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Journal articles on the topic 'MIDBRAIN ORGANOIDS'

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Author: Grafiati

Published: 9 March 2023

Last updated: 10 March 2023

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1

Tejchman, Anna, Agnieszka Znój, Paula Chlebanowska, Aneta Frączek-Szczypta, and Marcin Majka. "Carbon Fibers as a New Type of Scaffold for Midbrain Organoid Development." International Journal of Molecular Sciences 21, no. 17 (August 19, 2020): 5959. http://dx.doi.org/10.3390/ijms21175959.

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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.

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2

Mohamed, Nguyen-Vi, Meghna Mathur, Ronan V. da Silva, Rhalena A. Thomas, Paula Lepine, Lenore K. Beitel, Edward A. Fon, and Thomas M. Durcan. "Generation of human midbrain organoids from induced pluripotent stem cells." MNI Open Research 3 (February 11, 2021): 1. http://dx.doi.org/10.12688/mniopenres.12816.2.

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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.

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3

Mohamed, Nguyen-Vi, Meghna Mathur, Ronan V. da Silva, Lenore K. Beitel, Edward A. Fon, and Thomas M. Durcan. "Generation of human midbrain organoids from induced pluripotent stem cells." MNI Open Research 3 (April 3, 2019): 1. http://dx.doi.org/10.12688/mniopenres.12816.1.

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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.

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4

Chlebanowska, Paula, Anna Tejchman, Maciej Sułkowski, Klaudia Skrzypek, and Marcin Majka. "Use of 3D Organoids as a Model to Study Idiopathic Form of Parkinson’s Disease." International Journal of Molecular Sciences 21, no. 3 (January 21, 2020): 694. http://dx.doi.org/10.3390/ijms21030694.

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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.

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5

Smits, Lisa M., Stefano Magni, Kaoru Kinugawa, Kamil Grzyb, Joachim Luginbühl, Sonia Sabate-Soler, Silvia Bolognin, et al. "Single-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids." Cell and Tissue Research 382, no. 3 (July 31, 2020): 463–76. http://dx.doi.org/10.1007/s00441-020-03249-y.

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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.

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6

Zanetti, Cristian, Sarah Spitz, Emanuel Berger, Silvia Bolognin, Lisa M. Smits, Philipp Crepaz, Mario Rothbauer, et al. "Monitoring the neurotransmitter release of human midbrain organoids using a redox cycling microsensor as a novel tool for personalized Parkinson's disease modelling and drug screening." Analyst 146, no. 7 (2021): 2358–67. http://dx.doi.org/10.1039/d0an02206c.

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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.

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7

Tieng, Vannary, Luc Stoppini, Sabrina Villy, Marc Fathi, Michel Dubois-Dauphin, and Karl-Heinz Krause. "Engineering of Midbrain Organoids Containing Long-Lived Dopaminergic Neurons." Stem Cells and Development 23, no. 13 (July 2014): 1535–47. http://dx.doi.org/10.1089/scd.2013.0442.

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8

Monzel, Anna S., Kathrin Hemmer, Tony Kaoma, Lisa M. Smits, Silvia Bolognin, Philippe Lucarelli, Isabel Rosety, et al. "Machine learning-assisted neurotoxicity prediction in human midbrain organoids." Parkinsonism & Related Disorders 75 (June 2020): 105–9. http://dx.doi.org/10.1016/j.parkreldis.2020.05.011.

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9

Zagare, Alise, Matthieu Gobin, Anna S. Monzel, and Jens C. Schwamborn. "A robust protocol for the generation of human midbrain organoids." STAR Protocols 2, no. 2 (June 2021): 100524. http://dx.doi.org/10.1016/j.xpro.2021.100524.

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10

Lin, Yi, Benjamin Liou, Jason Hammonds, Christopher N. Mayhew, and Ying Sun. "Modeling neuronopathic Gaucher disease with human patient-specific midbrain organoids." Molecular Genetics and Metabolism 135, no. 2 (February 2022): S75. http://dx.doi.org/10.1016/j.ymgme.2021.11.191.

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11

Monzel, Anna S., Lisa M. Smits, Kathrin Hemmer, Siham Hachi, Edinson Lucumi Moreno, Thea van Wuellen, Javier Jarazo, et al. "Derivation of Human Midbrain-Specific Organoids from Neuroepithelial Stem Cells." Stem Cell Reports 8, no. 5 (May 2017): 1144–54. http://dx.doi.org/10.1016/j.stemcr.2017.03.010.

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12

Kim, Hongwon, Hyeok Ju Park, Hwan Choi, Yujung Chang, Hanseul Park, Jaein Shin, Junyeop Kim, Christopher J. Lengner, Yong Kyu Lee, and Jongpil Kim. "Modeling G2019S-LRRK2 Sporadic Parkinson's Disease in 3D Midbrain Organoids." Stem Cell Reports 12, no. 3 (March 2019): 518–31. http://dx.doi.org/10.1016/j.stemcr.2019.01.020.

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13

Ben-Reuven, Lihi, and Orly Reiner. "Toward Spatial Identities in Human Brain Organoids-on-Chip Induced by Morphogen-Soaked Beads." Bioengineering 7, no. 4 (December 18, 2020): 164. http://dx.doi.org/10.3390/bioengineering7040164.

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Recent advances in stem-cell technologies include the differentiation of human embryonic stem cells (hESCs) into organ-like structures (organoids). These organoids exhibit remarkable self-organization that resembles key aspects of in vivo organ development. However, organoids have an unpredictable anatomy, and poorly reflect the topography of the dorsoventral, mediolateral, and anteroposterior axes. In vivo the temporal and the spatial patterning of the developing tissue is orchestrated by signaling molecules called morphogens. Here, we used morphogen-soaked beads to influence the spatial identities within hESC-derived brain organoids. The morphogen- and synthetic molecules-soaked beads were interpreted as local organizers, and key transcription factor expression levels within the organoids were affected as a function of the distance from the bead. We used an on-chip imaging device that we have developed, that allows live imaging of the developing hESC-derived organoids. This platform enabled studying the effect of changes in WNT/BMP gradients on the expression of key landmark genes in the on-chip human brain organoids. Titration of CHIR99201 (WNT agonist) and BMP4 directed the expression of telencephalon and medial pallium genes; dorsal and ventral midbrain markers; and isthmus-related genes. Overall, our protocol provides an opportunity to study phenotypes of altered regional specification and defected connectivity, which are found in neurodevelopmental diseases.

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14

Zagare, Alise, Kyriaki Barmpa, Semra Smajic, Lisa M. Smits, Kamil Grzyb, Anne Grünewald, Alexander Skupin, Sarah L. Nickels, and Jens C. Schwamborn. "Midbrain organoids mimic early embryonic neurodevelopment and recapitulate LRRK2-p.Gly2019Ser-associated gene expression." American Journal of Human Genetics 109, no. 2 (February 2022): 311–27. http://dx.doi.org/10.1016/j.ajhg.2021.12.009.

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15

Nickels, Sarah Louise, Jennifer Modamio, Bárbara Mendes-Pinheiro, Anna Sophia Monzel, Fay Betsou, and Jens Christian Schwamborn. "Reproducible generation of human midbrain organoids for in vitro modeling of Parkinson’s disease." Stem Cell Research 46 (July 2020): 101870. http://dx.doi.org/10.1016/j.scr.2020.101870.

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16

Sarrafha, Lily, Gustavo M. Parfitt, Ricardo Reyes, Camille Goldman, Elena Coccia, Tatyana Kareva, and Tim Ahfeldt. "High-throughput generation of midbrain dopaminergic neuron organoids from reporter human pluripotent stem cells." STAR Protocols 2, no. 2 (June 2021): 100463. http://dx.doi.org/10.1016/j.xpro.2021.100463.

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17

Jo, Junghyun, Lin Yang, Hoang‐Dai Tran, Weonjin Yu, Alfred Xuyang Sun, Ya Yin Chang, Byung Chul Jung, et al. "Lewy Body–like Inclusions in Human Midbrain Organoids Carrying Glucocerebrosidase and α‐Synuclein Mutations." Annals of Neurology 90, no. 3 (August 10, 2021): 490–505. http://dx.doi.org/10.1002/ana.26166.

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18

Jo, Junghyun, Yixin Xiao, Alfred Xuyang Sun, Engin Cukuroglu, Hoang-Dai Tran, Jonathan Göke, Zi Ying Tan, et al. "Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons." Cell Stem Cell 19, no. 2 (August 2016): 248–57. http://dx.doi.org/10.1016/j.stem.2016.07.005.

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19

Kim, Seung Won, Hye-Ji Woo, Eun Hee Kim, Hyung Sun Kim, Han Na Suh, Soo-hyun Kim, Jae-Jin Song, et al. "Neural stem cells derived from human midbrain organoids as a stable source for treating Parkinson’s disease." Progress in Neurobiology 204 (September 2021): 102086. http://dx.doi.org/10.1016/j.pneurobio.2021.102086.

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20

Kwak, Tae Hwan, Ji Hyun Kang, Sai Hali, Jonghun Kim, Kee-Pyo Kim, Chanhyeok Park, Ju-Hyun Lee, et al. "Generation of homogeneous midbrain organoids with in vivo- like cellular composition facilitates neurotoxin-based Parkinson's disease modeling." STEM CELLS 38, no. 6 (February 28, 2020): 727–40. http://dx.doi.org/10.1002/stem.3163.

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21

Becerra-Calixto, Andrea, Abhisek Mukherjee, Santiago Ramirez, Sofia Sepulveda, Tirthankar Sinha, Rabab Al-Lahham, Nicole De Gregorio, Camila Gherardelli, and Claudio Soto. "Lewy Body-like Pathology and Loss of Dopaminergic Neurons in Midbrain Organoids Derived from Familial Parkinson’s Disease Patient." Cells 12, no. 4 (February 15, 2023): 625. http://dx.doi.org/10.3390/cells12040625.

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Progressive accumulation of α-Synuclein (αSyn) in Lewy bodies (LBs) and loss of dopaminergic (DA) neurons are the hallmark pathological features of Parkinson’s disease (PD). Although currently available in vitro and in vivo models have provided crucial information about PD pathogenesis, the mechanistic link between the progressive accumulation of αSyn into LBs and the loss of DA neurons is still unclear. To address this, it is critical to model LB formation and DA neuron loss, the two key neuropathological aspects of PD, in a relevant in vitro system. In this study, we developed a human midbrain-like organoid (hMBO) model of PD. We demonstrated that hMBOs generated from induced pluripotent stem cells (hiPSCs), derived from a familial PD (fPD) patient carrying αSyn gene (SNCA) triplication accumulate pathological αSyn over time. These cytoplasmic inclusions spatially and morphologically resembled diverse stages of LB formation and were composed of key markers of LBs. Importantly, the progressive accumulation of pathological αSyn was paralleled by the loss of DA neurons and elevated apoptosis. The model developed in this study will complement the existing in vitro models of PD and will provide a unique platform to study the spatiotemporal events governing LB formation and their relation with neurodegeneration. Furthermore, this model will also be beneficial for in vitro screening and the development of therapeutic compounds.

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22

Boussaad, Ibrahim, Carolin D. Obermaier, Zoé Hanss, Dheeraj R. Bobbili, Silvia Bolognin, Enrico Glaab, Katarzyna Wołyńska, et al. "A patient-based model of RNA mis-splicing uncovers treatment targets in Parkinson’s disease." Science Translational Medicine 12, no. 560 (September 9, 2020): eaau3960. http://dx.doi.org/10.1126/scitranslmed.aau3960.

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Parkinson’s disease (PD) is a heterogeneous neurodegenerative disorder with monogenic forms representing prototypes of the underlying molecular pathology and reproducing to variable degrees the sporadic forms of the disease. Using a patient-based in vitro model of PARK7-linked PD, we identified a U1-dependent splicing defect causing a drastic reduction in DJ-1 protein and, consequently, mitochondrial dysfunction. Targeting defective exon skipping with genetically engineered U1-snRNA recovered DJ-1 protein expression in neuronal precursor cells and differentiated neurons. After prioritization of candidate drugs, we identified and validated a combinatorial treatment with the small-molecule compounds rectifier of aberrant splicing (RECTAS) and phenylbutyric acid, which restored DJ-1 protein and mitochondrial dysfunction in patient-derived fibroblasts as well as dopaminergic neuronal cell loss in mutant midbrain organoids. Our analysis of a large number of exomes revealed that U1 splice-site mutations were enriched in sporadic PD patients. Therefore, our study suggests an alternative strategy to restore cellular abnormalities in in vitro models of PD and provides a proof of concept for neuroprotection based on precision medicine strategies in PD.

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23

Veszelka, Szilvia, Mária Mészáros, Gergő Porkoláb, Anikó Szecskó, Nóra Kondor, Györgyi Ferenc, Tamás F. Polgár, et al. "A Triple Combination of Targeting Ligands Increases the Penetration of Nanoparticles across a Blood-Brain Barrier Culture Model." Pharmaceutics 14, no. 1 (December 30, 2021): 86. http://dx.doi.org/10.3390/pharmaceutics14010086.

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Nanosized drug delivery systems targeting transporters of the blood-brain barrier (BBB) are promising carriers to enhance the penetration of therapeutics into the brain. The expression of solute carriers (SLC) is high and shows a specific pattern at the BBB. Here we show that targeting ligands ascorbic acid, leucine and glutathione on nanoparticles elevated the uptake of albumin cargo in cultured primary rat brain endothelial cells. Moreover, we demonstrated the ability of the triple-targeted nanovesicles to deliver their cargo into midbrain organoids after crossing the BBB model. The cellular uptake was temperature- and energy-dependent based on metabolic inhibition. The process was decreased by filipin and cytochalasin D, indicating that the cellular uptake of nanoparticles was partially mediated by endocytosis. The uptake of the cargo encapsulated in triple-targeted nanoparticles increased after modification of the negative zeta potential of endothelial cells by treatment with a cationic lipid or after cleaving the glycocalyx with an enzyme. We revealed that targeted nanoparticles elevated plasma membrane fluidity, indicating the fusion of nanovesicles with endothelial cell membranes. Our data indicate that labeling nanoparticles with three different ligands of multiple transporters of brain endothelial cells can promote the transfer and delivery of molecules across the BBB.

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24

Mészáros, Mária, Thi Ha My Phan, Judit P. Vigh, Gergő Porkoláb, Anna Kocsis, Emese K. Páli, Tamás F. Polgár, et al. "Targeting Human Endothelial Cells with Glutathione and Alanine Increases the Crossing of a Polypeptide Nanocarrier through a Blood–Brain Barrier Model and Entry to Human Brain Organoids." Cells 12, no. 3 (February 3, 2023): 503. http://dx.doi.org/10.3390/cells12030503.

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Nanoparticles (NPs) are the focus of research efforts that aim to develop successful drug delivery systems for the brain. Polypeptide nanocarriers are versatile platforms and combine high functionality with good biocompatibility and biodegradability. The key to the efficient brain delivery of NPs is the specific targeting of cerebral endothelial cells that form the blood–brain barrier (BBB). We have previously discovered that the combination of two different ligands of BBB nutrient transporters, alanine and glutathione, increases the permeability of vesicular NPs across the BBB. Our aim here was to investigate whether the combination of these molecules can also promote the efficient transfer of 3-armed poly(l-glutamic acid) NPs across a human endothelial cell and brain pericyte BBB co-culture model. Alanine and glutathione dual-targeted polypeptide NPs showed good cytocompatibility and elevated cellular uptake in a time-dependent and active manner. Targeted NPs had a higher permeability across the BBB model and could subsequently enter midbrain-like organoids derived from healthy and Parkinson’s disease patient-specific stem cells. These results indicate that poly(l-glutamic acid) NPs can be used as nanocarriers for nervous system application and that the right combination of molecules that target cerebral endothelial cells, in this case alanine and glutathione, can facilitate drug delivery to the brain.

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25

Berger, Emanuel, Chiara Magliaro, Nicole Paczia, Anna S. Monzel, Paul Antony, Carole L. Linster, Silvia Bolognin, Arti Ahluwalia, and Jens C. Schwamborn. "Millifluidic culture improves human midbrain organoid vitality and differentiation." Lab on a Chip 18, no. 20 (2018): 3172–83. http://dx.doi.org/10.1039/c8lc00206a.

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26

Yeap, Yee Jie, Tng J. W. Teddy, Mok Jung Lee, Micaela Goh, and Kah Leong Lim. "From 2D to 3D: Development of Monolayer Dopaminergic Neuronal and Midbrain Organoid Cultures for Parkinson’s Disease Modeling and Regenerative Therapy." International Journal of Molecular Sciences 24, no. 3 (January 28, 2023): 2523. http://dx.doi.org/10.3390/ijms24032523.

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Parkinson’s Disease (PD) is a prevalent neurodegenerative disorder that is characterized pathologically by the loss of A9-specific dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain. Despite intensive research, the etiology of PD is currently unresolved, and the disease remains incurable. This, in part, is due to the lack of an experimental disease model that could faithfully recapitulate the features of human PD. However, the recent advent of induced pluripotent stem cell (iPSC) technology has allowed PD models to be created from patient-derived cells. Indeed, DA neurons from PD patients are now routinely established in many laboratories as monolayers as well as 3D organoid cultures that serve as useful toolboxes for understanding the mechanism underlying PD and also for drug discovery. At the same time, the iPSC technology also provides unprecedented opportunity for autologous cell-based therapy for the PD patient to be performed using the patient’s own cells as starting materials. In this review, we provide an update on the molecular processes underpinning the development and differentiation of human pluripotent stem cells (PSCs) into midbrain DA neurons in both 2D and 3D cultures, as well as the latest advancements in using these cells for drug discovery and regenerative medicine. For the novice entering the field, the cornucopia of differentiation protocols reported for the generation of midbrain DA neurons may seem daunting. Here, we have distilled the essence of the different approaches and summarized the main factors driving DA neuronal differentiation, with the view to provide a useful guide to newcomers who are interested in developing iPSC-based models of PD.

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27

Chlebanowska, Paula, Maciej Sułkowski, Klaudia Skrzypek, Anna Tejchman, Agata Muszyńska, Rezvan Noroozi, and Marcin Majka. "Origin of the Induced Pluripotent Stem Cells Affects Their Differentiation into Dopaminergic Neurons." International Journal of Molecular Sciences 21, no. 16 (August 9, 2020): 5705. http://dx.doi.org/10.3390/ijms21165705.

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Neuronal differentiation of human induced pluripotent stem (iPS) cells, both in 2D models and 3D systems in vitro, allows for the study of disease pathomechanisms and the development of novel therapies. To verify if the origin of donor cells used for reprogramming to iPS cells can influence the differentiation abilities of iPS cells, peripheral blood mononuclear cells (PBMC) and keratinocytes were reprogrammed to iPS cells using the Sendai viral vector and were subsequently checked for pluripotency markers and the ability to form teratomas in vivo. Then, iPS cells were differentiated into dopaminergic neurons in 2D and 3D cultures. Both PBMC and keratinocyte-derived iPS cells were similarly reprogrammed to iPS cells, but they displayed differences in gene expression profiles and in teratoma compositions in vivo. During 3D organoid formation, the origin of iPS cells affected the levels of FOXA2 and LMX1A only in the first stages of neural differentiation, whereas in the 2D model, differences were detected at the levels of both early and late neural markers FOXA2, LMX1A, NURR1, TUBB and TH. To conclude, the origin of iPS cells may significantly affect iPS differentiation abilities in teratomas, as well as exerting effects on 2D differentiation into dopaminergic neurons and the early stages of 3D midbrain organoid formation.

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Sam, Richard, Yu Chen, Barbara Stubblefield, and Ellen Sidransky. "Development of a human 3D midbrain organoid model for investigating the link between glucocerebrosidase and Parkinson's disease." Molecular Genetics and Metabolism 129, no. 2 (February 2020): S142. http://dx.doi.org/10.1016/j.ymgme.2019.11.375.

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29

Eze, Ugomma C., Aparna Bhaduri, Maximilian Haeussler, Tomasz J. Nowakowski, and Arnold R. Kriegstein. "Single-cell atlas of early human brain development highlights heterogeneity of human neuroepithelial cells and early radial glia." Nature Neuroscience 24, no. 4 (March 15, 2021): 584–94. http://dx.doi.org/10.1038/s41593-020-00794-1.

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AbstractThe human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.

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30

Fiorenzano, Alessandro, Edoardo Sozzi, Marcella Birtele, Janko Kajtez, Jessica Giacomoni, Fredrik Nilsson, Andreas Bruzelius, et al. "Single-cell transcriptomics captures features of human midbrain development and dopamine neuron diversity in brain organoids." Nature Communications 12, no. 1 (December 2021). http://dx.doi.org/10.1038/s41467-021-27464-5.

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AbstractThree-dimensional brain organoids have emerged as a valuable model system for studies of human brain development and pathology. Here we establish a midbrain organoid culture system to study the developmental trajectory from pluripotent stem cells to mature dopamine neurons. Using single cell RNA sequencing, we identify the presence of three molecularly distinct subtypes of human dopamine neurons with high similarity to those in developing and adult human midbrain. However, despite significant advancements in the field, the use of brain organoids can be limited by issues of reproducibility and incomplete maturation which was also observed in this study. We therefore designed bioengineered ventral midbrain organoids supported by recombinant spider-silk microfibers functionalized with full-length human laminin. We show that silk organoids reproduce key molecular aspects of dopamine neurogenesis and reduce inter-organoid variability in terms of cell type composition and dopamine neuron formation.

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Abbott, Joshua, Mitali Tambe, Ivan Pavlinov, Atena Farkhondeh, Ha Nam Nguyen, Miao Xu, Manisha Pradhan, et al. "Generation and characterization of NGLY1 patient-derived midbrain organoids." Frontiers in Cell and Developmental Biology 11 (February 16, 2023). http://dx.doi.org/10.3389/fcell.2023.1039182.

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NGLY1 deficiency is an ultra-rare, autosomal recessive genetic disease caused by mutations in the NGLY1 gene encoding N-glycanase one that removes N-linked glycan. Patients with pathogenic mutations in NGLY1 have complex clinical symptoms including global developmental delay, motor disorder and liver dysfunction. To better understand the disease pathogenesis and the neurological symptoms of the NGLY1 deficiency we generated and characterized midbrain organoids using patient-derived iPSCs from two patients with distinct disease-causing mutations–one homozygous for p. Q208X, the other compound heterozygous for p. L318P and p. R390P and CRISPR generated NGLY1 knockout iPSCs. We demonstrate that NGLY1 deficient midbrain organoids show altered neuronal development compared to one wild type (WT) organoid. Both neuronal (TUJ1) and astrocytic glial fibrillary acid protein markers were reduced in NGLY1 patient-derived midbrain organoids along with neurotransmitter GABA. Interestingly, staining for dopaminergic neuronal marker, tyrosine hydroxylase, revealed a significant reduction in patient iPSC derived organoids. These results provide a relevant NGLY1 disease model to investigate disease mechanisms and evaluate therapeutics for treatments of NGLY1 deficiency.

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32

Lee, Youngsun, Ji Su Kang, On-Ju Ham, Mi-Young Son, and Mi-Ok Lee. "Gut metabolite trimethylamine N-oxide induces aging-associated phenotype of midbrain organoids for the induced pluripotent stem cell-based modeling of late-onset disease." Frontiers in Aging Neuroscience 14 (August 16, 2022). http://dx.doi.org/10.3389/fnagi.2022.925227.

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Brain organoids are valuable research models for human development and disease since they mimic the various cell compositions and structures of the human brain; however, they have challenges in presenting aging phenotypes for degenerative diseases. This study analyzed the association between aging and the gut metabolite trimethylamine N-oxide (TMAO), which is highly found in the midbrain of elderly and Parkinson’s disease (PD) patients. TMAO treatment in midbrain organoid induced aging-associated molecular changes, including increased senescence marker expression (P21, P16), p53 accumulation, and epigenetic alterations. In addition, TMAO-treated midbrain organoids have shown parts of neurodegeneration phenotypes, including impaired brain-derived neurotrophic factor (BDNF) signaling, loss of dopaminergic neurons, astrocyte activation, and neuromelanin accumulation. Moreover, we found TMAO treatment-induced pathophysiological phosphorylation of α-synuclein protein at Ser-129 residues and Tau protein at Ser202/Thr205. These results suggest a role of TMAO in the aging and pathogenesis of the midbrain and provide insight into how intestinal dysfunction increases the risk of PD. Furthermore, this system can be utilized as a novel aging model for induced pluripotent stem cell (iPSC)-based modeling of late-onset diseases.

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Shang, Jia, Bin Li, Han Fan, Peidi Liu, Wen Zhao, Tao Chen, Pu Chen, and Longqiu Yang. "Sevoflurane promotes premature differentiation of dopaminergic neurons in hiPSC-derived midbrain organoids." Frontiers in Cell and Developmental Biology 10 (September 13, 2022). http://dx.doi.org/10.3389/fcell.2022.941984.

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Background: Conventional animal models used in corresponding basic studies are distinct from humans in terms of the brain’s development trajectory, tissue cytoarchitecture and cell types, making it difficult to accurately evaluate the potential adverse effects of anesthetic treatments on human fetal brain development. This study investigated the effects of sevoflurane on the midbrain’s development and cytopathology using human physiologically-relevant midbrain organoids.Methods: Monolayer human induced pluripotent stem cells (hiPSC)-derived human floor plate cells and three-dimensional hiPSC-derived midbrain organoids (hMBOs) were exposed to 2% (v/v) sevoflurane for 2 or 6 h, followed by expansion or differentiation culture. Then, immunofluorescence, real-time PCR, EdU assay, Tunnel assay, and transcriptome sequencing were performed to examine the effects of sevoflurane on the midbrain’s development.Results: We found that 2% sevoflurane exposure inhibited hFPCs’ proliferation (differentiation culture: 7.2% ± 0.3% VS. 13.3% ± 0.7%, p = 0.0043; expansion culture: 48% ± 2.2% VS. 35.2% ± 1.4%, p = 0.0002) and increased their apoptosis, but did not affect their differentiation into human dopaminergic neurons After 6 h, 2% sevoflurane exposure inhibited cell proliferation (62.8% ± 5.6% VS. 100% ± 5.5%, p = 0.0065) and enhanced the premature differentiation of hMBOs (246% ± 5.2% VS. 100% ± 28%, p = 0.0065). The RNA-seq results showed long-term exposure to sevoflurane up regulates some transcription factors in the differentiation of dopaminergic neurons, while short-term exposure to sevoflurane has a weak up-regulation effect on these transcription factors.Conclusion: This study revealed that long-term exposure to sevoflurane could promote the premature differentiation of hMBOs, while short-term exposure had negligible effects, suggesting that long-term exposure to sevoflurane in pregnant women may lead to fetals’ midbrain development disorder.

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Mohamed, Nguyen-Vi, Julien Sirois, Janani Ramamurthy, Meghna Mathur, Paula Lépine, Eric Deneault, Gilles Maussion, et al. "Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy." Brain Communications, September 25, 2021. http://dx.doi.org/10.1093/braincomms/fcab223.

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Abstract SNCA, the first gene associated with Parkinson’s disease, encodes the α-synuclein protein, the predominant component within pathological inclusions termed Lewy bodies. The presence of Lewy bodies is one of the classical hallmarks found in the brain of patients with Parkinson’s disease, and Lewy bodies have also been observed in patients with other synucleinopathies. However, the study of α-synuclein pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids, differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These human midbrain organoids recapitulate key features of α-synuclein pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication human midbrain organoids express elevated levels of α-synuclein and exhibit an age-dependent increase in α-synuclein aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-synuclein. These phosphorylated α-synuclein aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, human midbrain organoids from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinson’s disease and provide a powerful system to study the pathogenesis of synucleinopathies.

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Smits, Lisa M., Lydia Reinhardt, Peter Reinhardt, Michael Glatza, Anna S. Monzel, Nancy Stanslowsky, Marcelo D. Rosato-Siri, et al. "Modeling Parkinson’s disease in midbrain-like organoids." npj Parkinson's Disease 5, no. 1 (April 5, 2019). http://dx.doi.org/10.1038/s41531-019-0078-4.

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Renner, Henrik, Martha Grabos, Katharina J. Becker, Theresa E. Kagermeier, Jie Wu, Mandy Otto, Stefan Peischard, et al. "A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids." eLife 9 (November 3, 2020). http://dx.doi.org/10.7554/elife.52904.

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Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates (‘organoids’) or 3D structures with less physiological relevance (‘spheroids’). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.

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Renner, Henrik, Martha Grabos, Hans Schöler, and Jan Bruder. "Generation and Maintenance of Homogeneous Human Midbrain Organoids." BIO-PROTOCOL 11, no. 11 (2021). http://dx.doi.org/10.21769/bioprotoc.4049.

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Renner, Henrik, Katharina J. Becker, Theresa E. Kagermeier, Martha Grabos, Farsam Eliat, Patrick Günther, Hans R. Schöler, and Jan M. Bruder. "Cell-Type-Specific High Throughput Toxicity Testing in Human Midbrain Organoids." Frontiers in Molecular Neuroscience 14 (July 15, 2021). http://dx.doi.org/10.3389/fnmol.2021.715054.

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Toxicity testing is a crucial step in the development and approval of chemical compounds for human contact and consumption. However, existing model systems often fall short in their prediction of human toxicity in vivo because they may not sufficiently recapitulate human physiology. The complexity of three-dimensional (3D) human organ-like cell culture systems (“organoids”) can generate potentially more relevant models of human physiology and disease, including toxicity predictions. However, so far, the inherent biological heterogeneity and cumbersome generation and analysis of organoids has rendered efficient, unbiased, high throughput evaluation of toxic effects in these systems challenging. Recent advances in both standardization and quantitative fluorescent imaging enabled us to dissect the toxicities of compound exposure to separate cellular subpopulations within human organoids at the single-cell level in a framework that is compatible with high throughput approaches. Screening a library of 84 compounds in standardized human automated midbrain organoids (AMOs) generated from two independent cell lines correctly recognized known nigrostriatal toxicants. This approach further identified the flame retardant 3,3′,5,5′-tetrabromobisphenol A (TBBPA) as a selective toxicant for dopaminergic neurons in the context of human midbrain-like tissues for the first time. Results were verified with high reproducibility in more detailed dose-response experiments. Further, we demonstrate higher sensitivity in 3D AMOs than in 2D cultures to the known neurotoxic effects of the pesticide lindane. Overall, the automated nature of our workflow is freely scalable and demonstrates the feasibility of quantitatively assessing cell-type-specific toxicity in human organoids in vitro.

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Zhu, Wanying, Mengdan Tao, Yuan Hong, Shanshan Wu, Chu Chu, Zhilong Zheng, Xiao Han, et al. "Dysfunction of vesicular storage in young-onset Parkinson’s patient-derived dopaminergic neurons and organoids revealed by single cell electrochemical cytometry." Chemical Science, 2022. http://dx.doi.org/10.1039/d2sc00809b.

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Electrochemical cytometry based on nano-tip microelectrodes was used to quantify the vesicular storage at the single-cell level in human neurons and midbrain organoids which acted as disease models of young-onset...

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Smits, Lisa Maria, and Jens Christian Schwamborn. "Midbrain Organoids: A New Tool to Investigate Parkinson’s Disease." Frontiers in Cell and Developmental Biology 8 (May 19, 2020). http://dx.doi.org/10.3389/fcell.2020.00359.

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Wahlin, Karl J., Jie Cheng, Shawna L. Jurlina, Melissa K. Jones, Nicholas R. Dash, Anna Ogata, Nawal Kibria, et al. "CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids." Frontiers in Cell and Developmental Biology 9 (November 16, 2021). http://dx.doi.org/10.3389/fcell.2021.764725.

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Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced SIX6 expression and promoted eye formation. SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide “retina-like” structures we generated a SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting SIX6 + /POU4F2- and SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.

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Lacalle-Aurioles, María. "Matisse and the Organoids: The Art of Science." Neuroscientist, October 3, 2020, 107385842096136. http://dx.doi.org/10.1177/1073858420961362.

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Over the past few decades, scientists have transformed the way they do and understand science. They are now exploring their more creative side. This approach has accelerated fascinating discoveries such as human induced pluripotent stem cells and brain organoids; however, they have not been able to jump over the communication barrier with society. La Danse des Astrocytes, a scene observed during a routine microscopy session working on midbrain organoids, has motivated this essay, which urges scientists to find new forms of science communication and to work as a community to achieve the consolidation of a scientific culture.

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Magliaro, Chiara, and Arti Ahluwalia. "Clarifying mid-brain organoids: Application of the CLARITY protocol to unperfusable samples." Biomedical Science and Engineering, February 14, 2020. http://dx.doi.org/10.4081/bse.2019.113.

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The aim of this study was to apply a workflow, integrating delipidation methods and advanced 3D imaging techniques for mapping of the global neuronal organization of brain organoids. These are self-organizing constructs in vitro generated from human pluripotent stem cells encased in a Matrigel shell, which resemble downscaled structural and functional features of human brains. In particular, we focused on midbrain organoids, widely considered a promising tool for studying dopaminergic neuron degeneration in Parkinson’s Disease. The evaluation of the microanatomical alterations at a patient-level will potentially guide future research of this neuropathy, providing meaningful human specific data in line with the European Directives and the 3Rs principles.

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Kano, Masayoshi, Masashi Takanashi, Genko Oyama, Asako Yoritaka, Taku Hatano, Kahori Shiba-Fukushima, Makiko Nagai, et al. "Reduced astrocytic reactivity in human brains and midbrain organoids with PRKN mutations." npj Parkinson's Disease 6, no. 1 (November 13, 2020). http://dx.doi.org/10.1038/s41531-020-00137-8.

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AbstractParkin (encoded by PRKN) is a ubiquitin ligase that plays an important role in cellular mitochondrial quality control. Mutations in PRKN cause selective dopaminergic cell loss in the substantia nigra and are presumed to induce a decrease in mitochondrial function caused by the defective clearance of mitochondria. Several studies have demonstrated that parkin dysfunction causes mitochondrial injury and astrocytic dysfunction. Using immunohistochemical methods, we analyzed astrocytic changes in human brains from individuals with PRKN mutations. Few glial fibrillary acidic protein- and vimentin-positive astrocytes were observed in the substantia nigra in PRKN-mutated subjects compared with subjects with idiopathic Parkinson’s disease. We also differentiated patient-specific induced pluripotent stem cells into midbrain organoids and confirmed decreased numbers of glial fibrillary acidic protein-positive astrocytes in PRKN-mutated organoids compared with age- and sex-matched controls. Our study reveals PRKN-mutation-induced astrocytic alteration and suggests the possibility of an astrocyte-related non-autonomous cell death mechanism for dopaminergic neurons in brains of PRKN-mutated patients.

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Sozzi, Edoardo, Fredrik Nilsson, Janko Kajtez, Malin Parmar, and Alessandro Fiorenzano. "Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells." Current Protocols 2, no. 9 (September 2022). http://dx.doi.org/10.1002/cpz1.555.

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Galet, Benjamin, Hélène Cheval, and Philippe Ravassard. "Patient-Derived Midbrain Organoids to Explore the Molecular Basis of Parkinson's Disease." Frontiers in Neurology 11 (September 4, 2020). http://dx.doi.org/10.3389/fneur.2020.01005.

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Sabate‐Soler, Sonia, Sarah Louise Nickels, Cláudia Saraiva, Emanuel Berger, Ugne Dubonyte, Kyriaki Barmpa, Yan Jun Lan, et al. "Microglia integration into human midbrain organoids leads to increased neuronal maturation and functionality." Glia, March 9, 2022. http://dx.doi.org/10.1002/glia.24167.

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Hou, Yuxin, Chang Li, Chaemin Yoon, On Wah Leung, Sikun You, Xiaoming Cui, Jasper Fuk-Woo Chan, Duanqing Pei, Hoi Hung Cheung, and Hin Chu. "Enhanced replication of SARS-CoV-2 Omicron BA.2 in human forebrain and midbrain organoids." Signal Transduction and Targeted Therapy 7, no. 1 (November 20, 2022). http://dx.doi.org/10.1038/s41392-022-01241-2.

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Boussaad, Ibrahim, Gérald Cruciani, Silvia Bolognin, Paul Antony, Claire M. Dording, Yong-Jun Kwon, Peter Heutink, Eugenio Fava, Jens C. Schwamborn, and Rejko Krüger. "Integrated, automated maintenance, expansion and differentiation of 2D and 3D patient-derived cellular models for high throughput drug screening." Scientific Reports 11, no. 1 (January 14, 2021). http://dx.doi.org/10.1038/s41598-021-81129-3.

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AbstractPatient-derived cellular models become an increasingly powerful tool to model human diseases for precision medicine approaches. The identification of robust cellular disease phenotypes in these models paved the way towards high throughput screenings (HTS) including the implementation of laboratory advanced automation. However, maintenance and expansion of cells for HTS remains largely manual work. Here, we describe an integrated, complex automated platform for HTS in a translational research setting also designed for maintenance and expansion of different cell types. The comprehensive design allows automation of all cultivation steps and is flexible for development of methods for variable cell types. We demonstrate protocols for controlled cell seeding, splitting and expansion of human fibroblasts, induced pluripotent stem cells (iPSC), and neural progenitor cells (NPC) that allow for subsequent differentiation into different cell types and image-based multiparametric screening. Furthermore, we provide automated protocols for neuronal differentiation of NPC in 2D culture and 3D midbrain organoids for HTS. The flexibility of this multitask platform makes it an ideal solution for translational research settings involving experiments on different patient-derived cellular models for precision medicine.

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Rodrigues, Paulla Vieira, João Vitor Pereira de Godoy, Beatriz Pelegrini Bosque, Dionísio Pedro Amorim Neto, Katiane Tostes, Soledad Palameta, Sheila Garcia-Rosa, Celisa Caldana Costa Tonoli, Hernandes Faustino de Carvalho, and Matheus de Castro Fonseca. "Transcellular propagation of fibrillar α-synuclein from enteroendocrine to neuronal cells requires cell-to-cell contact and is Rab35-dependent." Scientific Reports 12, no. 1 (March 9, 2022). http://dx.doi.org/10.1038/s41598-022-08076-5.

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AbstractParkinson’s disease (PD) is a neurodegenerative condition featured by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (αSyn) aggregates. Growing evidence suggests that PD diagnosis happens late in the disease progression and that the pathology may originate much earlier in the enteric nervous system (ENS) before advancing to the brain, via autonomic fibers. It was recently described that a specific cell type from the gut epithelium named enteroendocrine cells (EECs) possess many neuron-like properties including αSyn expression. By facing the gut lumen and being directly connected with αSyn-containing enteric neurons in a synaptic manner, EECs form a neural circuit between the gastrointestinal tract and the ENS, thereby being a possible key player in the outcome of PD in the gut. We have characterized the progression and the cellular mechanisms involved in αSyn pre-formed fibrils (PFFs) transfer from EECs to neuronal cells. We show that brain organoids efficiently internalize αSyn PFF seeds which triggers the formation of larger intracellular inclusions. In addition, in the enteroendocrine cell line STC-1 and in the neuronal cell line SH-SY5Y, αSyn PFFs induced intracellular calcium (Ca2+) oscillations on an extracellular Ca2+ source-dependent manner and triggered αSyn fibrils internalization by endocytosis. We characterized the spread of αSyn PFFs from enteroendocrine to neuronal cells and showed that this process is dependent on physical cell-to-cell contact and on Rab35 GTPase. Lastly, inhibition of Rab35 increases the clearance of αSyn fibrils by redirecting them to the lysosomal compartment. Therefore, our results reveal mechanisms that contribute to the understanding of how seeded αSyn fibrils promote the progression of αSyn pathology from EECs to neuronal cells shifting the focus of PD etiology to the ENS.

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