area are critical to development of hESC as biomedical tools for treatment of disease. Culture media that support growth without a layer of feeder cells and that are chemically defined are necessary to facilitate work in this field and for future therapeutic applications of stem cells to human disease. Improvements in culturing technology and maintenance of stability are necessary to advance research in this area and will potentially affect all applications of hESC to treatment of disease. In conducting our work, we expect to find multiple chemicals that will become valuable additives in hESC culture media and to contribute to our basic understanding of hESC survival and self renewal in culture.
Statement of Benefit to California: 
In November 2004 the voters of California passed Proposition 71 by a large margin indicating the importance of stem cell technology to citizens of our state. Stem cell technology provides one of the most optimistic technologies in biomedical research for the future and has the potential to address treatments for many major diseases such as diabetes and Parkinson's disease. Our country has always been at the forefront of biomedical technology, and California now offers the promise of keeping our country in that position through funding of stem cell research. The development of better technologies for the growth and maintenance of hESC in culture is fundamental to any forward achievements in the application of stem cells to treatment of human disease. Before hESC can be used therapeutically, they will need to be cultured in media that support self renewal and at the same time maintain chromosomal stability and pluripotency. Ideally these media would not have animal products and feeder cells would not be used. Development of suitable media is a complex problem that needs to be explored from multiple perspectives. Improvements need to be thoroughly tested to ensure cells remain capable of self renewal, chromosomally stable, and pluripotent. Our proposed work builds off of our preliminary data that identified a small organic chemical capable of greatly enhancing survival and self renewal of mouse embryonic stem cells (mESC) grown in the absence of a feeder layer or protein attachment substrate. We propose to examine the ability of this and related chemicals to enhance growth, stability, and maintenance of pluripotency of hESC. The data obtained in our project should lead to identification and characterization of small organic molecules that can be added to hESC culture media to improve survival, growth, and maintenance of pluripotency. Addition of such chemicals to culture media will streamline media and make them more defined. Our work may also lead to improvements in methods for culturing other types of stem cells, such as adult stem cells. Moreover many of the chemicals that we will screen are considered safe and are already approved as additives to human consumer products. Improvements in hESC culture media are fundamental to any therapeutic work with stem cells. Data obtained on our project will improve the technology for culturing hESCs and thereby move us a step closer to being able to use stem cells to treat disease. The use of hESC therapeutically will be directly or indirectly extremely important to the citizens of California. Even young citizens who currently do not have major medical problems could benefit in the event of future illness or accidents. Development of hESC technologies and their application to medical problems will also contribute to the overall economy of the state by creating opportunities for the formation of new biotechnology companies and by stimulating growth of existing companies.
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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