Towards Rapid Label-free Enrichment of Specific Stem Cell Populations for Autologous Cell Therapies

Autologous mesenchymal stem cell (MSC) therapies have huge potential in addressing clinical challenges for otherwise intractable diseases. Label-free, intra-operative separation and enrichment of MSC subpopulations would provide a step change in delivery of such therapies. The long term goal of this research is to use binding proteins to provide a surface with switchable affinity, coupled with microfluidics to selectively bind and subsequently collect released cells. The specific aim of this thesis was to take the first steps towards achieving this goal, by identifying the most suitable binding proteins for cell capture and release in a prototype device and determining the feasibility of cell enrichment from complex clinical samples such as bone marrow aspirate. A prototype device was developed exploiting the cell surface marker CD271 to select for MSCs. Affimer binding proteins and a commercially available antibody were investigated for specific cell capture and release. Specificity for CD271+ cells was demonstrated via flow cytometry using two different cell types. CD271 binding proteins were immobilised to a low-fouling substrate in a microfluidic channel and known mixtures of the two cell populations used to demonstrate specific cell capture. Increased flow rates allowed for bound cells to be released, collected and analysed, providing evidence that cells remained viable and minimally manipulated after enrichment. Clinical samples of bone marrow aspirate were then used in the same way and the results compared to gold standard methods of cell sorting. Results showed that the percentage of CD271+ cells selected from bone marrow mononuclear cell populations using the prototype device was similar to results obtained using established cell sorting methodologies. This work demonstrated that affinity capture via antibody technology, together with a surface designed to provide a controlled release mechanism, offers a high-throughput, minimally manipulative approach to select and enrich MSC populations for therapeutic applications

The Impact of Nanopulse Treatment on the Tumor Microenvironment in Breast Cancer: Overturning the Treg Immunosuppressive Dominance

Nanopulse treatment (NPT) is a high-power electric engineering modality that has been shown to be an effective local tumor treatment approach in multiple cancer models. Our previous studies on the orthotopic 4T1-luc breast cancer model demonstrated that NPT ablated local tumors. The treatment consequently conferred protection against a second live tumor challenge and minimized spontaneous metastasis. This study aims to understand how NPT mounts a potent immune response in a predominantly immunosuppressive tumor. NPT changed the local and systemic dynamics of immunosuppressive cells by significantly reducing the numbers of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) that contribute to the dominant immunosuppressive environment in the 4T1-luc mouse breast cancer model. The Treg suppression capacity and activation markers, including 4-1BB and TGFβ, were diminished post-treatment, and the Treg activation profile shifted from a predominantly activated (CD44+CD62L-) to a naïve (CD44-CD62L+) profile. Furthermore, we observed an increase in apoptosis among Tregs and TAMs followed by a concomitant M1-macrophage polarization of the surviving TAMs. Meanwhile, a continuing rise in the effector T cell (Teff) / Treg ratio and among resident memory CD8 T cells hinted at the expansion of antitumor specific cytotoxic T cells. Overall, these findings suggest that NPT is a potent tumor microenvironment (TME) modifier that can effectively reverse the tumor’s immunosuppressive barrier by decreasing MDSCs, TAMs and functionally suppressive Tregs. Thus, the TME modification by NPT confers cytotoxic T cell function and immune memory formation contributing to the tumor challenge rejection and reduction in metastasis risk. Future studies will investigate the underlying mechanisms of NPT-induced T cell immunity by determining the changes it creates among Treg and Teff T cell receptor (TCR) clonality in both mice and humans. Control studies on human blood indicated that peripheral Tregs are predominantly polyclonal in nature among healthy donors. Overall, these findings on NPT-induced immunity can help develop novel immunotherapeutic approaches to effectively treat poorly immunogenic cancers, such as breast and pancreatic cancers, that are largely resistant to current systemic immunotherapies

Silk fibroin nanoparticles : in vitro performance of a putative anticancer nanomedicine

Despite the advantages of nanoparticle-based carriers for anticancer drug delivery, their translation into the clinic has been limited by factors including: (i) poor endocytic uptake and intracellular routing, (ii) macrophage clearance and (iii) a disregard of the tumour microenvironment governing nanoparticle uptake. As a result, there is a continued demand to explore the performance of polymer-based nanoparticles.;The principle hypothesis of this thesis is that silk fibroin nanoparticles can be used as anticancer nanomedicines. To validate this, the mechanisms governing drug release from silk fibroin nanoparticles are explored in Chapter 3. Next, the immunogenicity of silk fibroin nanoparticles towards macrophages is assessed (Chapter 4). Finally, Chapter 5 investigates the endocytosis of silk fibroin nanoparticles in response to the cell cycle and culture substrate mechanics.;This thesis provided the first experimental proof of lysosomotropic anticancer drug delivery from silk fibroin nanoparticles in single human breast cancer cells (Totten et al. 2017. J. Drug Target. 25, pp 865-872) (Chapter 3). Drug loaded silk fibroin nanoparticles were endocytosed by MCF-7 cells and a combination of the acidic lysosomal pH and enzymatic degradation facilitated drug release and subsequent nuclear translocation of the payload within 5 hours of dosing.;Next, nanoparticle-macrophage interactions were studied (Chapter 4). Silk fibroin nanoparticles exerted similar immunogenicity to silica and poly(lactic-co-glycolic acid) nanoparticles (Saborano, Wongpinyochit, Totten, Johnston, Seib and Duarte. 2017. Adv. Healthc. Mater. 6, 1601240). This indicated that silk fibroin nanoparticles can compete with leading healthcare materials in pre-clinical and clinical use.;Further assessment into immunomodulatory potential of silk fibroin nanoparticles revealed that they drive macrophage polarisation towards a pro-inflammatory M1-like state (Totten et al. 2019. ACS Appl. Mater. Interfaces. in press). This effect could be fine-tuned with surface modification (i.e. PEGylation). This observation is important because silk fibroin nanoparticles could act both as carriers for chemotherapeutics and as synergistic attenuators of tumour-associated macrophages in the tumour site.;Finally, advanced analysis of silk fibroin nanoparticle endocytosis was conducted (Chapter 5) by assessing intracellular trafficking in a time-dependent manner. Endocytosis of silk fibroin nanoparticles by breast cancer (MCF-7) cells was influenced by cell cycle progression, but not substrate mechanics. However, substrate mechanics were found to modulate the endocytic behaviour of healthy human (MCF-10A) breast epithelial cells. This relationship warrants further investigation with regard to the cellular response of nanomedicines.;Overall, this thesis accomplished in vitro analysis of silk fibroin nanoparticle drug delivery performance, macrophage interactions and endocytic uptake. These findings indicate that silk fibroin nanoparticles are emerging as an interesting biopolymer for anticancer applications. Work presented in this thesis provides a foundation to now move to pre-clinical in vivo studies.Despite the advantages of nanoparticle-based carriers for anticancer drug delivery, their translation into the clinic has been limited by factors including: (i) poor endocytic uptake and intracellular routing, (ii) macrophage clearance and (iii) a disregard of the tumour microenvironment governing nanoparticle uptake. As a result, there is a continued demand to explore the performance of polymer-based nanoparticles.;The principle hypothesis of this thesis is that silk fibroin nanoparticles can be used as anticancer nanomedicines. To validate this, the mechanisms governing drug release from silk fibroin nanoparticles are explored in Chapter 3. Next, the immunogenicity of silk fibroin nanoparticles towards macrophages is assessed (Chapter 4). Finally, Chapter 5 investigates the endocytosis of silk fibroin nanoparticles in response to the cell cycle and culture substrate mechanics.;This thesis provided the first experimental proof of lysosomotropic anticancer drug delivery from silk fibroin nanoparticles in single human breast cancer cells (Totten et al. 2017. J. Drug Target. 25, pp 865-872) (Chapter 3). Drug loaded silk fibroin nanoparticles were endocytosed by MCF-7 cells and a combination of the acidic lysosomal pH and enzymatic degradation facilitated drug release and subsequent nuclear translocation of the payload within 5 hours of dosing.;Next, nanoparticle-macrophage interactions were studied (Chapter 4). Silk fibroin nanoparticles exerted similar immunogenicity to silica and poly(lactic-co-glycolic acid) nanoparticles (Saborano, Wongpinyochit, Totten, Johnston, Seib and Duarte. 2017. Adv. Healthc. Mater. 6, 1601240). This indicated that silk fibroin nanoparticles can compete with leading healthcare materials in pre-clinical and clinical use.;Further assessment into immunomodulatory potential of silk fibroin nanoparticles revealed that they drive macrophage polarisation towards a pro-inflammatory M1-like state (Totten et al. 2019. ACS Appl. Mater. Interfaces. in press). This effect could be fine-tuned with surface modification (i.e. PEGylation). This observation is important because silk fibroin nanoparticles could act both as carriers for chemotherapeutics and as synergistic attenuators of tumour-associated macrophages in the tumour site.;Finally, advanced analysis of silk fibroin nanoparticle endocytosis was conducted (Chapter 5) by assessing intracellular trafficking in a time-dependent manner. Endocytosis of silk fibroin nanoparticles by breast cancer (MCF-7) cells was influenced by cell cycle progression, but not substrate mechanics. However, substrate mechanics were found to modulate the endocytic behaviour of healthy human (MCF-10A) breast epithelial cells. This relationship warrants further investigation with regard to the cellular response of nanomedicines.;Overall, this thesis accomplished in vitro analysis of silk fibroin nanoparticle drug delivery performance, macrophage interactions and endocytic uptake. These findings indicate that silk fibroin nanoparticles are emerging as an interesting biopolymer for anticancer applications. Work presented in this thesis provides a foundation to now move to pre-clinical in vivo studies

"Studies on the cardiomyocytic potential of dermal stem cells”

In Europe, diseases of the heart and circulatory system are the main cause of death; 48% of all deaths are from coronary heart disease, accounting for over 4.30 million deaths each year (European cardiovascular disease statistics 2008). Identifying stem cell types that can potentially contribute to the regeneration of cardiac tissue is one route to addressing this global problem. The follicular dermis has been proposed as a tissue containing cells with adult stem cell properties that could be used for regenerative medicine. This study showed that follicular dermal structures demonstrated spontaneous synchronised contractions in vitro, indicative of cardiomyocytic commitment. In contrast, isolated populations of follicular dermis, the dermal papilla and sheath, did not demonstrate spontaneous cardiomyocytic commitment in vitro. Co-culture of isolated follicular dermal populations with embryonic cardiomyocytes, a cell type previously reported to support cardiomyocytic commitment of mesenchymal stem cell types, induced expression of a panel of gene products indicative of cardiomyocytic commitment, but only when the inducing cell and the target cell were of the same species. The level of gene induction indicated that the number of cells affected were likely to be very modest. In order to better identify the stem cell types in the follicular dermis, this study used a novel mTert-GFP transgenic mouse to identify candidate stem cells on the basis of their expression of the stem cell phenotypic marker, telomerase. A small population of GFP-expressing non-haematopoietic cells present in the dermis (0.17- 0.49%) were identified and found associated with the follicular dermis and bulge region. This minor sub-population of GFP-expressing dermal cells contained all the detectable gene expression of four key markers of stem cells: Tert; Oct4; Nanog and Sox2, both at time of isolation and after 2 weeks in vitro culture. Intriguingly, non- haematopoietic, GFP-expressing cells were also detectable in the adult heart at low level (0.08%). After cryo-induced infarct, these non-haematopoietic GFP-expressing cells were associated with the epicardium, concentrated close to the infarct site, in a fashion consistent with other reports of candidate cardiac stem cells. This study indicates that the population of stem cells within the follicular dermis may be very modest. Further, it shows that phenotypic markers of stem cells from different organs may be a suitable approach for the investigation of the stem cell phenotype

Isoform-specific tau antibodies and degrabodies to study 4R tau function in MAPT mutant iPSC-derived neurons

Hyperphosphorylated, insoluble aggregates of the microtubule-associated protein tau are a pathological hallmark of a group of clinically diverse diseases termed the tauopathies. There are six protein isoforms of tau protein separated into two groups; 3-repeat (3R) and 4-repeat (4R) tau, based on the number of repeats within the microtubule-binding region. Within neurons under normal healthy conditions, the ratio of expression of 3R and 4R tau is equal. However, in several tauopathies there is an imbalance of 3R and 4R tau, suggesting a disruption in tau splicing may be associated with disease. This is observed in Frontotemporal dementia and parkinsonism linked to chromosome-17, where intronic and splice-site mutations in MAPT increase the relative amount of 4R tau and cause early-onset neurodegeneration. This altered tau splicing has been recapitulated in induced pluripotent stem cell (iPSC) derived neurons, which recapitulate increased 4R tau levels along with other phenotypes including hyperpolarised mitochondrial membranes and an associated increase in reactive oxygen species (ROS) production. This highly relevant to neurodegeneration as ROS-induced cell death is a key feature of many neurodegenerative diseases. The mechanisms by which increased 4R results in neuronal dysfunction and neurodegeneration are not fully understood, and progress has been limited in part by a lack of suitable tools to investigate tau isoform imbalance. In this project, I generated and characterised novel 3R and 4R tau specific antibodies. I then used these antibodies to develop a multiplexed flow cytometry assay to show a correlation between 4R tau levels and mitochondrial membrane polarisation in iPSC-derived neurons containing the 10+16 MAPT splice mutation. Finally, I generated a 4R tau specific degrabody (target degrading intracellular antibody). I demonstrated this causes the specific degradation of 4R tau to achieve phenotypic restoration of non-mutant levels of mitochondrial membrane polarisation within the mutant neurons