Formation of the Satellite Cell Niche in Engineered Human Skeletal Muscle: The Roles of Vascularization and Notch Signaling

Abstract

Tissue engineering methodologies have enabled generation of sophisticated 3-dimensional (3D) cell culture systems for in vitro studies of tissue development, physiology, disease, and drug response. Specifically, engineered skeletal muscle tissues can be produced to contain both contractile muscle fibers and a specialized population of cells that closely resemble the native adult muscle stem cells, known as satellite cells (SCs). SCs are essential founder cells that endow skeletal muscle with the capacity to seamlessly regenerate in response to everyday injuries and lacerations. In homeostatic muscle, SCs are quiescent, reside in specialized microenvironments, called “niches”, and only proliferate in response to injury to repair damaged muscle fibers. SC quiescence within the niche is regulated by a myriad of cell-cell and cell-matrix interactions including endothelial cell (EC) mediated Notch signaling. While these interactions could be studied in vitro, traditional co-culture of ECs and skeletal muscle cells is limited by media incompatibility, which results in either impaired skeletal muscle differentiation and function or requires compartmentalization of skeletal muscle and endothelial components, limiting interactions between ECs and SCs. Furthermore, despite reported therapeutic effects of Notch signaling upregulation in dystrophic animals, the effects of Notch signaling upregulation on human SCs remain understudied. To overcome these limitations, we set the following dissertation goals: 1) develop and optimize a high-fidelity in vitro model of human vascularized skeletal muscle to study the effects of ECs on SC abundance and quiescence and 2) study the roles of Notch signaling in SC abundance, quiescence, and myogenic capacity using a tissue-engineered muscle model with inducible Notch ligand overexpression. To achieve these goals, we first optimized media and initial cell ratios for co-culture to generate highly functional vascularized human skeletal muscle tissues (“myovascular bundles”) with contractile properties (~10 mN/mm2) equaling those of avascular, muscle-only tissues (“myobundles”). Within one week of muscle differentiation, ECs in these tissues formed a dense network of capillaries that co-aligned with muscle fibers and underwent initial lumenization. Incorporating vasculature within myobundles increased the total SC number by 82%, with SC density and quiescence signature being increased proximal (≤20μm) to EC networks. In vivo, at two weeks post-implantation into dorsal window chambers in nude mice, vascularized myobundles exhibited improved calcium handling compared to avascular implants. Next, we sought to utilize the 3D myobundle platform to evaluate the effects of Notch signaling upregulation on human SC behavior in a biomimetic model of human skeletal muscle. Specifically, we examined the effects of temporally controlled expression of the Notch ligand Delta-like ligand 1 (DLL1) on undifferentiated and differentiated myobundles. Early DLL1 expression enhanced Notch signaling and resulted in increased SC abundance and an elevated quiescence-associated transcriptomic signature, while impairing muscle differentiation and contractile function. Intriguingly, muscle progenitor cells isolated from myobundles with increased Notch signaling were able to form “secondary” myobundles that exhibited normal muscle differentiation and contractile function, while also showing increased abundance of Pax7+ SC-like cells. In summary, this thesis has developed new methodologies for vascularization of human myobundles and perturbation of SC niche signaling aimed at enhancing our understanding of mechanisms governing SC behavior. We describe for the first time highly functional myovascular tissues that can be utilized to study the roles of EC-SC crosstalk in human muscle development, physiology, and disease. Additionally, we present a novel in vitro platform that enables time-dependent modulation of Notch signaling within a 3D engineered human SC niche, effectively increasing SC abundance and inducing a shift in SCs towards a more quiescent state. We anticipate that these findings will pave the way for future studies of cellular and signaling constituents of the SC niche and facilitate understanding of human skeletal muscle biology and discovery of new therapies for muscle disease.