Optimized creation of lineage-specific reporter iPS cell lines
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.
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