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Long Term Gene Expression in Human Induced Pluripotent Stem Cells and Cerebral Organoids to Model a Neurodegenerative Disease.

ABSTRACT: Human brain organoids (mini-brains) consist of self-organized three-dimensional (3D) neural tissue which can be derived from reprogrammed adult cells and maintained for months in culture. These 3D structures manifest substantial potential for the modeling of neurodegenerative diseases and pave the way for personalized medicine. However, as these 3D brain models can express the whole human genetic complexity, it is critical to have access to isogenic mini-brains that only differ in specific and controlled genetic variables. Genetic engineering based on retroviral vectors is incompatible with the long-term modeling needed here and implies a risk of random integration while methods using CRISPR-Cas9 are still too complex to adapt to stem cells. We demonstrate in this study that our strategy which relies on an episomal plasmid vector derived from the Epstein-Barr virus (EBV) offers a simple and robust approach, avoiding the remaining caveats of mini-brain models. For this proof-of-concept, we used a normal tau protein with a fluorescent tag and a mutant genetic form (P301S) leading to Fronto-Temporal Dementia. Isogenic cell lines were obtained which were stable for more than 30 passages expressing either form. We show that the presence of the plasmid in the cells does not interfere with the mini-brain differentiation protocol and obtain the development of a pathologically relevant phenotype in cerebral organoids, with pathological hyperphosphorylation of the tau protein. Such a simple and versatile genetic strategy opens up the full potential of human organoids to contribute to disease modeling, personalized medicine and testing of therapeutics.

SUBMITTER: Nassor F

PROVIDER: S-EPMC7026130 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Long Term Gene Expression in Human Induced Pluripotent Stem Cells and Cerebral Organoids to Model a Neurodegenerative Disease.

Nassor Ferid F Jarray Rafika R Biard Denis S F DSF Maïza Auriane A Papy-Garcia Dulce D Pavoni Serena S Deslys Jean-Philippe JP Yates Frank F

Frontiers in cellular neuroscience 20200211

Human brain organoids (mini-brains) consist of self-organized three-dimensional (3D) neural tissue which can be derived from reprogrammed adult cells and maintained for months in culture. These 3D structures manifest substantial potential for the modeling of neurodegenerative diseases and pave the way for personalized medicine. However, as these 3D brain models can express the whole human genetic complexity, it is critical to have access to isogenic mini-brains that only differ in specific and c ...[more]

PMID: 32116560

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Project description:Maternal alcohol exposure during pregnancy can substantially impact the development of the fetus, causing a range of symptoms, known as fetal alcohol spectrum disorders (FASDs), such as cognitive dysfunction and psychiatric disorders, with the pathophysiology and mechanisms largely unknown. Recently developed human cerebral organoids from induced pluripotent stem cells are similar to fetal brains in the aspects of development and structure. These models allow more relevant in vitro systems to be developed for studying FASDs than animal models. Modeling binge drinking using human cerebral organoids, we sought to quantify the downstream toxic effects of alcohol (ethanol) on neural pathology phenotypes and signaling pathways within the organoids. The results revealed that alcohol exposure resulted in unhealthy organoids at cellular, subcellular, bioenergetic metabolism, and gene expression levels. Alcohol induced apoptosis on organoids. The apoptotic effects of alcohol on the organoids depended on the alcohol concentration and varied between cell types. Specifically, neurons were more vulnerable to alcohol-induced apoptosis than astrocytes. The alcohol-treated organoids exhibit ultrastructural changes such as disruption of mitochondria cristae, decreased intensity of mitochondrial matrix, and disorganized cytoskeleton. Alcohol exposure also resulted in mitochondrial dysfunction and metabolic stress in the organoids as evidenced by (1) decreased mitochondrial oxygen consumption rates being linked to basal respiration, ATP production, proton leak, maximal respiration and spare respiratory capacity, and (2) increase of non-mitochondrial respiration in alcohol-treated organoids compared with control groups. Furthermore, we found that alcohol treatment affected the expression of 199 genes out of 17,195 genes analyzed. Bioinformatic analyses showed the association of these dysregulated genes with 37 pathways related to clinically relevant pathologies such as psychiatric disorders, behavior, nervous system development and function, organismal injury and abnormalities, and cellular development. Notably, 187 of these genes are critically involved in neurodevelopment, and/or implicated in nervous system physiology and neurodegeneration. Furthermore, the identified genes are key regulators of multiple pathways linked in networks. This study extends for the first time animal models of binge drinking-related FASDs to a human model, allowing in-depth analyses of neurotoxicity at tissue, cellular, subcellular, metabolism, and gene levels. Hereby, we provide novel insights into alcohol-induced pathologic phenotypes, cell type-specific vulnerability, and affected signaling pathways and molecular networks, that can contribute to a better understanding of the developmental neurotoxic effects of binge drinking during pregnancy.

Project description:ImportanceThree-dimensional cerebral organoids generated from patient-derived induced pluripotent stem cells (iPSCs) may be used to interrogate cellular-molecular underpinnings of schizophrenia.ObjectiveTo determine transcriptomic profiles and functional characteristics of cerebral organoids from patients with schizophrenia using gene expression studies, complemented with investigations of mitochondrial function through measurement of real-time oxygen consumption rate, and functional studies of neuronal firing with microelectrode arrays.Design, setting, and participantsThis case-control study was conducted at Massachusetts General Hospital between 2017 and 2019. Transcriptomic profiling of iPSC-derived cerebral organoids from 8 patients with schizophrenia and 8 healthy control individuals was undertaken to identify cellular pathways that are aberrant in schizophrenia. Induced pluripotent stem cells and cerebral organoids were generated from patients who had been diagnosed as having schizophrenia and from heathy control individuals.Main outcomes and measuresTranscriptomic analysis of iPSC-derived cerebral organoids from patients with schizophrenia show differences in expression of genes involved in synaptic biology and neurodevelopment and are enriched for genes implicated in schizophrenia genome-wide association studies (GWAS).ResultsThe study included iPSC lines generated from 11 male and 5 female white participants, with a mean age of 38.8 years. RNA sequencing data from iPSC-derived cerebral organoids in schizophrenia showed differential expression of genes involved in synapses, in nervous system development, and in antigen processing. The differentially expressed genes were enriched for genes implicated in schizophrenia, with 23% of GWAS genes showing differential expression in schizophrenia and control organoids: 10 GWAS genes were upregulated in schizophrenia organoids while 15 GWAS genes were downregulated. Analysis of the gene expression profiles suggested dysregulation of genes involved in mitochondrial function and those involved in modulation of excitatory and inhibitory pathways. Studies of mitochondrial respiration showed lower basal consumption rate, adenosine triphosphate production, proton leak, and nonmitochondrial oxygen consumption in schizophrenia cerebral organoids, without any differences in the extracellular acidification rate. Microelectrode array studies of cerebral organoids showed no differences in baseline electrical activity in schizophrenia but revealed a diminished response to stimulation and depolarization.Conclusions and relevanceInvestigations of patient-derived cerebral organoids in schizophrenia revealed gene expression patterns suggesting dysregulation of a number of pathways in schizophrenia, delineated differences in mitochondrial function, and showed deficits in response to stimulation and depolarization in schizophrenia.

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Long Term Gene Expression in Human Induced Pluripotent Stem Cells and Cerebral Organoids to Model a Neurodegenerative Disease.

Publications

Long Term Gene Expression in Human Induced Pluripotent Stem Cells and Cerebral Organoids to Model a Neurodegenerative Disease.

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