Cardiovascular disease (CVD) represents a global medical and economic problem with high morbidity and mortality rates. CVD is often associated with partial occlusion of the blood vessels and tissue ischemia, and restoring blood supply to ischemic tissues is critical to prevent irreversible tissue damage. Therapeutic angiogenesis aims to stimulate the growth of new blood vessels from pre-existing vessels, which offers a valuable tool for treating CVD. Several strategies have been developed to promote angiogenesis, including growth factor delivery and gene therapy. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth. However, it is limited by short half-lives in vivo, and uncontrolled diffusion of angiogenic factors may also cause undesirable side effects. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but previous approaches often require the use of viral vectors for efficient gene delivery, and are limited by safety concerns such as immunogenicity. The goal of this thesis research is to develop novel strategies for stimulating therapeutic angiogenesis by harnessing stem cells as drug delivery vehicles and to validate the efficacy of non-viral engineered stem cells in vivo using mouse models of hindlimb ischemia. Our strategy takes advantage of the natural homing capacity of stem cells towards ischemic tissues in vivo, and their ability to secrete paracrine signals to stimulate blood vessel growth. Specifically we developed two strategies including: (1) isolating and transfecting stem cells ex vivo using biodegradable polymeric nanoparticles to overexpress therapeutic genes, followed by transplanting non-viral engineered stem cells back to ischemic tissues; and (2) recruiting and programming endogenous stem cells in situ using biomaterials-mediated delivery of biologics. In the first strategy, we have chosen adipose-derived stem cells (ADSCs), an abundantly available autologous cell source that can be easily obtained in a minimally invasive manner. To enhance the paracrine signaling of ADSCs for therapeutic angiogenesis, we transfected ADSCs using in-house developed biodegradable polymeric nanoparticles, which eliminate the dependence on viruses for efficient gene delivery. Using the optimized polymeric vectors, we examined the efficacy of ADSCs overexpressing various angiogenic factors or homing factors on therapeutic angiogenesis in vitro and in vivo. Transplantation of non-viral engineered ADSCs led to significantly enhanced tissue salvage in a murine model of hindlimb ischemia with faster restoration of blood reperfusion and muscle regeneration. Our results suggest that stem cells programmed with biodegradable polymeric nanoparticles can serve as delivery vehicles to express therapeutic factors in situ to promote therapeutic angiogenesis. In the second strategy, we seek to circumvent the need of isolating and manipulating stem cells ex vivo by directly recruiting and transfecting endogenous progenitor cells in situ at the site of ischemia. To achieve this, we developed a biomaterials-mediated delivery platform for sequential release of stem cell homing factors and DNA encoding therapeutic genes. Our results show that biomaterials-mediated release of homing factors followed by delayed DNA delivery enhanced recruitment of endogenous progenitor cells and improved limb salvage in a mouse model of hindlimb ischemia. Finally, we demonstrate the potential of using microfluidic-synthesized microspheres to aid therapeutic angiogenesis using a biomaterials-mediated drug delivery depot.

Topic Editor RL is a patent inventor on exosome-related patents, PCT/AU2017/050821 and PCT/AU2016/050468. All other Topic Editors declare no competing interests with regards to the Research Topic subject.

The repair of damaged tissues through cell and growth factor administration is a promising strategy in regenerative medicine. However, clinical success has been limited due to the lack of effective delivery methods. While deemed minimally invasive, the conventional approach of direct injection in saline compromises the efficacy of the biological payloads. Post-injection cell survival is often dismally low, owing to the combination of shear forces during injection and hostile environments at the injury site. Growth factors delivered by bolus injections face rapid clearance and distribution to off-target sites, leading to suboptimal concentrations at the sites of therapy and potential side effects in normal tissues. These are major therapeutic hurdles because symptomatic relief following cell therapy is often correlated with the number of surviving donor cells. In the case of growth factor therapy, spatiotemporal control over growth factor distribution is critical for normal recovery, as tissue regeneration is a tightly coordinated process. This thesis presents a biomaterials approach to overcome these hurdles by harnessing polymer and protein engineering strategies to design injectable hydrogel carries for optimal cell and drug delivery. A broad survey of protein-engineered biomaterials is first given in Chapter 1 to provide a foundation for the design principles, synthesis, and characterization methods employed in subsequent chapters. Chapter 2 describes a mechanistic investigation into the physical forces imposed on cells during injection, using alginate polysaccharides as model carriers. This study reveals extensional stresses to be the dominant cause of cell death, and that cell viability can be restored by pre-encapsulation in hydrogel carriers that exhibit thixotropy, or the ability to shear-thin and self-heal. Subsequent chapters describe the design and characterization of a thixotropic protein-engineered hydrogel system called MITCH, or Mixing-Induced Two-Component Hydrogels, and their applications as cell and drug delivery vehicles in regenerative medicine. The crosslinking of the MITCH network relies on the reversible binding between complementary peptide domains, enabling gelation and cell encapsulation by simple mixing at physiological conditions. The design rationale and demonstration of thixotropy, cyto-compatibility, and three-dimensional cell encapsulation are discussed in Chapter 3. In Chapter 4, the specificity and stoichiometric precision of the peptide-peptide crosslinking interactions are highlighted as a distinguishing feature of MITCH. Polymer physics considerations are combined with protein science methodologies to enable the predictable tuning of macroscopic-level gel mechanics through molecular-level variations of component concentration and stoichiometric ratio. The remainder of the thesis focuses on the utility of the MITCH material as a delivery carrier for cell and drug regenerative therapy. Cell protection is demonstrated in Chapter 5, where adipose-derived stem cells injected subcutaneously in mice exhibit improved retention when encapsulated and delivered in MITCH, relative to saline and control biomatrices. Moreover, histological analyses of explants show endogenous cell invasion and signs of native extracellular matrix remodeling at day 3. The simple mixing protocol allows the encapsulation and release of peptide drugs and growth factors in their bioactive state. Chapter 6 describes the engineering of an affinity- and avidity-based peptide drug delivery system, developed by using the molecular recognition domains in MITCH. Fusion of angiogenic peptides to MITCH-specific affinity tags enables drug immobilization, sustained release, and prolonged local drug availability. This controlled release strategy induces higher levels of endothelial cell migration and matrix invasion compared to delivery from saline alone. Chapter 7 presents a therapeutic angiogenesis strategy by dual delivery of human induced stem cell-derived endothelial cells (hiPSC-ECs) and vascular endothelial growth factor (VEGF). This chapter also introduces MITCH 2.0, a new class of protein polymer/synthetic polymer hybrid hydrogel system created to improve tunability and ease of synthesis. Similar to the original version, MITCH 2.0 protects cells during injection and delivers drugs with tunable kinetics. In a murine hindlimb ischemia model, hiPSC-ECs co-delivered with VEGF in MITCH show improved post-transplantation viability and restore blood perfusion to the ischemic limb. All in all, by improving the delivery of cells and biochemical factors, the biomaterials work completed here provides enabling tools to advance other biological research endeavors, to ultimately realize the clinical translation and success of regenerative medicine.

Mesenchymal stem cell-derived exosomes are at the forefront of research in two of the most high profile and funded scientific areas – cardiovascular research and stem cells. Mesenchymal Stem Cell Derived Exosomes provides insight into the biofunction and molecular mechanisms, practical tools for research, and a look toward the clinical applications of this exciting phenomenon which is emerging as an effective diagnostic. Primarily focused on the cardiovascular applications where there have been the greatest advancements toward the clinic, this is the first compendium for clinical and biomedical researchers who are interested in integrating MSC-derived exosomes as a diagnostic and therapeutic tool. - Introduces the MSC-exosome mediated cell-cell communication - Covers the major functional benefits in current MSC-derived exosome studies - Discusses strategies for the use of MSC-derived exosomes in cardiovascular therapies

This book is a compilation of the bench experience of leading experts from various research labs involved in the cutting edge area of research. The authors describe the use of stem cells both as part of the combinatorial therapeutic intervention approach and as tools (disease model) during drug development, highlighting the shift from a conventional symptomatic treatment strategy to addressing the root cause of the disease process. The book is a continuum of the previously published book entitled "Stem Cells: from Drug to Drug Discovery" which was published in 2017.

A state-of-the-art primer on the role of pharmacological sciences in regenerative medicine, for advanced students, postdoctoral fellows, and researchers.

The book provides and intensive overview on exosomes in cardiovascular diseases, its potential as biomarkers, as well as pathological and therapeutic effects. It firstly describes the general aspects of exosomes including the definition, formation and secretion of exosomes and highlight their roles as biomarkers and pathological and therapeutic effects in cardiovascular diseases as well. Secondly, basic aspects of exosomes including the purification methods of exosomes, exosomes content, and functional roles of the cardiovascular exosomes are summarized. Thirdly, exosomes as biomarkers of cardiovascular diseases are overviewed including their roles in diagnosis, prognosis and reaction to therapy. Fourthly, pathological effects of exosomes and therapeutic effects of exosomes are highlighted. Finally, future prospects of exosomes in cardiovascular research would be provided. This is an essential reference for researchers working in cell biology and regeneration, as well as clinicians such as cardiologist.