Funding opportunities

Engineering Platforms for The Creation Of Lineage-Rainbow Reporter Lines In Human Embryonic Stem Cells

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01096
Funds requested: 
$598 752
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
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.
Statement of Benefit to California: 
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.
Review Summary: 
The goal of this proposal is to engineer a multi-reporter human embryonic stem cell (hESC) line that can be used to optimize differentiation procedures and to produce homogenous populations of functionally mature derivatives. The applicants propose to use the pancreatic endocrine lineage as a model for developing and optimizing this tool. In the first aim, hESC lines will be generated that express three different reporter proteins from promoters that are active at sequential stages of the endoderm/pancreatic lineage. The reporter constructs will be stably inserted into a specific chromosomal site using a recently published, non-viral integrating methodology. For the second aim, the applicants propose to validate their new tool by using published protocols to perform staged differentiation of the reporter line and produce mature, insulin-producing beta cells. The reviewers were enthusiastic about the proposed technology and its potential impact on the field as it addresses a key roadblock in stem cell biology: the difficulty of achieving efficient and homogeneous differentiation into desired lineages. If successful, the proposed work may provide useful tools for fine-tuning of pancreatic differentiation protocols. Furthermore, while this application focuses on pancreatic cells, the technology is described as a platform that could be applied to any number of lineages and as such could have a larger impact on the stem cell field. With regard to Aim 1, the reviewers were enthusiastic about the strategy for engineering and integrating the reporter constructs, a strategy they characterized as innovative, straightforward, realistic and supported by encouraging preliminary data. Reviewers were convinced that this aspect of the proposal would be likely to succeed. However, some reviewers expressed concern about the approaches relating to the application of this technology to the pancreatic lineage in Aim 2. One reviewer felt strongly that the promoters chosen to represent different stages of pancreatic differentiation were not ideal, and was particularly concerned about the final differentiation step, since functional maturity of beta cells is not only defined by expression of the gene whose promoter will drive reporter expression. Thus it remained unclear how the applicants will assess functional maturity of in vitro differentiated cells. Additional criticism was leveraged against the choice of cell line for validation of pancreatic expression of the reporter constructs, since that cell line does not represent the beta cell lineage. Based on these and additional concerns, the reviewers were not convinced that the proposed experiments would lead to the generation of mature beta cells. The reviewers felt that the applicants were well qualified to generate the proposed reporter cell lines, and that the research environment is exceptionally appropriate for this task. However, the investigators lack expertise in pancreatic cell biology, and reviewers suggested that the team would benefit greatly from a collaborator or consultant who is an expert in this area. The budget was judged to be slightly excessive. In summary, this proposal describes a powerful new tool that seems technically feasible, but several weaknesses in the validation strategy raised doubts about its feasibility and significantly tempered the enthusiasm of the reviewers.
Conflicts: 

© 2013 California Institute for Regenerative Medicine