Type-I hypersensitivity is a mast cell-mediated degranulation process induced by IgE-antigen recognition. Inspired by this feature, we genetically modified mouse mast cell line MC/9 to overexpress perforin and granzyme B (PG-MCs) with lentiviral particles. In our melanoma model utilizing B16 cells expressing membrane-bound OVA, we observed a polarized degranulation of mast cells towards cancer cells upon the recognition of antigens by anti-OVA IgE. PG-MCs could selectively kill cancer cells via IgE-antigen recognition. The IgE-sensitized PG-MCs administered through an intertumoral route successfully delivered perforin and granzyme B toward target cells to induce anti-tumor efficacy. Moreover, we demonstrated the tuning effect of tumor microenvironment by mast cell-induced hypersensitivity, skewing the tumor milieu toward a ‘hot’ state with more lymphocyte infiltration. In addition, this mast cell therapy, in combination with 5- fluorouracil (5-Fu), induced a more pronounced inhibition of tumor growth, which is likely associated with the depletion of myeloid-derived suppressor cells (MDSCs).

The immune system serves as a vital defense mechanism, protecting the body against exogenous pathogens and endogenous aberrations through a complex network of cells and signaling pathways. Many diseases arise as a consequence of imbalanced or ineffective immune responses. However, altering the immune system systematically can lead to a range of complications and negative outcomes. Thus, targeted and controlled modulation of the local immune cells and their responses is highly desirable, which minimizes side effects throughout the body and provide precision medicine with the lowest effective dose at local tissues. My thesis harnesses principles of immunoengineering, drug delivery and regenerative medicine to achieve local immune modulation via engineered microneedle patches (MNP) that enables minimally invasive tissue penetration and local retention. To address the critical role of destructive immune cells in tissue loss and achieve periodontal tissue regeneration, I engineered a modular MNP that allows a multistage and controlled release of antibiotics and cytokines. My work demonstrated that the dual delivery of antibiotics and cytokines modulated immune cells, promoted pro-regenerative signals locally, and reversed the degeneration of soft tissue and bone. I further simplified the formulation by identifying a bifunctional antibiotic that not only suppresses bacterial growth but also modulates the macrophages to suppress inflammation, and utilized a fast-degrading MNP loaded with biodegradable PLGA microparticles. Significant therapeutic effects, including suppression of pro-inflammatory cytokine secretion, induction of pro-regenerative cytokines and promotion of tissue/bone regeneration markers were observed in rat periodontitis model. In another application, to suppress the immunogenicity of viral vectors during in vivo gene delivery and boost gene transduction efficiency, I optimized a dissolvable MNP that maintains the virus integrity and releases immunomodulatory agents slowly via microparticles that can be integrated into MNP, and showed that local and slow release of immunosuppressants (i.e. dexamethasone) enhances gene transfer efficiency. Our results in mouse ischemia-reperfusion model demonstrated a significant increase of gene transduction efficiency in the heart. Overall, this work uses MNP as a drug delivery strategy for its minimal invasiveness, local penetration, sustained release and controlled delivery. It reveals the feasibility and efficacy to locally modulate immune responses for more efficient tissue regeneration and gene therapy.

The objective of this doctoral thesis was to investigate the effects of laminar fluid shear stress on the function three types of adult stem cells: mesenchymal stem cells, neural crest stem cells and multipotent vascular stem cells. These three types of stem cells represent potential cell sources for vascular tissue engineering. Before these stem cells can be used to create tissue engineered vascular grafts, a thorough understanding of the effects of hemodynamic forces on their function is necessary. Much like cyclical stretch and normal pressure, fluid shear stress plays a major role in the microenvironment of the blood vessel wall but the effects of this mechanical force on the function of these adult stem cells is not well understood.

First, we began by investigating the effects of laminar fluid shear stress on TGF-beta1/SMAD2 signaling in human mesenchymal stem cells. Human mesenchymal cells (hMSCs) are multipotent fibroblast-like cells, which are found primarily in the bone marrow. MSCs are a potential cell source for tissue engineering because of their ease of isolation and expansion, their multipotency and their low immunogenicity. We found that exposing hMSCs to fluid flow promotes transforming growth factor 1 (TGF-beta1) signaling in a receptor-dependent manner. The mechanism explaining this phenomenon, however, was unclear. Based on our results, we rejected several hypotheses to explain the observed phenomenon: shear force transmission through the glycocalyx; changes in membrane fluidity; and changes in the internalization of TGF-beta; receptors. We were able to show that the increase in TGF-beta1/Smad2 signaling when hMSC are exposed to fluid flow was not caused by shear stress but instead, by an increase in the flow rate. We were also able to show that shear stress inhibits TGF-beta1/Smad2 signaling.

Second, we examined the effects of laminar fluid shear stress on the function of human neural crest stem cells. Neural crest stem cells (NCSCs) are multipotent cells that give rise to various tissues during the embryonic development of vertebrates. NCSCs were derived from induced pluripotent stem cells and, then, exposed to a laminar fluid shear stress of 10 dynes/cm2 for various time periods. We found that laminar fluid shear stress increased NCSC proliferation. We also showed that fluid shear stress increased the activation of ERK1/2 in a time dependent manner. In addition, we observed that exposure to laminar fluid shear stress did not affect myogenic, neurogenic or osteogenic differentiation in NCSCs. We found, however, that exposure to fluid shear stress prevented adipogenic differentiation.

Third, we explored the effects of laminar fluid shear stress on the function of multipotent vascular stem cells. Multipotent vascular stem cells (MVSCs) are adult stem cells that have recently been discovered in the medial layer of blood vessels. MVSCs have been shown to give rise to a variety of cell types including: schwann cells, peripheral neurons, smooth muscle cells, adipocytes, chondrocytes and osteocytes. MVSCs were isolated from rat carotid arteries. These cells were then expanded without differentiation and exposed to a laminar fluid shear stress of 6 dynes/cm2. We found that laminar fluid shear stress increased rat MVSC proliferation. We also showed that fluid shear stress increased the activation of ERK1/2 in a time dependent manner in MVSCs. Laminar fluid shear stress also caused a decrease in the gene expression of smooth muscle cell markers and an increase in the expression of osteoblastic differentiation genes. In addition, we observed that exposure to fluid shear stress did not affect myogenic, osteogenic and neurogenic cell differentiation in MVSCs.

The results of the aforementioned studies provide new clues in efforts to elucidate the mechanobiology of adult stem cells but further investigation into the effects of mechanical stimulation on the function of human MSCs, NCSCs and MVSCs will be necessary to provide a rational basis for the use of these cells in tissue engineering applications.

Traditional self-administered treatment of type 1 diabetes typically requires multiple injections on a daily basis, which is painful and inconvenient and often results in poor compliance and glucose regulation. Closed-loop insulin systems have been proposed such as electronic based glucose-responsive pumps and synthetic glucose-sensitive materials. In this report, erythrocyte membrane-coated nanoparticles with GLUT receptors on the surface of the particles and bonded synthetic glucose-conjugated insulin (Glu-insulin) were created for mounting glucose-sensitive release of insulin. Multiple biomaterial delivery systems were tested in vitro to achieve optimal extended and sustained release. In hyperglycemic conditions, high concentrations of glucose in interstitial fluid can displace Glu-Insulin via a competitive interaction with GLUT, leading to a quick release of Glu-Insulin and subsequent regulation of blood glucose (BG) levels.

Smooth muscle cells (SMCs) have been heavily implicated in the progression of vascular disease: aberrant proliferation of SMCs leads to narrowing of the blood vessel, and deposition of ectopic calcified deposits compromises the structural integrity of the vessel wall. Since the 1960s, scientists have characterized SMCs as a largely inactive homogeneous population that dedifferentiates to become proliferative and migratory upon vascular injury. However, other studies have suggested that the tunica media layer is comprised of separate subpopulations of SMCs that are not interchangeable. Furthermore, the source of SMCs in atherosclerotic plaques and neointima formation has been proven to be oligoclonal, or derived from a few cells; dedifferentiation of SMCs, which experts describe as a widespread and escalating process undergone by SMCs, should result in a distinctly polyclonal origin of SMCs in neointima and plaques. To investigate heterogeneity among SMCs, single-cell analysis was necessary. For our experiments, RFP+ SMCs were dissociated from the aorta of SMMHC-CreERT2/LoxP-tdTomato transgenic mice and immediately used for strict primary culture or lysis of cell contents. Single-cell analysis of functional contractility showed that although all SMCs were contractile, the level of contractile force was heterogeneous among SMCs. Moreover, the levels of cell traction force and contractile force after exposure to a vasoconstrictor peptide did not correlate with the expression level of essential contractile proteins, α-SMA, CNN-1, or SMMHC. These results indicated that the common practice of characterizing phenotypes of SMCs based on contractile state may be misguided, and direct assessment of the pathogenic behavior of SMCs, such as proliferation and differentiation, may be more appropriate to defining subpopulations of SMCs. To optimize the efficiency of obtaining single-cell clones, we integrated optoelectronic tweezers (OET) with a micropatterned substrate designed for clonal culture. Using the light-induced dielectrophoretic force of OET, single SMCs were selected and positioned in an array of ECM-conjugated islands surrounded by PEG. Through behavioral analysis of single-cell clonal colonies, two subpopulations of SMCs were distinguished: proliferative and migratory SMCs that were capable of osteogenic differentiation and calcium-phosphate deposition as well as non-proliferative SMCs with extensive cell spreading and no differentiation potential. Additionally, the protein expression of SMCs from the native vessel and from primary culture were compared through immunostaining of cultured clones and single-cell Western blotting of cells that were dissociated from tissue and directly analyzed. From the protein expression profiles of SMMHC and α-SMA, we determined that clustering individual SMCs based on proliferative potential, protein expression of SMCs from normal vasculature, or protein expression of SMCs in primary culture all outlined the same subpopulations of SMCs. Therefore, SMCs are heterogeneous within the normal blood vessel wall. Our body of evidence suggests that only a minority subpopulation of SMCs is capable of undergoing dedifferentiation to proliferate or osteogenic differentiation to deposit ectopic calcium. Consequently, upon perturbation to the vessel wall, these phenotypically plastic SMCs may launch the response of proliferation and differentiation that leads to the progression of vascular disease.

Tissue specific adult stem cells can be isolated from various tissues and show great capacity of self-renewal and multipotency, thus making it a valuable cell source for regenerative medicine. However, despite its function in homeostasis and tissue repair, it is not clear whether normal adult stem cells could be involved in diseases, like cancer stem cells. Therefore, we investigated this issue in the cardiovascular system, wound healing model and knee joint system. Cardiovascular disease is the number one killer worldwide, which is responsible for approximately 40% of annual deaths in United States. Although various vascular stem cells and progenitor cells were isolated from blood vessel wall, it is not clear whether and how these stem cells contribute to diseases. In the general wound healing, although bone marrow cells and hematopoietic stem cells were identified as the sources of myofibroblasts, which are generally believed to be the culprit of scar tissue formation, it is not clear whether local tissue specific stem cells could be another source of myofibroblasts. In knee joint, osteoarthritis represents structural breakdown of the synovial joint, affecting 70 million people in the United States. However previous identified synovial stem cells are only characterized by non-specific surface markers and their response to biomaterials are not well understood. Therefore, in this dissertation work, we will address these fundamental issues to elucidate the role of adult stem cells in tissue remodeling and diseases.

It is generally accepted that the de-differentiation of smooth muscle cells (SMCs) from contractile to proliferative or synthetic phenotype plays an important role during vascular remodeling and diseases. In the first 3 chapters, we provide evidence that challenges this dogma. We identify a new type of multipotent vascular stem cell (MVSC) in blood vessel wall. MVSCs express markers including Sox17, Sox10 and S100β, are cloneable, have telomerase activity, and can differentiate into neural cells and mesenchymal stem cell (MSC)-like cells that subsequently differentiate into SMCs. On the other hand, we use lineage tracing with smooth muscle myosin heavy chain (SM-MHC) as a marker to show that MVSCs and proliferative or synthetic SMCs do not arise from the de-differentiation of mature SMCs. Upon vascular injuries, MVSCs, instead of SMCs, become proliferative, and MVSCs can differentiate into SMCs and chondrogenic cells, thus contributing to vascular remodeling and neointimal hyperplasia. Moreover, we also isolated MVSCs from neointima and plaque from patients with atherosclerosis. These findings support a new hypothesis that the differentiation of MVSCs rather than the de-differentiation of SMCs contributes to vascular remodeling and diseases. The MVSCs instead of SMCs should be treated as the targets for drug screening and development.

In the second part of the work, we investigated the general wound healing process and remodeling of implanted artificial biomaterials. It is generally accepted that myofibroblasts play a retractile role in wound contraction and are involved in the synthesis of extracellular matrix components to form the scar tissues. We performed detailed characterization and found a novel type of stem cells showing similar feature with MVSCs, which express markers including Sox10, Sox17 and S100β, can spontaneously differentiate into myofibroblasts and eventually SMCs. In vivo studies also identified Sox10+ cells at early stage of wound healing, remodeling of vascular grafts and implanted biomaterials. This work identified a new precursor of myofibroblasts and provided new tools for further research.

In the last part, we identified and characterized a new type of stem cells from the synovial membrane of knee joint, named neural crest cell-like synovial stem cells (NCCL-SSCs). NCCL-SSCs showed the characteristics of neural crest stem cells: they expressed markers such as Sox10, Sox17 and S100, were clonable, and could differentiate into neural lineages as well as mesenchymal lineages. However, lineage tracing with Wnt-1 as marker showed that NCCL-SSCs were not derived from neural crest. When treated with transforming growth factor 1 (TGF-1), NCCL-SSCs differentiated into MSCs, lost the expression of Sox17, and lost the differentiation potential into neural lineages, but retained the potential of differentiating into mesenchymal lineages. To determine the responses of NCCL-SSCs to microfibrous scaffolds for tissue engineering, electrospun composite scaffolds with various porosities were fabricated by co-electrospinning of structural and sacrificial microfibers. Interestingly, microfibrous scaffolds with higher porosity increased the expression of chondrogenic and osteogenic genes but suppressed smooth muscle and adipogenic genes. These results suggest that NCCL-SSCs have tremendous potential for tissue engineering and their differentiation can be controlled by both soluble chemical factors and the biophysical factors such as the porosity of the scaffold.

In summary, this dissertation work identified new tissue specific adult stem cells and performed detailed characterization. This work demonstrated the role of adult stem cells in vascular diseases and scar formation for the first time, indicating that many diseases might be stem cell diseases like cancer. Stem cells instead of somatic cells might be the target for the design of novel therapeutic strategy. Furthermore, we also provide new insight into the response of stem cells on physical properties of biomaterials, thus providing guidance for the design of suitable scaffolds for tissue engineering. We hope this dissertation work could promote the research in investigating the function of stem cells in not only regenerative medicine, but also diseases, thus contributing to our healthcare system.

Artificial antigen presenting cells (aAPCs) are engineered platforms for T-cell activation. It has been widely used for the ex vivo generation of tumor-reactive lymphocytes to ample numbers for effective Adopted Cell Transfer (ACT) therapies including Chimeric Antigen Receptor (CAR) T cell therapy.Viscoelastic materials with stress relaxation are closer to living tissues than pure elastic materials, which can be used to tune cell spreading, proliferation, or differentiation. By controlling molecular weight in combination with crosslinker concentration, we can adjust the stress relaxation properties of materials and therefore modulate cell behavior that is cocultured with viscoelastic biomaterials. It has also been found that T cells could sense the mechanical properties of the substrate and show different morphology when cultured on the substrate with different stiffness. Based on the modulation of cell behavior by viscoelastic materials as well as the mechanosensing properties of T cells, a hypothesis could be made that viscoelastic materials may help improve T cell expansion and generate more proliferative T cell subgroups. There are already publications about the cell response to the matrix with different viscoelasticity using alginate hydrogel modified with adhesion molecules. However, no study has been conducted to investigate the T cell response to the viscoelastic properties of aAPCs. This is due to several technical issues including the fabrication of homogeneous, cell-sized alginate microgel, as well as the conjugation of T cell- specific antibodies to the microgel. In this study, we overcame some of the technical issues and successfully fabricated viscoelastic aAPCs using ionically-crosslinked alginate, which can be applied to in vitro T cell activation and expansion. The alginate microbeads are generated using a microfluidic platform and then conjugated with T-cell-specific antibodies by click chemistry. These viscoelastic aAPCs show homogenous size distribution, low cytotoxicity and can be easily dissolved and removed after coculture. The T cell activation rate of these aAPCs is close to widely-used commercial aAPCs like Dynabeads. Therefore, this kind of viscoelastic aAPCs can serve as the platform to study how viscoelastic properties could affect T cell stimulation, and possess the potential to improve the quality of in vitro expanded T cells.

Directed genetic reprogramming of cells from one identity to another offers tremendous potential in regenerative medicine, disease modelling and drug testing. However, its application is limited by the low efficiency at which it occurs, and existing methods to improve efficiency mostly utilize additional molecular biology and biochemical manipulations. This thesis explored an alternative paradigm for improving reprogramming efficiency: presentation of physical cues. To this end, I first showed that simply agitating an adherent culture with an orbital shaker enhanced its efficiency of reprogramming to induced pluripotent stem cells (iPSCs). I further demonstrated that convective mixing of the culture medium by orbital agitation blunted the upregulation of CDK inhibitor p57/Kip2 that was caused by the culture becoming overconfluent, which in turn enhanced the efficiency of reprogramming to iPSCs. Next, I showed that culturing reprogramming cells on solid supports scored with microgrooves enhanced their reprogramming into cardiomyocytes. I demonstrated that the microgrooves caused upregulation of the activity of the transcription factor megakaryoblastic leukemia-1 (Mkl1) / myocardin-related transcription factor A (Mrtf-a) and also enhanced organization of sarcomeric structure, with both effects contributing to better reprogramming efficiency. In addition to physical cues, I also explored whether treatment with only small molecules could reprogram fibroblasts into skeletal muscle cells. Indeed, I found that an optimized basal medium (10% FBS in DMEM with 50 μg/ml of ascorbic acid and 50 ng/ml of basic fibroblast growth factor (bFGF)) containing just 2 small molecules— 616452 [an inhibitor of the protein kinase activity of the transforming growth factor-beta (TGF-β) type I receptor (R1)] and forskolin (a plant diterpene that stimulates adenylyl cyclase and elevates the intracellular level of 3',5'-cyclic-AMP)—was sufficient to achieve reprogramming at high efficiency. In summary, this thesis described how both physical and chemical cues can contribute to enhancing the reprogramming of cell identity.

Tissue engineering and regenerative medicine is a multidisciplinary field that combines the knowledge and technologies of biology, materials, chemistry and medicine among others. Within the realm of tissue repair and regeneration, vascular reconstruction, in particular, is influenced by both biophysical and biochemical cues and it is critical to fully understand how these cues interact with the physiological microenvironment. In this dissertation, we demonstrated how host vascular regenerative potential can be harnessed by optimizing vascular grafts from both the biomaterial and biochemical perspectives. We utilized and optimized electrospinning technology to create microfibrous vascular grafts. We showed that incorporating a fast degrading polymer into the vascular grafts effectively enhances cell infiltration without majorly compromising its mechanical and structural properties. Our in vivo data suggests that although the optimized vascular graft was able to induce the initiation of vascular regeneration, the process was accelerated and enhanced with the help of biochemical cues presented in the form of stromal cell-derived factor-1α (SDF-1α). In the presence of chemokine SDF-1α, the vascular grafts demonstrated better wall remodeling and long-term patency. This observation again highlights the significance of both biomaterial and biochemical effects. While the vascular grafts in our studies might recruit inflammatory cells, calcification which is a destructive complication typically associated with inflammatory response was not observed. The final part of this dissertation details our efforts in using protein engineering to broaden the biological application of SDF-1α by introducing a free cysteine residue on its C-terminus. We showed that the cysteine-modified SDF-1α exhibits a bioactivity different from its unmodified counterpart, the underlying mechanism of which needs to be elucidated in future studies. Taken together, this work is expected to provide insights into optimization of vascular graft platforms for broad therapeutic applications.