Leveraging In Vivo and Ex vivo Approaches to Alter Adult Muscle and Blood Stem Cell Fates

Abstract

Adult stem cells play a central role in maintaining tissue homeostasis throughout life, with the blood and skeletal muscle systems relying heavily on hematopoietic stem cells (HSCs) and muscle stem cells (satellite cells), respectively. These specialized cell populations sustain tissue function through their ability to self-renew and differentiate into mature cell types. However, the regenerative capacity of stem cells can be compromised by aging, genetic alterations, and disease, ultimately leading to tissue dysfunction. Understanding how adult stem cells maintain, and in some cases lose, their regenerative potential is critical for advancing cell-based therapeutic strategies. In this thesis, I sought to apply both chemical and genetic approaches to interrogate the regulators of adult stem cell function, in health and in disease. I first worked to improve the regenerative potential of satellite stem cells by exposure to small molecules that would enhance their engraftment following transplantation. Using zebrafish and mouse model systems, I worked collaboratively to investigate the impact of ex vivo treatment with FDA-approved lipid biomolecules on satellite cell survival and function. Our studies showed that treatment with either lysophosphatidic acid (LPA) or niflumic acid (NFA) significantly enhances the engraftment efficiency of muscle stem cells, in part by promoting cell survival via a calcium-dependent mechanism. These findings highlight the importance of ex vivo manipulation as a strategy to improve stem cell-based therapies. As it is likely to be challenging to establish small molecule modulators for all stem cell-relevant gene targets, I additionally evaluated genetic approaches to stem cell manipulation. As a first step, I focused on overcoming a major limitation in the field: the ability to efficiently modify stem cell genomes in situ. This effort specifically utilized recombinant adeno-associated viruses (AAVs) as vectors for delivery of genetic cargo to endogenous hematopoietic stem cells (HSCs). AAVs have emerged as leading tools for in vivo gene delivery due to their strong safety profile and adaptability, but vectors with robust tropism for HSCs, and particularly human HSCs have yet to be identified. Using an in vivo screening approach, I worked collaboratively to assess a pool of novel AAV variants to identify those with enhanced transduction efficiency for hematopoietic cells. This effort resulted in the identification of several variant capsids with improved performance both ex vivo and in vivo. These findings demonstrate the potential to tailor AAV vectors for more effective targeting of endogenous stem cell populations. Building on these findings, I finally expanded application of the AAV-mediated gene delivery approaches I’d developed to create a new tool to study stem cell biology in vivo. By combining AAV-mediated delivery of guide RNAs with transgenic Cas9 mouse models, I established a modular system to introduce targeted genetic perturbations into HSC genomes. This approach enabled direct interrogation of gene function within the native hematopoietic environment and provides a new platform for modeling early stages of stem cell dysfunction. Together, this work highlights the complementary roles of ex vivo and in vivo strategies in advancing cell-based therapies. By improving satellite cell engraftment and developing AAV-based tools for targeting and modifying HSCs, this thesis underscores the potential of stem cells both as therapeutic agents and as platforms for studying tissue regeneration and disease.