Project Summary
The short lifespan of human somatic cells, including adult stem cells, in ex vivo settings is a significant
hurdle for many clinical and research applications. Therefore, the development of novel approaches that can
facilitate the expansion of adult cells and at the same time restore the young-like characteristics of these cells
without permanent immortalization is of high priority for regenerative medicine. Telomere attrition is one of the
well-characterized mechanisms responsible for the decline in proliferation and function of somatic cells in
culture and can be counteracted by the activity of the enzyme telomerase. Mitochondrial dysfunctions and
epigenetic changes are also among mechanisms responsible for cellular aging, senescence and decline in
functionality. In our preliminary experiments, we developed a patent-pending non-integrating RNA-based
cocktail of factors that upon transfection into human somatic cells increases the length of telomeres to that
observed in pluripotent stem cells, improves the proliferation rate of cells, restores mitochondrial DNA content
and allows for a clonal expansion of individually seeded adult human fibroblasts (FBs). We now propose to use
this rejuvenating RNA cocktail to develop two novel cellular technology platforms. One will improve the
expansion of human primary somatic cells and promote their rejuvenation in a clinically relevant manner
without permanent immortalization, while maintaining the proliferative capacity, functionality and normal
characteristics of these primary cells. The second platform will allow for the efficient clinically relevant genetic
engineering directly in primary somatic cells. In the latter, the use of our rejuvenating RNA cocktail will allow for
the clonal expansion of genetically modified primary somatic cells, while preserving the functionality of these
cells for subsequent transplantation. To develop the cell expansion platform, in Specific Aims 1 and 2, we will
further characterize the effect of our cocktail on three human cell types commonly used in research and clinical
settings: FBs, keratinocytes (KCs) and mesenchymal stromal/stem cells (MSCs). We will perform detailed
molecular and functional characterizations of low and high passage human FBs, KCs and MSCs treated with
our cocktail with a focus on the restoration of young-like characteristics of these cells. In Specific Aim 3, we will
employ our rejuvenating cocktail to develop a platform for the generation of genetically modified human
somatic cell lines. Using Cas9-mediated gene targeting, we will generate a panel of primary human FB, KC
and MSC lines with the site-specific knock-in of fluorescence reporters and luciferase. The derived lines will be
tested ex vivo and in vivo to confirm their functionality and safety, providing feasibility data for the development
of novel somatic cell gene therapies for many diseases. If successful, the studies will serve as a proof of
principle for developing rejuvenating strategies and genetic engineering platforms for other somatic cell types
used for transplantation, such as hematopoietic and muscle stem cells.
