Engineering Platforms for The Creation Of Lineage-Rainbow Reporter Lines In Human Embryonic Stem Cells
One of the more innovative areas of the stem cell research has been the effort to harness the value of the stem cell itself as a research tool for drug testing, basic developmental research, and potential therapeutic applications. Success in such efforts largely rely on development of tools and technologies. Once such area that remains a challenge is uniform efficient differentiation of cell types of desired lineages.
Current methods of differentiation often results in cells that lack mature cell-specific markers and fail to show functionality. Development of complex, multi-stage differentiation protocols that mimic developmental stages are promising but laborious and require monitoring of appropriate marker expression as the differentiation progresses along the various stages. An ideal tool that would aid in this process would be creation of reporter ESC lines that allows for live monitoring of transition of cells from its stem cell state to the desired lineage.
This proposal describes the use of a novel platform technology to engineer stem cells that enables the insertion of multiple genetic elements into a single defined chromosomal locus. The platform will provide a more relevant and easier-to-use way to create novel stem cell reporter lines that can be genetically programmed to produce desired readouts for gene expression from live cells. As proof of principle, we propose the creation of hESC lines expressing three reporters independently driven by three promoters specific for pancreatic lineage differentiation. Such reporter lines will aid in the development of homogeneous differentiation methods and enrichment methods to enrich cells at various stages of specific lineages.
The importance of human embryonic stem cells in basic biology and regenerative medicine has led to its rapid growth since first isolated a decade ago. Further progress towards it application largely relies on development of new and novel technologies.
While most ventures are focused on generating lineage specific stem cells for treatment of various diseases, our focus is to develop tools to aid researchers in such ongoing efforts. The core technologies proposed here involve novel methods such as Multisite Gateway and site specific integration. Combination of these two methods will provide a platform technology with clear competitive advantage in efficiently assembling multiple promoter reporter systems for insertion into specific locations within the genome. The resulting reporter ESC lines will aid in development of efficient methods to generate cells of desired lineage, a critical objective that needs to be fulfilled to realize their therapeutic potential in regenerative medicine.