The human brain is a complex, three‐dimensional structure. To better recapitulate brain complexity, recent efforts have focused on the development of human‐specific midbrain organoids. Human ...iPSC‐derived midbrain organoids consist of differentiated and functional neurons, which contain active synapses, as well as astrocytes and oligodendrocytes. However, the absence of microglia, with their ability to remodel neuronal networks and phagocytose apoptotic cells and debris, represents a major disadvantage for the current midbrain organoid systems. Additionally, neuroinflammation‐related disease modeling is not possible in the absence of microglia. So far, no studies about the effects of human iPSC‐derived microglia on midbrain organoid neural cells have been published. Here we describe an approach to derive microglia from human iPSCs and integrate them into iPSC‐derived midbrain organoids. Using single nuclear RNA Sequencing, we provide a detailed characterization of microglia in midbrain organoids as well as the influence of their presence on the other cells of the organoids. Furthermore, we describe the effects that microglia have on cell death and oxidative stress‐related gene expression. Finally, we show that microglia in midbrain organoids affect synaptic remodeling and increase neuronal excitability. Altogether, we show a more suitable system to further investigate brain development, as well as neurodegenerative diseases and neuroinflammation.
Main Points
Microglia were efficiently integrated into midbrain organoids.
Oxidative stress‐related genes are downregulated in organoids with microglia.
Gene expression and electrophysiology suggest a better neuronal functionality upon coculture.
Human 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.
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.
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.
RATIONALE:The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion ...of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown.
OBJECTIVE:We investigated LDL contributions to the m-RCT pathway in hypercholesterolemic mice.
METHODS AND RESULTS:Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (human APOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B–containing lipoproteins.
CONCLUSIONS:Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces.
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