Chronic kidney disease has restricted treatment options and leads to a lower quality of life, even before the end stage of renal disease is reached. The ability to generate new transplantable... Show moreChronic kidney disease has restricted treatment options and leads to a lower quality of life, even before the end stage of renal disease is reached. The ability to generate new transplantable kidney tissue could help millions of people worldwide and is therefore of great interest. This thesis explores options within regenerative medicine to develop new transplantable kidney tissue from a bio-engineering point of view. Show less
Wiersma, L.E.; Avramut, M.C.; Lievers, E.; Rabelink, T.J.; Berg, C.W. van den 2022
Background: The generation of human induced pluripotent stem cells (hiPSCs) has opened a world of opportunities for stem cell-based therapies in regenerative medicine. Currently, several human... Show moreBackground: The generation of human induced pluripotent stem cells (hiPSCs) has opened a world of opportunities for stem cell-based therapies in regenerative medicine. Currently, several human kidney organoid protocols are available that generate organoids containing kidney structures. However, these kidney organoids are relatively small ranging up to 0.13 cm(2) and therefore contain a small number of nephrons compared to an adult kidney, thus defying the exploration of future use for therapy. Method: We have developed a scalable, easily accessible, and reproducible protocol to increase the size of the organoid up to a nephron sheet of 2.5 cm(2) up to a maximum of 12.6 cm(2) containing a magnitude of nephrons. Results: Confocal microscopy showed that the subunits of the nephrons remain evenly distributed throughout the entire sheet and that these tissue sheets can attain similar to 30,000-40,000 glomerular structures. Upon transplantation in immunodeficient mice, such nephron sheets became vascularized and matured. They also show reuptake of injected low-molecular mass dextran molecules in the tubular structures, indicative of glomerular filtration. Furthermore, we developed a protocol for the cryopreservation of intermediate mesoderm cells during the differentiation and demonstrate that these cells can be successfully thawed and recovered to create such tissue sheets. Conclusion: The scalability of the procedures, and the ability to cryopreserve the cells during differentiation are important steps forward in the translation of these differentiation protocols to future clinical applications such as transplantable auxiliary kidney tissue. Show less
Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic,... Show moreDifferentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular and extracellular compositions, and their developmental plateau need to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a threedimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on an air-liquid interface. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application. Show less
Within this thesis several novel strategies to regenerate the human kidney are exploited. The first strategy is to improve kidney function is by mesenchymal stromal cell (MSC) therapy. In chapter 2... Show moreWithin this thesis several novel strategies to regenerate the human kidney are exploited. The first strategy is to improve kidney function is by mesenchymal stromal cell (MSC) therapy. In chapter 2 the current status of clinical trials with MSC therapy are discussed. In chapter 3 we show an extensive characterization of MSCs derived from human kidney (hkPSCs) compared to bone marrow derived MSCs (bmMSCs) and show that hkPSCs show organotypic expression signatures and functionality. For fluent clinical translation, we developed a clinical grade acceptable standard operation procedure (SOP) (chapter 4). In chapter 5 we show that the cytokine secretion profile of both hkPSCs and bmMSCs was closely related to cell morphology adaptation to culture surface topography and was stromal cell type specific. In chapter 6 we show that not only the kidney cortex but also the kidney capsule contains a stromal cell population. In chapter 7 we report the regeneration of kidney vasculature by repopulating the vascular compartment of human and rat kidney matrices with hiPSC-derived endothelial cells. We show efficient cell delivery, adherence and survival of these endothelial cells as a first, but critical, step towards a human bioengineered kidney. Show less
The aim of this thesis was to explore cellular modification processes associated with heart disease, as well as harnessing its potential for treatment and prevention of detrimental... Show more The aim of this thesis was to explore cellular modification processes associated with heart disease, as well as harnessing its potential for treatment and prevention of detrimental electrophysiological consequences of heart disease. For regenerative cell replacement therapies, optimal differentiation strategies can give rise to efficient generation of mature cardiomyocytes. Insuline-like growth factor (IGF) was found to selectively expand early mesodermal cardiac precursor cell populations in an embryonic stem cell model. Timed exposure of differentiating stem cells to IGF can significantly increase the efficiency of cardiac differentiation. In addition to cell replacement therapy, scar substrate can be modified in order to alter its electrophysiological properties. Fusion of fibroblastic scar cells with surrounding cardiomyocytes reduced pro-arrhtyhmic features through several mechanisms, including more negative mean diastolic potential, enhanced outward Kv current and increased connexin43 expression. Remodelled ventricular tissue is characterized by fibrosis and connexin43 downregulation. This creates an arrhythmogenic substrate, as it gives rise to action potential duration (APD) alternans. Organization of discrete areas of APD alternans is associated with stable reentry formation. By optogenetic modification of cardiomyocytes, re-entry could be effectively terminated by brief light stimuli. Cellular modification is a promising treatment strategy for heart disease. Show less
Bax, N.A.M.; Marion, M.H. van; Shah, B.; Goumans, M.J.; Bouten, C.V.C.; Schaft, D.W.J. van der 2012
The general introduction in Chapter 1 of this thesis provides an overview of the emerging field of regenerative medicine, and its different areas of technology. Contemporary approaches to heart... Show moreThe general introduction in Chapter 1 of this thesis provides an overview of the emerging field of regenerative medicine, and its different areas of technology. Contemporary approaches to heart valve repair and replacement by mechanical and biological prosthesis, as well as their limitations are discussed. Furthermore, the novel approach of (stem)cell-based therapy in ischemic heart disease and a variety of potential cell types are described. In Part I of the thesis different methods to produce completely acellular porcine and rat aortic valve scaffolds for the purpose of creating a biological substitute that can restore, maintain, or improve normal function. In addition, the in vivo behavior of these valves was studied in a rat model. Subsequently, Part II of the thesis studied the in vivo behavior and functional improvement after transplantation of different human cell types in an animal model of acute myocardial infarction. Furthermore, the feasibility of combining cell therapy with gene-therapy was studied in this same model. Finally, in Part III different methods for cardiac phenotyping in mice were assessed and compared. Show less
