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