A proper immune system is essential to fight off pathogens such as viruses, bacteria, and fungi. The immune system also plays a huge role in the protection against cancer, as it can eradicate tumor... Show moreA proper immune system is essential to fight off pathogens such as viruses, bacteria, and fungi. The immune system also plays a huge role in the protection against cancer, as it can eradicate tumor cells. All immune cells are derived from hematopoietic stem cells (HSC) that undergo differentiation in a highly regulated succession of developmental steps. Each of the cell types from the immune system perform a unique specialized role, and where most of these lineages develop in the bone marrow, the T cells that make part of our adaptive immunity, develop in the thymus within a specialized environment. To achieve this, the development of each of these cell types is regulated by a variety of transcription factors.In Chapter 2 of this thesis, we reviewed the complexity of one of the important signaling pathways of hematopoietic development, the Wnt pathway. While this serves as an introduction to the fundamental research we performed, it also shines light onto potential therapeutic targets within the Wnt pathway. For further study of the Wnt pathway, we generated a novel reporter mouse, which is described in Chapter 3 of this thesis. Here we developed a reporter mouse for the Axin2 gene with the fluorescent tag mTurquoise2 with CRISPR/Cas9 genome editing tools. Based on how the genetic engineering was done to create this reporter mouse, mice that are homozygous for this reporter knock-in are also a functional knockout for Axin2. For proper functional studies, the heterozygous mice should be used.The Axin2-mTurquoise2 mouse was used in Chapter 4 of this thesis to study Wnt involvement in hematopoiesis and T cell development. We observed an increase of canonical Wnt-signaling in thymocytes from mice that have a loss of Axin2 (Axin2-TQtg/tg mice). This confirms the Wnt dosage effect that was reported previously in literature. Conclusively, these results indicate that Axin2 is required to fine-tune Wnt activity to the levels that are “just right” and cannot be maintained by Wnt activator Axin1 alone.Chapters 2, 3 and 4 focused on fundamental research on hematopoiesis and T cell development. Chapter 5 is more translational oriented and is an introductory review to thymic regenerative therapies. In Chapter 6 of this thesis, we describe the development of a combined cell and gene therapy effort to regenerate a functional thymus transplant from human Induced Pluripotent Stem Cells (iPSCs). We generated an iPSC-derived thymus by directed differentiation of human iPSCs towards thymic epithelial progenitor cells (TEPCs) using FOXN1, formation of 3-D structures from these cells which we named iPSC-derived TEPCs, or iTEPCs, and transplantation of these organoids into mice that lack a functional thymus. Functionality was demonstrated by reconstitution of functional T cells from iPSC-derived grafts, which was introduced by FOXN1 gene therapy (FOXN1 iTEPCs).Chapter 7 is the final translational research chapter of this thesis and investigated the use of iPSCs for the modeling of PIDs and the initial steps towards T cell regeneration in SCID patients. This chapter describes the iPSC generation, and its repair to use gene-corrected iPSCs from a RAG2 SCID patient to repair their disrupted immune system. The resulting iPSC model was used for disease modelling and provided novel insights into the T cell development in these RAG2-SCID patients, as we observed developmental blocks at every investigated stage of T cell development. The findings in this chapter also provide a proof-of-principle to treat a variety of SCID patients by utilizing ex vivo cell and gene therapy.Altogether, this thesis tackles two sides of the same coin: fundamentals of hematopoiesis and T cell development, and regenerative therapies for the immune system. The fundamental tools and findings in this thesis can lead to important insights to find new treatment options or improve existing therapies. Furthermore, we provide the basis for two potential therapies to treat patients with a variety of immune disorders, including DiGeorge Syndrome, SCID, age-related immune deficiencies and (post-transplant) leukemia patients that received ablative therapies. Show less
The immune system consists of a complex network that protects the body from pathogens and cancer. Autoimmune diseases and inflammatory conditions are caused by a disturbed and unbalanced immune... Show moreThe immune system consists of a complex network that protects the body from pathogens and cancer. Autoimmune diseases and inflammatory conditions are caused by a disturbed and unbalanced immune system in which the body's own cells are destroyed by immune cells as a result of an inflammatory reaction. In this thesis, I focused on type 1 diabetes as an autoimmune disease to investigate the derailment of the immune system and explored strategies involving cell therapy with tolerogenicdendritic cells to restore immune tolerance. In addition, I studied the application of cell therapy in other inflammatory disorders such as rheumatoid arthritis and graft-versus-host disease. Show less
Although mesenchymal stromal cells (MSCs) from primary tissues have been successfully applied in the clinic, their expansion capabilities are limited and results are variable. MSCs derived from... Show moreAlthough mesenchymal stromal cells (MSCs) from primary tissues have been successfully applied in the clinic, their expansion capabilities are limited and results are variable. MSCs derived from human-induced pluripotent stem cells (hiMSCs) are expected to overcome these limitations and serve as a reproducible and sustainable cell source. We have explored characteristics and therapeutic potential of hiMSCs in comparison to hBMSCs. RNA sequencing confirmed high resemblance, with average Pearson correlation of 0.88 and Jaccard similarity index of 0.99, and similar to hBMSCs the hiMSCs released extracellular vesicles with in vitro immunomodulatory properties. Potency assay with TNF alpha and IFN gamma demonstrated an increase in well-known immunomodulatory genes such as IDO1, CXCL8/IL8, and HLA-DRA which was also highlighted by enhanced secretion in the media. Notably, expression of 125 genes increased more than 1000-fold. These genes were predicted to be regulated by NFKB signaling, known to play a central role in immune response. Altogether, our data qualify hiMSCs as a promising source for cell therapy and/or cell-based therapeutic products. Additionally, the herewith generated database will add to our understanding of the mode of action of regenerative cell-based therapies and could be used to identify relevant potency markers. Show less
Particles are omnipresent in biopharmaceutical products. In protein-based therapeutics such particles are generally associated with impurities, either derived from the drug product itself (e.g.... Show moreParticles are omnipresent in biopharmaceutical products. In protein-based therapeutics such particles are generally associated with impurities, either derived from the drug product itself (e.g. protein aggregates), or from extrinsic contaminations (e.g. cellulose fibers). These impurities can affect product stability, as well as cause adverse effects once introduced into the human body. Particulate impurities are present over a wide range of sizes (from nanometers to millimeters) making them difficult to characterize by using a single method.Novel drug products may also contain particles that act as the active pharmaceutical ingredient (e.g., living cells) or a drug delivery vehicle (e.g., lipid nanoparticles). Unwanted immunotoxicity and inconsistent in vivo functionality can result from particle instability and aggregate formation. Therefore, the efficacy and safety of these therapeutics is dependent on the particle composition, quantity and size distribution.Consequently, well-established methods are required to quantify and characterize particles in the submicron- and micron-size ranges. In this thesis, we developed new approaches which allow for comprehensive characterization of the particle populations present in biopharmaceutical products, both as impurities or as API. Furthermore, the performed work focused on comparing different particle characterization techniques to allow a better understanding of the limitations and strengths of each method applied. Show less
Corneal transplantation still represents the elected method for the treatment of corneal endothelial pathologies. However, the worldwide shortage of donor corneas induced the exploration of... Show moreCorneal transplantation still represents the elected method for the treatment of corneal endothelial pathologies. However, the worldwide shortage of donor corneas induced the exploration of approaches to use the donor tissue more efficiently or to be more independent from donor tissue. This thesis will illustrate the improvements of new strategies for cell-based corneal endothelial regeneration, alternative to corneal endothelial surgical transplantation, by bridging the gap between in vitro experiments and clinical models. In the studies described, we first address the establishment of a GMP-compliant protocol for in vitro hCEC culture for clinical application and then we focus on endothelial cell sheet transplantation, describing both in vitro and in vivo applications of expanded CEC-carriers constructs made by biocompatible materials. Show less
Atherosclerosis is the main underlying pathology of cardiovascular disease, the largest single cause of death in industrialized countries, and current treatment is still largely insufficient. In... Show moreAtherosclerosis is the main underlying pathology of cardiovascular disease, the largest single cause of death in industrialized countries, and current treatment is still largely insufficient. In recent years it has become evident that immune responses contribute to atherosclerosis. Therefore, during my PhD studies I focused on developing a therapy to induce and expand anti-inflammatory immune cells to reduce ongoing immune responses and atherosclerosis. I used the approach of cellular therapy and examined the effect of several different anti-inflammatory immune cells. For example, I made use of mesenchymal stem cells, which have previously been used to improve cardiac repair after myocardial infarction and were found to have anti-inflammatory properties. Additionally, I used drugs, e.g. inhibitors of protein degradation, and biologics, e.g. components of heat-killed bacteria, to directly increase the amount of anti-inflammatory immune cells. An interesting side-effect of some treatments was that they additionally reduced cholesterol levels. In summary, I have shown in pre-clinical models that immune cell-based therapies are promising for the treatment of atherosclerosis. As atherosclerosis is determined by both high cholesterol levels and inflammation reducing immune responses will greatly contribute to a better treatment of cardiovascular patients in the (near) future. Show less
Coronary artery disease is a major cause of mortality and morbidity in the western world. Despite successive revascularization procedures, a large number of patients ends up with end-stage coronary... Show moreCoronary artery disease is a major cause of mortality and morbidity in the western world. Despite successive revascularization procedures, a large number of patients ends up with end-stage coronary artery disease, not amenable for conventional revascularization. These patients often have stress-inducible myocardial ischemia, resulting in disabling complaints of angina, refractory to medical treatment. Intramyocardial bone marrow cell injection is currently under investigation as a new therapeutic option for these patients. This treatment aims to improve myocardial perfusion and contractile function (and decrease anginal complaints) through administration of bone marrow cells into ischemic myocardium. In the current thesis, the use of intramyocardial bone marrow cell injection for the treatment of chronic myocardial ischemia is evaluated several studies. First, the results of a randomized, placebo controlled, double-blinded trial are described, demonstrating that bone marrow cell injection is associated with a reduction in angina complaints, improved myocardial perfusion and increased left ventricular function. Furthermore, the effects of intramyocardial bone marrow cell injection were further explored by evaluating changes in diastolic function, cardiac innervation, and left ventricular synchronicity. In addition, the long term effects of bone marrow cell injection are described. Show less
Stem cell-based therapies represent promising approaches for the treatment of incurable diseases and tissue injury due to the capacity of these cells to self-renew and differentiate into... Show moreStem cell-based therapies represent promising approaches for the treatment of incurable diseases and tissue injury due to the capacity of these cells to self-renew and differentiate into specialized mature cells. Understanding the interaction of stem cells with the disease/injured environment and their contribution to the repair process by release of regeneration-promoting signals or by differentiation into the lost cell types is crucial for the advancement of their clinical application. This thesis focuses on one stem cell population, the mesenchymal stem cells (MSCs) from human and in particular on their role and utility in skeletal muscle regeneration. For this purpose, several in vivo tissue damage models were employed (i.e. cardiotoxin-induced injury, pressure ulcers, and subcutaneous implants of minced skeletal muscle). Most of the experiments were performed in immunocompromised mice (i.e. NOD/SCID) to avoid immunological rejection of the human cells. Furthermore, we described the downregulation of MHC class I protein expression on the surface of human MSCs by retroviral vectors encoding a herpesviral immunoevasin (i.e. the US11). This renders human MSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make human MSCs non-immunogenic and thereby universally transplantable. Show less
Hirsch, A.; Nijveldt, R.; Vleuten, P.A. van der; Tijssen, J.G.P.; Giessen, W.J. van der; Tio, R.A.; ... ; HEBE Investigators 2011
