NCE Year 4

Pluripotent stem cell research promises to revolutionize biomedical research by providing a human cell-based model system to study disease progression, test drug efficacy and safety and generate mature cells suitable for cell transplantation. However, progress in this direction is impeded by several deficiencies. First, differentiation of human pluripotent stem cells (hPSCs, both embryonic and induced pluripotent stem cells) is often inefficient and incomplete, generating only limited numbers of cells with the desired properties surrounded by an excess of undesirable and even tumor-forming cell populations. Second, culture conditions to promote differentiation into specific cell types are often undefined and contain animal products. Third, while specific mature cell populations have been obtained in relatively small numbers sufficient for basic science research, culture conditions for scale-up are limited.
The goal of the proposed research is to develop a technology that enables stem cell research and provides solutions to these shortcomings. Specifically, we are generating hPSC lines carrying genetic elements that will allow us to observe and track when a cell type has achieved a particular state of differentiation. This technology will facilitate the development of cell isolation and purification methods. This is especially important so that undesirable cell types—such as tumor forming cells—can be efficiently separated from the cells of interest.
To generate “marker cell lines”—cell lines carrying reporter genes under control of tissue-specific promoters—we used a viral gene transduction method that relied on a genetically modified adeno-associated virus (AAV). This virus can efficiently infect human cells while having no known adverse effects. We developed a large collection of viral DNA constructs that we used to target genes encoding fluorescent proteins to specific locations of the genome. More recently, we have integrated another method, called CRISPR/Cas9, with the AAV-based method to increase the efficiency of generating marker cell lines. The successful completion of this research will provide the scientific community with a large panel of these marker cell lines that can be employed to develop, optimize and define differentiation protocols. In addition, this research will provide a streamlined technology for site-specific alteration and correction of the genome.
A recent publication from our laboratory describes the successful use of one such marker cell line (Brafman et al. Stem Cell Reports, 2013, PMID: 24286033). Importantly, this engineered cell line allowed us to define conditions to derive lung progenitor cells. Such cells have the potential to replace damaged lung tissue. Ongoing experiments are leveraging these findings for additional funding and to develop therapeutic strategies for the treatment of diseases in which lung tissue is damaged, defective or dead.