The study of human 3D cell culture models not only bridges the gap between traditional 2D
experiments and
animal models, it also addresses processes that cannot be recapitulated by either of these ...traditional models. Therefore, it offers an opportunity to better understand complex biology including brain development. The brain organoid technology provides a physiologically relevant context, which holds great potential for its application in modeling neurological diseases. Here, we compare different methods to obtain highly specialized structures that resemble specific features of the human midbrain. Regionally patterned neural stem cells (NSCs) were utilized to derive such human midbrain-specific organoids (hMO). The resulting neural tissue exhibited abundant neurons with midbrain dopaminergic neuron identity, as well as astroglia and oligodendrocyte differentiation. Within the midbrain organoids, neurite myelination, and the formation of synaptic connections were observed. Regular neuronal fire patterning and neural network synchronicity were determined by multielectrode array recordings. In addition to electrophysiologically functional neurons producing and secreting dopamine, responsive neuronal subtypes, such as GABAergic and glutamatergic neurons were also detected. In order to model disorders like Parkinson's disease (PD)
, midbrain organoids carrying a disease specific mutation were derived and compared to healthy control organoids to investigate relevant neurodegenerative pathophysiology. In this way midbrain-specific organoids constitute a powerful tool for human-specific
modeling of neurological disorders with a great potential to be utilized in advanced therapy development.
The study of human midbrain development and midbrain related diseases, like Parkinson’s disease (PD), is limited by deficiencies in the currently available and validated laboratory models. Three ...dimensional midbrain organoids represent an innovative strategy to recapitulate some aspects of the complexity and physiology of the human midbrain. Nevertheless, also these novel organoid models exhibit some inherent weaknesses, including the presence of a necrotic core and batch-to-batch variability. Here we describe an optimized approach for the standardized generation of midbrain organoids that addresses these limitations, while maintaining key features of midbrain development like dopaminergic neuron and astrocyte differentiation. Moreover, we have established a novel time-efficient, fit for purpose analysis pipeline and provided proof of concept for its usage by investigating toxin induced PD.
We describe the generation of an isogenic control cell line DJ-1-delP GC13 from an induced pluripotent stem cell (iPSC) line DJ-1-delP LCSBi008-A that was derived from fibroblasts obtained from a ...Parkinson’s disease (PD) patient. Using CRISPR/Cas9 technology, we corrected the disease causing c.471_473delGCC homozygous mutation in the PARK7 gene leading to p.158P deletion in the encoded protein DJ-1. The generated isogenic pair will be used for phenotypic analysis of PD-patient derived neurons and astrocytes.
Genome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue ...therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.
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•Fluorescent protein-SNP pairs enable deterministic genotypes for genome editing•Repetitive elements modulate off-targeting random integration•Parkinson's disease NESCs present impaired mitochondrial energy performance
In this article, Arias-Fuenzalida and colleagues show a platform to achieve deterministic genotypes for genome editing. The combinatorial use of fluorescent proteins and defined SNPs facilitates the genome editing endeavor. Using neuroepithelial stem cells, they demonstrate early mitochondrial phenotypes in SNCA mutants.
Abstract
The study of complex diseases relies on large amounts of data to build models toward precision medicine. Such data acquisition is feasible in the context of high-throughput screening, in ...which the quality of the results relies on the accuracy of the image analysis. Although state-of-the-art solutions for image segmentation employ deep learning approaches, the high cost of manually generating ground truth labels for model training hampers the day-to-day application in experimental laboratories. Alternatively, traditional computer vision-based solutions do not need expensive labels for their implementation. Our work combines both approaches by training a deep learning network using weak training labels automatically generated with conventional computer vision methods. Our network surpasses the conventional segmentation quality by generalising beyond noisy labels, providing a 25% increase of mean intersection over union, and simultaneously reducing the development and inference times. Our solution was embedded into an easy-to-use graphical user interface that allows researchers to assess the predictions and correct potential inaccuracies with minimal human input. To demonstrate the feasibility of training a deep learning solution on a large dataset of noisy labels automatically generated by a conventional pipeline, we compared our solution against the common approach of training a model from a small manually curated dataset by several experts. Our work suggests that humans perform better in context interpretation, such as error assessment, while computers outperform in pixel-by-pixel fine segmentation. Such pipelines are illustrated with a case study on image segmentation for autophagy events. This work aims for better translation of new technologies to real-world settings in microscopy-image analysis.
Degenerative diseases affecting bone tissues and the brain represent important problems with high socio-economic impact. Certain bone diseases, such as osteoporosis, are considered risk factors for ...the progression of neurological disorders. Often, patients with neurodegenerative diseases have bone fractures or reduced mobility linked to osteoarthritis. The bone is a dynamic tissue involved not only in movement but also in the maintenance of mineral metabolism. Bone is also associated with the generation of both hematopoietic stem cells (HSCs), and thus the generation of the immune system, and mesenchymal stem cells (MSCs). Bone marrow is a lymphoid organ and contains MSCs and HSCs, both of which are involved in brain health via the production of cytokines with endocrine functions. Hence, it seems clear that bone is involved in the regulation of the neuronal system and vice versa. This review summarizes the recent knowledge on the interactions between the nervous system and bone and highlights the importance of the interaction between nerve and bone cells. In addition, experimental models that study the interaction between nerve and skeletal cells are discussed, and innovative models are suggested to better evaluate the molecular interactions between these two cell types.
Abstract
Microglia, the immune cells of the brain, are a focus of studies in neurodegenerative diseases. Similarly, research about induced pluripotent stem cell (iPSC)-derived whole brain and ...region-specific organoids is increasing. In organoids, the complexity of the culture systems increases, mimicking better the actual scenario in the human brain. Furthermore, animal models do not always recapitulate human neurodegeneration, and they imply more ethical concerns compared to organoid systems. Recently the integration of iPSC-derived microglia into brain organoids has been achieved, and on-chip technologies have been focusing on microglia interaction with neural cells. In this review, we discuss the achievements on integrating microglia into brain organoids. We study the cell organization, ultrastructure and cell signalling of microglia with respect to other cell types in organoids as well as their functionality in the system. A particular focus here is on the interaction with the midbrain and dopaminergic systems. Finally, we discuss the achievements until now concerning neuroinflammation and disease modelling, and the possible therapeutic approaches targeting microglia and neuroinflammation in 3D systems.
Nurr1 Spathis, Athanasios D.; Asvos, Xenophon; Ziavra, Despina ...
Proceedings of the National Academy of Sciences,
04/2017, Letnik:
114, Številka:
15
Journal Article
Recenzirano
Odprti dostop
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons in the substantia nigra and the gradual depletion of dopamine (DA). ...Current treatments replenish the DA deficit and improve symptoms but induce dyskinesias over time, and neuroprotective therapies are nonexistent. Here we report that Nuclear receptor-related 1 (Nurr1):Retinoid X receptor α (RXRα) activation has a double therapeutic potential for PD, offering both neuroprotective and symptomatic improvement. We designed BRF110, a unique in vivo active Nurr1:RXRα-selective lead molecule, which prevents DAergic neuron demise and striatal DAergic denervation in vivo against PD-causing toxins in a Nurr1-dependent manner. BRF110 also protects against PD-related genetic mutations in patient induced pluripotent stem cell (iPSC)-derived DAergic neurons and a genetic mouse PD model. Remarkably, besides neuroprotection, BRF110 up-regulates tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and GTP cyclohydrolase I (GCH1) transcription; increases striatal DA in vivo; and has symptomatic efficacy in two postneurodegeneration PD models, without inducing dyskinesias on chronic daily treatment. The combined neuroprotective and symptomatic effects of BRF110 identify Nurr1:RXRα activation as a potential monotherapeutic approach for PD.
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