Funding opportunities

Fluorescent reporters for monitoring key transcriptional control steps in the differentiation of human embryonic stem cells into T cells

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01002
Funds requested: 
$907 204
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human embryonic stem (hES) are defined by their ability to generate all of the different cells in the body, from brain cells to infection-fighting white blood cells, and everything in between. The environment around the hES cells signals them to become certain cell types. This process, dubbed differentiation, involves the step-wise generation of multiple intermediate cell types that, over time, become increasingly restricted to making just one cell type. Through this series of steps, a hES cell becomes a functionally mature cell, capable of performing its specialized function(s) in the body. Of particular therapeutic interest, is the ability to generate a subset of white blood cells, called T cells, from hES cells in a culture dish. T cells are best known as cells exquisitely poised to defend us against infections. T cells can also destroy tumor cells, and their activities can be directed to alleviate autoimmune diseases such as type I diabetes, rheumatoid arthritis, and multiple sclerosis. We are currently optimizing conditions to direct hES cells through several developmental intermediates into functional T cells. These developmental intermediates, from hES cell to T cell, can be broadly classified as mesoderm, hemangioblast, hematopoietic stem cell, common lymphoid progenitor, and T cell precursor and can be identified by the sequential expression of identifiable genes. In this grant application, we propose to tag developmentally important genes along the T cell differentiation pathway with unique fluorescent proteins (red or green) in established hES cell lines. The fluorescent “tag” can be used to “report” when a particular gene or genes for cells with double “tags” are turned on in the cell. The genes chosen to “tag” are those known to appear at particular developmental stages, allowing us to visualize, without disruption of the cell or further cell manipulation, the differentiation process from hES cell to T cell precursor. The availability of these engineered cells will further enable us to determine the requirements of individual differentiation step in this pathway and provide invaluable information for the eventual goal to generate mature and functional T cells in a culture dish. In addition, these fluorescent cells will also give us the opportunity to develop video imaging technique using advanced microscopy to observe the differentiation of particular T cell developmental intermediates in space and time. Successful completion of the proposed research will not only generate invaluable information as to how, where, and when specific signals are sent and received by cells in their decisions to develop into white blood cells but also provide tools for studies of differentiation for other cell types.
Statement of Benefit to California: 
Genetic modification of existing human embryonic stem (ES) cell lines to report, via fluorescent proteins, developmental milestones during the differentiation from stem cell to T cell, will have a major impact on both basic and clinical/translational research. These novel tools will significantly improve our ability to screen cell culture conditions in order to develop the optimal protocol for generating T cells from hES cells in a culture dish. This, in turn, will facilitate the production of T cells from this potentially limitless source that could provide much needed therapies for many Californians suffering from AIDS, cancer, and autoimmune disorders. In addition, the hES reporter lines generated in this proposal will significantly increase our basic understanding of the development of the human immune system. Also, hES reporter lines are amenable for high-throughput screens, which are of great interest to other researchers and biotech companies in California for a number of uses including, but not limited to, drug screening for a variety of blood cell disorders such as leukemia and lymphoma.
Review Summary: 
This proposal focuses on the creation of fluorescent, hESC reporter lines that will be used to optimize differentiation towards the T cell lineage. The Principal Investigator (PI) proposes to use homologous recombination to target reporter constructs to three different loci that have been shown to be important for T cell development in rodent studies. In addition to single insertions, the applicants will attempt to create cell lines with reporters at two loci for use in subsequent optimization of differentiation procedures. Finally, the applicants propose to use the reporter lines to develop video imaging technologies to observe T cell differentiation under conditions that might more closely mimic the natural environment in which the cells develop. The reviewers were enthusiastic about the scope of this proposal and the capabilities of the research team but felt that the PI did not provide a sufficiently compelling case that the technical challenges could be met. In addition, the third aim appeared ancillary to the theme of the proposal. If successful, the impact that this technology would have on the field is substantial. Stem cell studies have been hampered by the lack of stable transgenic lines such as that are proposed by the applicants. Researchers could use these reporter lines to screen for factors that drive T cell differentiation. Such differentiated cells could be used for a variety of applications including therapies for AIDS, cancer, and autoimmune disorders. In addition, these cells could prove useful for elucidating the mechanisms that underlie hematopoiesis and development of the human immune system. The reviewers felt that the third aim was incompatible with the overall goals and would not contribute substantially to the impact of this proposal. The reviewers judged the feasibility of this effort to be uncertain, despite their confidence in the qualifications of the research team. While the PI has had success with similar studies in murine cell lines, it is not clear that analogous approaches will be similarly successful in human cells. Reviewers pointed out that there is only one example of a successful, single knock-in line in this field, mainly due to difficulty of achieving homologous recombination (HR) and the inherent fragility of hESC. Attempting to obtain double-reporter strains is extremely ambitious, and the reviewers were uncertain that this could be readily achieved. The reviewers were very concerned that the final reporter lines might not be representative of the parent hESC population, particularly given the multiple cloning steps and selection strategies that would be required to achieve the correct gene targeting events. The reviewers were also uncertain of the extent to which the knock-in constructs might alter the ability of hESC to differentiate into T cells. The reviewers were confident in the capabilities of the research team. The PI is an established, proven immunologist and is well versed in gene targeting technology. The research team has successfully performed experiments in murine cells that are similar to those proposed here. The budget was determined to be excessive, mainly due to unnecessary personnel and equipment requested for Aim 3. Overall, while the scope and potential impact of this proposal are impressive, the effort is not adequately justified by the technically challenging, risky and ambitious approach, despite the team’s qualifications.
Conflicts: 

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