Tools and Technologies II
Human embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells have the ability to “differentiate” or turn into virtually any cell type found in the human body. This makes them very promising sources of cells to replace corresponding defective cells in patients suffering from conditions such as diabetes, Alzheimer’s, heart, and sickle cell disease. However, there are a number of challenges that will have to be overcome in order to move this technology from the lab bench to the clinic. Currently one of the bottlenecks in laboratory studies of human pluripotent stem cells is the lack of robust cell markers to track the behavior of the cells as they progress into the desired cell type. To address this bottleneck, we will create large bacterial artificial chromosome (BAC) constructs containing entire genes with full regulatory elements to ensure the faithful and timed expression of these reporter genes. We will then introduce these reporter constructs into human pluripotent stem cells at “safe” regions in the genome, areas that do not contain essential genes or regulatory elements. The success of our study has very important implications. Firstly, it will generate methods to introduce reporter genes that will allow tracking and recovery of desired cell types that arise from the original stem cells. Secondly, our study will generate materials that will be made available to other researchers. These materials will in turn help CIRM-funded groups and others in their efforts to further understand molecular mechanisms of stem cells and also boost the development of stem-cell based therapies.
Statement of Benefit to California:
Precise control and monitoring of development of specific tissues starting from human embryonic stem cells (ESCs) or induced-pluripotent stem cells (IPSC) is essential for regenerative medicine. Real time monitoring of live cells is facilitated by the availability of stem cell lines harboring lineage-specific “reporter” genes. Such reporter cell lines will, for instance, provide a fluorescent light signal when the proper tissue cell type is generated, thus enabling the identification of factors controlling cellular differentiation. This proposal aims to provide an “open-source” of protocols & resources to create stem cell lines equipped with stable tissue-specific reporters. We will develop a modular approach to efficiently integrate cell-type specific fluorescent markers into human embryonic stem cells. By creating readily available resources and procedures, we hope to expedite stem cell research in other stem cell laboratories. We will develop our protocols with an initial focus on hematopoietic (blood cell) lineage tracking. This preference reflects our interest in sickle-cell disease and CIRM-funded interests at [REDACTED] in the development of the immune response through T-cell development. We will seek the advice of CIRM and CIRM-funded researchers to nominate specific genes for lineage tracking to address neurological, cardiac, and hematopoietic diseases. A public website and publications will be used to report progress and disseminate protocols and resources to the research community
The goal of this proposal is to generate lineage-specific human pluripotent stem cell (hPSC) BAC reporter lines. The absence of stable cell-type specific reporters presents an impediment to developing robust methodologies for the specification and progression of stem cells to a desired cell type. To address this translational bottleneck, a series of three aims has been proposed. First, the applicant will create and optimize a set of modular protocols for preparing reporter genes based on bacterial artificial chromosomes (BACs). Next, a series of BAC constructs will be generated and integrated into “safe harbor” genomic sites to create transgenic hPSC reporter lines, the utility of which will be assessed by collaborators. For the final aim, a public website and other resources will be established to distribute the knowledge, tools and cell lines that emerge from this work. Reviewers agreed that a lack of robust, well-characterized hPSC lines with cell-type specific reporters represents a significant translational bottleneck to the development of cell therapies. If successful, the proposed tools could have considerable impact on the pace of discovery. Reviewers found the overall strategy of modular vector construction and delivery to be powerful and elegant, citing the ability to engineer large constructs with intact genomic regulatory sequences as an important advantage over other systems. The novelty, however, was considered moderate, as the BAC approach and other components have been previously established. Furthermore, while the reviewers appreciated the techniques, they were uncertain of the extent to which the reporter lines would prove useful. As only a limited number of hPSC lines were being considered for modification, they cautioned that the differentiation procedures developed through their use might not be broadly representative of other hPSC populations. Reviewers also questioned the rationale for targeting BAC constructs to safe harbor loci, particularly as the proposed reporter lines would be used for optimizing differentiation procedures rather than for therapeutic applications. While such targeting could minimize the risk of gene silencing or oncogenic activation, the trade off in terms of throughput and expense could prove significant. In addition, there were no rigorous plans to characterize the effect of integration site on reporter expression. Most reviewers believed that a concerted effort to randomly integrate BAC constructs into multiple lines and obtain convincing validation of expression would represent a simpler, more judicious approach forward. As presented, some felt the proposed outcomes resembled a core service to provide existing technologies rather than developing a specific and transformative tool for the research community. Overall the reviewers found the research plan to be feasible, with well-conceived aims and appropriate milestones. Although potential pitfalls and alternative strategies were only superficially acknowledged, reviewers agreed that the preliminary data strongly support the capabilities of the team to achieve the proposed goals. While confident in the underlying technology, reviewers were less certain of the plans for quality control and distribution, which they found poorly elaborated and lacking in detail. Most of them questioned the feasibility of maintaining and disseminating the generated cell lines with an appropriate level of quality control. Several reviewers indicated that an arrangement with an existing cell bank might prove more effective as a means for achieving this objective. Reviewers described the PI as extremely qualified, with a phenomenal track record and an unquestioned ability to supervise a project of this scope. The co-investigators were highly regarded for their respective expertise in BACs, gene modification, and hematopoietic differentiation. While they did not doubt the capabilities of the team, reviewers noted that the impact of this work rests on an assumption that collaborators and members of the scientific community will provide validation of emerging tools. Overall, while reviewers appreciated the outstanding research team, underlying technology and feasible research plan, they were not convinced that this effort would have significant impact on a translational bottleneck and therefore did not recommend this application for funding.