Disease modeling
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In vitro models allow us to answer outstanding questions on patient-specific drug effects and whether new disease targets can be identified that would lead to new therapeutic strategies. To do so it is necessary to generate better, and when necessary, more complex, models not only containing cells of the tissue of interest but also surrounding cells.
The validation of these human disease models and comparison with manifestations of the disease in animal models and patients are a key objective of this subtheme. The 3Rs (reduction, replacement, refinement) of animal experiments is among LUMC’s objectives. However, implementation and acceptance are only realistic if the human models provide at least equivalent or preferably better information on disease mechanisms and targets and are at the same time amenable for screening new therapies.
…In vitro models allow us to answer outstanding questions on patient-specific drug effects and whether new disease targets can be identified that would lead to new therapeutic strategies. To do so it is necessary to generate better, and when necessary, more complex, models not only containing cells of the tissue of interest but also surrounding cells.
The validation of these human disease models and comparison with manifestations of the disease in animal models and patients are a key objective of this subtheme. The 3Rs (reduction, replacement, refinement) of animal experiments is among LUMC’s objectives. However, implementation and acceptance are only realistic if the human models provide at least equivalent or preferably better information on disease mechanisms and targets and are at the same time amenable for screening new therapies.
Our researchers mainly work with two types of complex and innovative human in vitro models: organoids (lab grown 3D mini-organs) and Organs-on-Chip (OoC). These models are both usually based on iPSCs. Doing so, RegMedTO researchers have successfully generated models for diseases that affect a variety of organs including vascularized 3D cardiac microtissue on chip, vascularized kidney organoids and lung-on-chip models through which air can flow.
A full list of projects can be seen here:
Projects
Eye – Human retinal organoids
Ambassador: Jan Wijnholds (j.wijnholds@lumc.nl, OOG)
Genetic mouse models are most useful to examine retinal physiological function and the connection of the retina to the brain. Unfortunately, viral vectors that work very well in mouse models often work less efficiently in humans. Human retinal organoids can be derived from hiPSC isogenic controls can be obtained by genetic repair of patient cells using CRISPR/Cas engineering. The retinal organoids can be used to study the effects of loss of function of the retinal disease genes by advanced molecular and cellular techniques, and for testing gene augmentation, or gene editing, therapy vectors. Retinal organoids for CRB1, ABCA4, RPGR, USH2A and isogenic controls have been generated, whereas generation of others such as ND4 is ongoing. These studies will yield insight into human inherited retinal disease and generate candidate medicines to fight blindness.
Heart – Modeling genetic heart disease
Ambassador: Richard Davis (r.p.davis@lumc.nl, ANA)
Heart disease can be modeled using hiPSCs derived from patients with disease-causing mutations or engineered by inserting specific mutations into healthy cells. Our techniques enable us to create hiPSC lines with mutations in various genes, including those that can cause arrhythmias in patients, affect contractility of the heart, or affect metabolism.
We developed STRAIGHT-IN, a method for rapidly introducing large DNA sequences into hiPSCs, allowing us to generate numerous hiPSC lines with mutations and/or genetic (lineage) reporters, within just a few months.
These hiPSCs are used to create 3D cardiac models containing cardiac endothelial cells, fibroblasts and cardiomyocytes. In this environment, the cardiomyocytes show improved maturity, making them suitable for use in automated systems to test new drug therapies. We are now using this high-throughput approach to perform compound screens to identify promising new treatments for heart diseases.
Additionally, we are combining imaging data with machine learning to rapidly evaluate the toxicity profile of these compounds and identify which cardiac cell types are sensitive as well as the mechanism of action.
Heart - 3D multi-cell type cardiac microtissues for modeling inherited heart disease and drug response
Ambassadors: Milena Bellin (m.bellin@lumc.nl, ANA) and Christine Mummery (c.l.mummery@lumc.nl, ANA)
hiPSC technology enables modelling and predicting pro-arrhythmic effects of many genetic mutations associated with sudden cardiac death and cardiomyopathies. However, cardiomyocytes differentiated from hiPSCs are immature, limiting our ability to study postnatal cardiac phenotypes. We developed a cardiac microtissue system that integrates hiPSC-derived cardiomyocytes, cardiac fibroblasts and endothelial cells to induce structural, functional, and metabolic maturation of hiPSC-cardiomyocytes. We leverage this model 1) to understand mechanisms underlying cardiomyocyte maturation. We focus on signalling pathways and proteins that allow homotypic and heterotypic cell-cell crosstalk. 2) to study cardiac ion channel diseases in which ion channel genes are developmentally regulated, such as Brugada- and Long-QT syndrome type 1. 3) to model the differential expressivity of dilated cardiomyopathy (DCM) caused by mutations in the Lamin A/C (LMNA) gene. We have developed a library of genetically matched hiPSC lines carrying 11 distinct LMNA mutations (using STRAIGHT-IN developed by Dr. R.P. Davis). We are using them to better understand the molecular mechanisms of disease but also develop gene editing tools to correct the mutations in the microtissues. 4) to find ways to protect the heart from chemotherapy-induced damage. We are investigating the molecular mechanisms underlying cardiotoxicity, testing structural variants of doxorubicin with reduced cardiotoxicity (with Prof J. Neefjes, Academic Pharma) and building cardiac microtissues from patient hiPSC for whom cardiotoxicity has been observed. 5) to model the cardiorenal axis by combining hiPSC-derived cardiac and kidney organoids in vitro in a microfluidic chip (with Dr. C.W. van der Berg), and study cardiorenal syndrome.
Inner Ear – Human inner ear organoids
Ambassador: Heiko Locher (h.locher@lumc.nl, KNO)
Human inner ear organoids are a vital tool for exploring the complexities of the inner ear in a laboratory setting, providing a unique opportunity to study differentiated inner ear cells, which are otherwise challenging to access. To a certain extent, these organoids model the structural and functional characteristics of the inner ear, enabling detailed investigation into its development and associated disorders.
Our research includes several key objectives. 1) Model Improvement: by comparing the hiPSC or adult stem cell organoid models to normal human fetal inner ear during development, we are establishing ear organoid anatomical and functional fidelity. We thus aim to increase the efficiency of obtaining target key cell types and differentiate those missing. 2) Pathology Investigation: the organoids are employed to model various inner ear diseases, allowing investigation of the underlying pathophysiological mechanisms. Currently our focus includes congenital CMV infections, the ototoxic effects of platinum-based chemotherapy and aminoglycoside antibiotics, and genetic disorders such as USH2A and DFNA9. This approach helps in understanding these ailments and in developing and testing potential therapeutic strategies.
Joints and cartilage – Modeling human joint tissues with osteoarthritis
Ambassadors: Ingrid Meulenbelt (i.meultenbelt@lumc.nl, MOLEPI) and Yolande Ramos (y.f.m.ramos@lumc.nl, MOLEPI)
To reduce, refine, and replace animal studies regenerative medicines research in the RegMedTO theme is focusing on development of a wide variety of human biomimetic joint tissue models such as 3D in vitro organoids, human explants, or osteochondral compartment-on-chips while using primary and hiPSC derived joint tissue cells. The range of complexity (osteochondral compartment-on-chip) and throughput (3D in vitro cartilage organoids in 96 well plates) of the models allows flexible and efficient preclinical testing. Moreover, to capture relevant human OA disease pathophysiological aspects in our models perturbations such hyper-physiological mechanical cues, inflammation, hypertrophy, or aberrant OA risk gene function (CRISPR/Cas9) are validated and applied.
Kidney – Modeling human kidney disease and toxicity
Ambassador: Siebe Spijker (h.s.spijker@lumc.nl, NIER)
Kidney organoids can be applied as models to study inherited kidney disease or the effect of drug toxicity. We use either patient-derived hiPSCs with gene-corrected controls or knock out of specific genes in hiPSCs to study glomerular (Alport syndrome, APOL1 nephropathy) and tubular kidney disease (polycystic kidney disease). Disease models are established both in vitro as well as after transplantation into mice or chick embryos. Pathophysiological mechanisms are assessed by transcriptomics, metabolomics and functional assays. Furthermore, a platform to study the toxicity of antimicrobial drugs is in development using 2D culture, organ-on-chip and 3D organoids combined with fluorescent toxicity reporters.
Kidney – Modeling polycystic kidney disease
Ambassador: Dorien Peters (d.j.m.peters@lumc.nl, HG)
We apply a multidisciplinary state-of-the art program to get insight into the genetic, pathophysiologic and functional mechanisms of Polycystic Kidney Disease. Model systems are being used to perform in-depth analyses of the molecular alterations, to identify new drug targets and test potential therapies. In addition to inducible (knock out) mouse models and 3D-cultured spheroids, we study cystic tubular dilation and the effects of therapeutic interventions in a microfluidic model with adult renal cells. In general, wt, and (inducible) knock-out cells are being used.
Lung - Development of advanced pulmonary cell culture models to study tissue injury and repair of the lungs
Ambassador: Anne van der Does (a.van_der_does@lumc.nl, LONG)
Significant advances have been made in animal-free model development for lung but replicating its complex (3D) biology and accounting for the (mechano)biological and anatomical differences between humans and traditional animal models has been challenging. Collectively, lung diseases are the leading cause of death globally, their onset and advancement linked to environmental factors such as exposure to air pollutants, cigarette consumption and infection by pathogens. For this reason, creating realistic human lung models is of significant health importance. In particular, the progressive destruction of the alveoli—where vital gas exchange occurs—poses a significant threat to life. Alveoli loss is irreversible, persisting for example even after ceasing smoking, and diminishes the surface area crucial for efficient gas exchange. The absence of regenerative treatments to restore the alveolar compartment partly stems from failures in (pre-)clinical alveolar research. Addressing the pressing need for animal-free, human models that offer robust translatability to human physiology is the focus of researchers in RegMedTO who are actively developing advanced cell culture models to study tissue injury and regeneration in lung diseases such as COPD and pulmonary fibrosis.
Nervous system – Modeling symptomatic OA pain-on-chip
Ambassador: Yolande Ramos (y.f.m.ramos@lumc.nl, MOLEPI
To model symptomatic osteoarthritis (OA) hiPSC derived sensory neurons are generated. With this model we aim to unravel and study the intertwined processes of joint tissue homeostasis and sensitization to pain. This will allow us to ascertain evidence-based drugs that relieve joint pain without advancing development of OA. Henceforth, the symptomatic OA on chip is exploited to identify and test effective and safe drug to relief pain in OA.
Reproductive organs – in vitro gametogenesis to model infertility
Ambassador: Susana Chuva de Sousa Lopes (s.m.chuva_de_sousa_lopes@lumc.nl, ANA)
Gamete formation is a long and complex process, involving significant remodeling of the cytoplasm and nucleus of germ cells in a sex-specific manner. The aim of our project is to develop protocols to generate female and male gametes from hiPSC to model infertility. We investigate not only the differentiation to sex-specific gametes, but also to the sex-specific gonadal somatic niche.
Pancreas – Modeling pancreatic disease
Ambassador: Eelco de Koning (e.j.p.de_koning@lumc.nl, INT) and Françoise Carlotti (f.carlotti@lumc.nl, INT)
To model pancreatic disease, we use both primary human pancreatic tissue and islets generated from hiPSCs. The models help us understand the underlying mechanisms of the diseases and provide a platform to find targets for novel treatments and screen for new drugs. In the context of islets, we also study the interaction between the different islet cell types and how the ratio of these cell types influences the composition and functioning of the islets. We also create iPSC cell line models of MODY (maturity-onset of Diabetes of the Young), which are variants of diabetes caused by a single gene mutation. Furthermore, we are also working on an islet-on-a-chip model, where we can study individual islets, as opposed to pools of islets.
Skeletal Muscle – 3D tissue engineered skeletal muscles
Ambassador: Jessica de Greef (j.c.de_greef@lumc.nl, HG)
To improve disease modeling and develop novel therapies for skeletal muscle disorders like facioscapulohumeral muscular dystrophy, Duchenne muscular dystrophy, oculopharyngeal muscular dystrophy, and myasthenia gravis, we have established 3D tissue engineered skeletal muscles (3D-TESMs) from myogenic progenitors generated from hiPSC, allowing functional readout of contractile force and upon treatment with drugs these 3D-TESMs show similarity with clinical observations. We established an easy method based on 3D printing to fabricate PDMS chips with flexible pillars which can be used to generate 3D-TESMs. In addition, we co-developed the Cuore of Optics11 Life B.V., a cantilever-based system that uses integrated optical sensing and electrical pulse stimulation for continuous stimulation and real-time recording of the contractile activity of 3D-TESMs.
Skin – Modelling monogenic human skin diseases
Ambassador: Karine Raymond (k.i.raymond@lumc.nl, ANA)
Skin organoids can be used as models to study inherited skin diseases. We are developing mutant and control isogenic hiPSC-based models to study cellular and molecular mechanisms underlying inherited skin diseases and develop potential treatments. These diseases include disorders associated with skin fragility and hypersensitivity to UV radiation, i.e., certain types of Epidermolysis bullosa and Xeroderma pigmentosum. We develop stem cell-based models amenable to phenotypic screenings for repurposed drug therapy.
Skin – reconstructed human skin models: tools for cancer research and screening purposes
HSEs are in-vitro systems that are engineered by seeding fibroblasts into a three-dimensional dermal matrix onto which keratinocytes are seeded. After specific culture conditions a HSE is formed that recapitulates most of the in vivo characteristics including an epidermis and dermis and in which cellular processes may be normalized compared to the conventional monolayer cultures. HSEs are therefore an attractive tool to study cell-cell, cell-matrix, dermal-epidermal interactions and other processes that are involved in epidermal morphogenesis. The 3D- models that are mostly used within our research are the Leiden Epidermal model (LEM), the Full-Thickness model (FTM) and the Fibroblasts-Derived Matrix model FDM). The LEM consists of keratinocytes seeded on a non-cellular matrix (e.g. inert filter membrane or de-epidermized dermis). These epidermal models are suitable for e.g. skin toxicity, irritation, microbiome or to mimic eczema and psoriasis. The FTM consists of keratinocytes, melanocytes and a fibroblast-populated three-dimensional collagen matrix. FTMs are an excellent tool to mimic skin conditions (e.g. wound-healing, infection) or diseased skin disorders in vitro (e.g. Squamous Cell Carcinoma (SCC), head and neck SCC, Vulvar SCC, melanoma, Epidermolysis Bullosa Simplex) in order to test therapeutics. The FDM is similar to the FTM model, but the dermal compartment consists of human fibroblast-derived extracellular matrix and can kept in culture up to 20-weeks. This model can be used as a tool to evaluate the effect of e.g. ingredients on dermal processes, such as in skin aging. All these HSEs closely mimic many aspects of native human skin including a competent skin barrier. Currently all three models are being used for e.g. microbiome research in young and aged skin and to understand the host-microbiome interaction. In addition, to understand skin cancer processes, we are studying cancer-associated fibroblasts (CAFs) and their precursors using HSEs to unravel the underlying mechanism of CAF-tumor cell interplay and CAF differentiation process. Meanwhile, we are exploring potential CAF-targeting strategies to strengthen current tumor therapies in skin cancers. In a new project entitled “Next generation immunodermatology (NGID)”, funded NWO (NWA): From one-size-fits-all to high-tech personalized skin care, we aim to develop immune-competent skin models to better understand the process in skin lymphoma, psoriasis, atopic dermatitis, urticaria, lupus, and hidradentis.
Vascular system – vessels-on-chip from hiPSCs to model vascular disease
Hemorrhaging vascular disease exacerbated by inflammation can have serious effects on brain, heart, kidney, lung and other organs, leading to conditions varying from dementia, organ failure and death. We generate hiPSC from patients with these conditions that have mutations identified as causative. hiPSC can be differentiated to endothelial cells, pericytes and smooth muscle cells, the cellular components of blood (and lymph) vessels, and used in 2D, or in 3D vessels–on-chip under microfluidic flow. Under these conditions, the phenotype of diseases like hereditary hemorrhagic telangiectasia can be replicated and screened from drugs that reverse the phenotype. In addition, we use the model to investigate the effects on hiPSC-derived macrophages initiate vascular damage. In this way, we are working towards new therapies that can stabilize blood vessels and prevent sometimes devastating blood loss.
Other – Disease modeling using hiPSCs
LUMC is a major center in the Netherlands for the generation of hiPSC lines from patients. These cells can form any cells of the body and have the same genetic make-up as the donor. They can be genetically manipulated so that genetic mutations can be corrected, or disease mutations introduced into a healthy hiPSC line. Protocols have been developed and tested to create many cell types, including from the heart, brain, blood vessels, cells of the lung, kidney, eye, cartilage or bone, skeletal muscle, pancreas, and the reproductive and immune systems. This provides us with information on relevant changes in cells and tissue caused by mutations, drugs, toxic compounds, bacteria, and viruses. In combination with omics analyses, (live) imaging, and electrophysiology, it helps identify disease targets for phenotypic correction and future therapies.
Other – Creating realistic human tissue mimics using microfluidics
Ambassador: Hanna Lammertse (h.c.a.lammertse@lumc.nl, ANA), Valeria Orlova (v.orlova@lumc.nl, ANA) and Christine Mummery (c.l.mummery@lumc.nl, ANA)
Living tissues in the body are complex 3D structures composed of different cell types, with blood and lymph vessels through which fluids flow. The tissues undergo spatial constraints that result in pressure and load, and in the case of muscle, contraction, and relaxation in response to (patho)physiological stimuli. To create realistic tissue models, it is important to include at least some of these parameters. This is done in Organ-on-Chip devices (OoCs) and LUMC is one of a few centers in the Netherlands specialized in the use of OoCs to address biological questions on human tissue (dys)function based on stem cells. Heart-, lung-, eye-, sensory neurons-, brain-, lymph-, osteochondral-unit-, and blood vessels-on chip are already available or presently under development.
Other – Ethical evaluation of organ-on-chip models
Ambassadors: Jesse Weidema (j.j.weidema@lumc.nl, E&R) and Nienke de Graeff (n.de_graeff@lumc.nl, E&R)
Organ-on-chip (OoC) microdevices emulate the architecture and physiology of specific tissues and organs by growing human-derived (stem) cells on microfluidic chips. These chips can offer insights into human disease mechanisms, aid in discovering novel drugs, contribute to developing personalized medicine and potentially reduce the use of traditional animal testing models. Alongside its many promises for science, medicine and healthcare, OoC technology also poses ethical challenges because it makes use of human biological material, produces sensitive personal data, involves long-term storage mechanisms in biobanks, and could potentially create semi-biological entities with human-like features. By employing a combination of empirical and normative ethics research methodologies, this project intends to map the different views on OoC technologies, explore methodological and epistemological uncertainties apparent in OoC research, and assess the ethical implications of OoC models as they are translated from research to clinical use. As such, the project might effectively inform and potentially guide OoC model design and clinical implementation, as well as regulatory and policy decision-making procedures.
Contact
Email: RegMedTheme@lumc.nl
Sub theme lead: Christine Mummery (c.l.mummery@lumc.nl)