Mutations in the CRB1 gene can cause retinal dystrophies such as Retinitis Pigmentosa or Leber Congenital Amaurosis . These patients experience progressive vision loss which ultimately leads to... Show moreMutations in the CRB1 gene can cause retinal dystrophies such as Retinitis Pigmentosa or Leber Congenital Amaurosis . These patients experience progressive vision loss which ultimately leads to blindness. Currently, there are no treatment options available for these patients.This thesis provides novel information on AAV.hCRB gene augmentation therapy in CRB1 mutant animal and human-derived models. We show the phenotype of (1) a novel mouse model with CRB2 ablation specifically in rod photoreceptor cells with loss of retinal function (Chapter 2), (2) a Crb1 mutant brown Norway rat with severe and early onset progressive vision loss (Chapter 3), (3) CRB1 patient-derived retinal organoids (Chapter 4), and (4) CRB1KO and CRB1KOCRB2+/- LCA-like retinal organoids (Chapter 5). Next, AAV-mediated gene augmentation was explored in Crb1 mutant rats (Chapter 3) and CRB1 patient-derived and CRB1KO LCA retinal organoids (Chapter 4 and 5). Finally, single-cell RNA sequencing was performed on AAV.hCRB treated CRB1 patient-derived retinal organoids (Chapter 4). To our knowledge this is the first time that an improved phenotype after AAV.hCRB gene augmentation in CRB1 RP patient-derived and CRB1KO LCA retinal organoids is observed, providing essential information for future gene therapy possibilities in patients with a mutation in the CRB1 gene. Show less
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
KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further... Show moreKPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models.Kptn−/− mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants.Molecular and structural analysis of Kptn−/− mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1.By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity. Show less
Velden, J. van der; Asselbergs, F.W.; Bakkers, J.; Batkai, S.; Bertrand, L.; Bezzina, C.R.; ... ; Thum, T. 2022
Cardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies. All of... Show moreCardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies. All of which warrant experimental models that recapitulate human disease. The translation of basic science results to clinical practice is a challenging task. In particular for complex conditions such as cardiovascular diseases, which often result from multiple risk factors and co-morbidities. This difficulty might lead some individuals to question the value of animal research, citing the translational 'valley of death', which largely reflects the fact that studies in rodents are difficult to translate to humans. This is also influenced by the fact that new, human-derived in vitro models can recapitulate aspects of disease processes. However, it would be a mistake to think that animal models cannot provide a vital step in the translational pathway as they do provide important pathophysiological insights into disease mechanisms particularly on a organ and systemic level. While stem cell-derived human models have the potential to become key in testing toxicity and effectiveness of new drugs, we need to be realistic, and carefully validate all new human-like disease models. In this position paper, we highlight recent advances in trying to reduce the number of animals for cardiovascular research ranging from stem cell-derived models to in situ modelling of heart properties, bioinformatic models based on large datasets, and improved current animal models, which show clinically relevant characteristics observed in patients with a cardiovascular disease. We aim to provide a guide to help researchers in their experimental design to translate bench findings to clinical routine taking the replacement, reduction and refinement (3R) as a guiding concept. Show less
Duinen, V. van; Stam, W.; Mulder, E.; Famili, F.; Reijerkerk, A.; Vulto, P.; ... ; Zonneveld, A.J. van 2020
To advance pre-clinical vascular drug research,in vitroassays are needed that closely mimic the process of angiogenesisin vivo. Such assays should combine physiological relevant culture conditions... Show moreTo advance pre-clinical vascular drug research,in vitroassays are needed that closely mimic the process of angiogenesisin vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds. Show less
Metabolic disease has become pandemic in the developed world. Given our lack of understanding of its molecular pathology, we are often unable to diagnose patients before they reach an... Show moreMetabolic disease has become pandemic in the developed world. Given our lack of understanding of its molecular pathology, we are often unable to diagnose patients before they reach an irreversible state of diabetes or cardiovascular disease. Much research has been done on the role of insulin signaling in metabolic disease, as well as the resultant disturbed lipid homeostasis present in cardiovascular disease and atherosclerosis. Here we add to existing work by developing new tools and sketching out the pathology of dysregulated adipose insulin signaling. We discuss the mechanism of lipodystrophy by using adipocytes differentiated from patient-derived iPSCs. These cells mimic the clinical phenotype and hint at mechanism that reduced patients’ adipose tissue mass. In mice we find that if we knock out the adipose insulin receptor, there is disrupted adipose and liver metabolism. There is a protection from diet-induced obesity, but a dramatically reduced lifespan. We also establish a relationship between obesity and inflammation by transcriptomically assessing obese human adipocytes. We find that an immune factor is responsible for lipid droplet formation and content. Lastly, we develop a new differentiation and purification strategy for iPSC-derived hepatocytes, which we employ to in vitro model a SNP that protects against cardiovascular disease. Show less
