Littérature scientifique sur le sujet « MIDBRAIN ORGANOIDS »

The combination of induced pluripotent stem cell (iPSC) technology and 3D cell culture creates a unique possibility for the generation of organoids that mimic human organs in in vitro cultures. The use of iPS cells in organoid cultures enables the differentiation of cells into dopaminergic neurons, also found in the human midbrain. However, long-lasting organoid cultures often cause necrosis within organoids. In this work, we present carbon fibers (CFs) for medical use as a new type of scaffold for organoid culture, comparing them to a previously tested copolymer poly-(lactic-co-glycolic acid) (PLGA) scaffold. We verified the physicochemical properties of CF scaffolds compared to PLGA in improving the efficiency of iPSC differentiation within organoids. The physicochemical properties of carbon scaffolds such as porosity, microstructure, or stability in the cellular environment make them a convenient material for creating in vitro organoid models. Through screening several genes expressed during the differentiation of organoids at crucial brain stages of development, we found that there is a correlation between PITX3, one of the key regulators of terminal differentiation, and the survival of midbrain dopaminergic (mDA) neurons and tyrosine hydroxylase (TH) gene expression. This makes organoids formed on carbon scaffolds an improved model containing mDA neurons convenient for studying midbrain-associated neurodegenerative diseases such as Parkinson’s disease.

The development of brain organoids represents a major technological advance in the stem cell field, a novel bridge between traditional 2D cultures and in vivo animal models. In particular, the development of midbrain organoids containing functional dopaminergic neurons producing neuromelanin granules, a by-product of dopamine synthesis, represents a potential new model for Parkinson’s disease. To generate human midbrain organoids, we introduce specific inductive cues, at defined timepoints, during the 3D culture process to drive the stem cells towards a midbrain fate. In this method paper, we describe a standardized protocol to generate human midbrain organoids (hMOs) from induced pluripotent stem cells (iPSCs). This protocol was developed to demonstrate how human iPSCs can be successfully differentiated into numerous, high quality midbrain organoids in one batch. We also describe adaptations for cryosectioning of fixed organoids for subsequent histological analysis.

The development of brain organoids represents a major technological advance in the stem cell field, a novel bridge between traditional 2D cultures and in vivo animal models. In particular, the development of midbrain organoids containing functional dopaminergic neurons producing neuromelanin granules, a by-product of dopamine synthesis, represents a potential new model for Parkinson’s disease. To generate human midbrain organoids, we introduce specific inductive cues, at defined timepoints, during the 3D culture process to drive the stem cells towards a midbrain fate. In this method paper, we describe a standardized protocol to generate human midbrain organoids (hMOs) from induced pluripotent stem cells (iPSCs). This protocol was developed to demonstrate how human iPSCs can be successfully differentiated into numerous, high quality midbrain organoids in one batch. We also describe adaptations for cryosectioning of fixed organoids for subsequent histological analysis.

Organoids are becoming particularly popular in modeling diseases that are difficult to reproduce in animals, due to anatomical differences in the structure of a given organ. Thus, they are a bridge between the in vitro and in vivo models. Human midbrain is one of the structures that is currently being intensively reproduced in organoids for modeling Parkinson’s disease (PD). Thanks to three-dimensional (3D) architecture and the use of induced pluripotent stem cells (iPSCs) differentiation into organoids, it has been possible to recapitulate a complicated network of dopaminergic neurons. In this work, we present the first organoid model for an idiopathic form of PD. iPSCs were generated from peripheral blood mononuclear cells of healthy volunteers and patients with the idiopathic form of PD by transduction with Sendai viral vector. iPSCs were differentiated into a large multicellular organoid-like structure. The mature organoids displayed expression of neuronal early and late markers. Interestingly, we observed statistical differences in the expression levels of LIM homeobox transcription factor alpha (early) and tyrosine hydroxylase (late) markers between organoids from PD patient and healthy volunteer. The obtained results show immense potential for the application of 3D human organoids in studying the neurodegenerative disease and modeling cellular interactions within the human brain.

AbstractHuman stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organization and function of a respective organ or part of an organ. Human midbrain organoids (hMOs) have been described to contain midbrain-specific dopaminergic neurons that release the neurotransmitter dopamine. However, the human midbrain contains also additional neuronal cell types, which are functionally interacting with each other. Here, we analysed hMOs at high-resolution by means of single-cell RNA sequencing (scRNA-seq), imaging and electrophysiology to unravel cell heterogeneity. Our findings demonstrate that hMOs show essential neuronal functional properties as spontaneous electrophysiological activity of different neuronal subtypes, including dopaminergic, GABAergic, glutamatergic and serotonergic neurons. Recapitulating these in vivo features makes hMOs an excellent tool for in vitro disease phenotyping and drug discovery.

A novel dopamine targeted electrochemical detection strategy has enabled the phenotyping and non-invasive monitoring of human midbrain organoids (healthy and Parkinson's diseased), by employing a redox-cycling based microsensor.

ABSTRACT Background: Park2 mutations cause Autosomal Recessive Juvenile Parkinsonism (ARJP), characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Park2 encodes for a ubiquitin-ligase protein whose mutation upregulates Gluk2, a subunit of the glutamate kainate receptor (KAR), expressed in SNpc neurons. Park2 is highly expressed also in astrocytes and KARs upregulation may induce excitotoxicity both in DA neurons and glia, leading to an early synaptopathy, neuroinflamation and neurodegeneration. Aims and Objectives: 1. To generate Park2 induced pluripotent stem cells (iPSCs)-derived in vitro cellular models; 2. To characterize Park2 iPSCs-derived in vitro cellular models; 3. to test glutamate toxicity due to KAR upregulation in Park2 cellular models. Materials and Methods: Fibroblasts and lymphocytes from Park2 patients and age-matched controls were reprogrammed into iPSCs. The iPSCs were further differentiated into dopaminergic neurons, astrocytes and mesencephalic organoids were generated and differentiated. Protein expression profile was analysed through western blot (WB), qPCR and immunofluorescence (IF). Electrophysiology assessment was performed on dopaminergic neurons and midbrain organoids in order to better functionally profile these models. Results: Gluk2 levels resulted significantly increased in PARK2 midbrain organoids compared to CTR both at WB (p< 0.001) and qPCR analyses (p< 0.001). Gluk2 levels resulted also significantly enhanced in PARK2 astrocytes both at WB (p< 0.05) and qPCR analyses (p< 0.05). TH mRNA and protein levels were significantly increased both in PARK2 dopaminergic neurons (WB p< 0.01; qPCR p< 0.0001; IF p< 0.0001) and midbrain organoids (WB p< 0.01; qPCR p< 0.0001; IF p< 0.0001) compared to CTR. Glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, resulted enhanced in PARK2 astrocytes and especially in PARK2 midbrain organoids (WB p< 0.001; IF p< 0.01). EAAT2, the astrocytic glutamate transporter resulted reduced in mutated lines (WB p< 0.01). Calcium-imaging and HD-MEAs show an oscillatory augmented reactivity in PARK2 midbrain organoids. Conclusions and perspectives: Gluk2 expression was enhanced in PARK2 astrocytes and midbrain organoids, confirming the previous finding that Park2 mutations lead to KAR upregulation (Maraschi A, 2014). Neuronal reactivity was also found increased in PARK2 midbrain organoids at electrophysiology assessment, maybe linked to glutamate dysregulation. Two innovative findings emerged from this study. First of all, that TH expression resulted increased in PARK2, supporting previous finding that stated an augmented dopamine turnover and a reduced dopamine re-uptake (Jiang H., 2012). This is an impairment that happens early in the neurodegenerative process and that could consequently lead to an excessive oxidative stress and consequent neurodegeneration. The second original result is that PARK2 is associated to an increased astrocytic reactivity and a possible dysfunction of astrocytic glutamate transporter EAAT2. This finding means that astrocytes play a key role in neurodegeneration although it is not clear whether they contribute to the initiation or propagation of it. Their increased reactivity could be the consequence of a glutamate toxicity or glutamate toxicity could result from reactive astrocytes dysfunction, not able to process the excessive glutamate influx. Further studies are required in order to establish Park2 role in TH expression regulation, in astrocytic reactivity induction and in glutamate toxicity.