generation-of-human-brain-organoids-for-mitochondrial-disease-modeling

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

Heinrich Heine University

Max Delbrueck Center for Molecular Medicine (MDC)

The cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence. A number of studies have indeed observed a correlation between CCs gene expression, embryo quality, and final pregnancy outcome. Here, we isolated CCs from antral mouse oocytes of known developmental incompetence (NSN-CCs) or competence (SN-CCs) and compared their transcriptomes with the aim of identifying distinct marker transcripts.

Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes.

Genomic aberrations induced by somatic cell reprogramming are a major drawback for future applications of this technology in regenerative medicine. A new study by Ji et al. published in Stem Cell Reports suggests a counteracting strategy based on balancing the mitochondrial/oxidative stress pathway through antioxidant supplementation. 

A mitochondrial strategy for safeguarding the reprogrammed genome.

Alzheimer's disease (AD) is a complex, irreversible neurodegenerative disorder. At present there are neither reliable markers to diagnose AD at an early stage nor therapy. To investigate underlying disease mechanisms, induced pluripotent stem cells (iPSCs) allow the generation of patient-derived neuronal cells in a dish.

Induced pluripotent stem cell-derived neuronal cells from a sporadic Alzheimer's disease donor as a model for investigating AD-associated gene regulatory networks.

Erratum: Induced pluripotent stem cell-derived neuronal cells from a sporadic Alzheimer's disease donor as a model for investigating AD-associated gene regulatory networks.

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders.

The generation of new oligodendrocytes is essential for adult brain repair in diseases such as multiple sclerosis. We previously identified the multifunctional p57kip2 protein as a negative regulator of myelinating glial cell differentiation and as an intrinsic switch of glial fate decision in adult neural stem cells (aNSCs). In oligodendroglial precursor cells (OPCs), p57kip2 protein nuclear exclusion was recently found to be rate limiting for differentiation to proceed. Furthermore, stimulation with mesenchymal stem cell (MSC)-derived factors enhanced oligodendrogenesis by yet unknown mechanisms. To elucidate this instructive interaction, we investigated to what degree MSC secreted factors are species dependent, whether hippocampal aNSCs respond equally well to such stimuli, whether apart from oligodendroglial differentiation also tissue integration and axonal wrapping can be promoted and whether the oligodendrogenic effect involved subcellular translocation of p57kip2. We found that CC1 positive oligodendrocytes within the hilus express nuclear p57kip2 protein and that MSC dependent stimulation of cultured hippocampal aNSCs was not accompanied by nuclear p57kip2 exclusion as observed for parenchymal OPCs after spontaneous differentiation. Stimulation with human MSC factors was observed to equally promote rat stem cell oligodendrogenesis, axonal wrapping and tissue integration. As forced nuclear shuttling of p57kip2 led to decreased CNPase- but elevated GFAP expression levels, this indicates heterogenic oligodendroglial mechanisms occurring between OPCs and aNSCs. We also show for the first time that dominant pro-oligodendroglial factors derived from human fetal MSCs can instruct human induced pluripotent stem cell-derived NSCs to differentiate into O4 positive oligodendrocytes.

Human mesenchymal factors induce rat hippocampal- and human neural stem cell dependent oligodendrogenesis.

Spinocerebellar ataxia type-1 (SCA1) is caused by an abnormally expanded polyglutamine (polyQ) tract in ataxin-1. These expansions are responsible for protein misfolding and self-assembly into intranuclear inclusion bodies (IIBs) that are somehow linked to neuronal death. However, owing to lack of a suitable cellular model, the downstream consequences of IIB formation are yet to be resolved. Here, we describe a nuclear protein aggregation model of pathogenic human ataxin-1 and characterize IIB effects. Using an inducible Sleeping Beauty transposon system, we overexpressed the ATXN1(Q82) gene in human mesenchymal stem cells that are resistant to the early cytotoxic effects caused by the expression of the mutant protein. We characterized the structure and the protein composition of insoluble polyQ IIBs which gradually occupy the nuclei and are responsible for the generation of reactive oxygen species. In response to their formation, our transcriptome analysis reveals a cerebellum-specific perturbed protein interaction network, primarily affecting protein synthesis. We propose that insoluble polyQ IIBs cause oxidative and nucleolar stress and affect the assembly of the ribosome by capturing or down-regulating essential components. The inducible cell system can be utilized to decipher the cellular consequences of polyQ protein aggregation. Our strategy provides a broadly applicable methodology for studying polyQ diseases.

Nuclear inclusions of pathogenic ataxin-1 induce oxidative stress and perturb the protein synthesis machinery.

Mitochondria exhibit high degree of heterogeneity within various tissues, including differences in terms of morphology, quantity, or function. Mitochondria can even vary among distinct sub-compartments of the same cell. Emerging evidence suggest that the molecular diversity of mitochondria can influence the identity and functionality of a given cell type. Human pathologies affecting mitochondria typically cause tissue and cell-type-specific impairment. Mitochondrial diversity could thus play a contributing role not only in physiological cell fate specification but also during pathological disease development. In this review, we discuss the role of mitochondrial diversity in brain function during health and disease. Recent advances in induced pluripotent stem cells (iPSCs) research and the derivation of cerebral organoids could provide novel opportunities to unveil the role of mitochondrial heterogeneity for the function of the human brain. Mitochondrial diversity might be at the bases of the cell-type-specific vulnerability of mitochondrial disorders and may represent an underappreciated target of disease intervention.

Tackling mitochondrial diversity in brain function: from animal models to human brain organoids.

The establishment of protective humoral immunity is dependent on the ability of mature B cells to undergo antibody gene diversification while adjusting to the physiological stressors induced by activation with the antigen. Mature B cells diversify their antibody genes by class switch recombination (CSR) and somatic hypermutation (SHM), which are both dependent on efficient induction of activation-induced cytidine deaminase (AID). Here, we identified PDGFA-associated protein 1 (Pdap1) as an essential regulator of cellular homeostasis in mature B cells. Pdap1 deficiency leads to sustained expression of the integrated stress response (ISR) effector activating transcription factor 4 (Atf4) and induction of the ISR transcriptional program, increased cell death, and defective AID expression. As a consequence, loss of Pdap1 reduces germinal center B cell formation and impairs CSR and SHM. Thus, Pdap1 protects mature B cells against chronic ISR activation and ensures efficient antibody diversification by promoting their survival and optimal function.

PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification.

Biocompatible materials-topography could be used for the construction of scaffolds allowing the three-dimensional (3D) organization of human stem cells into functional tissue-like structures with a defined architecture.

Biocompatibility of α-AlO Ceramic Substrates with Human Neural Precursor Cells.

Primary mitochondrial diseases (PMD) refer to a group of severe, often inherited genetic conditions due to mutations in the mitochondrial genome or in the nuclear genes encoding for proteins involved in oxidative phosphorylation (OXPHOS). The mutations hamper the last step of aerobic metabolism, affecting the primary source of cellular ATP synthesis. Mitochondrial diseases are characterized by extremely heterogeneous symptoms, ranging from organ-specific to multisystemic dysfunction with different clinical courses. The limited information of the natural history, the limitations of currently available preclinical models, coupled with the large variability of phenotypical presentations of PMD patients, have strongly penalized the development of effective therapies. However, new therapeutic strategies have been emerging, often with promising preclinical and clinical results. Here we review the state of the art on experimental treatments for mitochondrial diseases, presenting "one-size-fits-all" approaches and precision medicine strategies. Finally, we propose novel perspective therapeutic plans, either based on preclinical studies or currently used for other genetic or metabolic diseases that could be transferred to PMD.

Therapeutic Approaches to Treat Mitochondrial Diseases: "One-Size-Fits-All" and "Precision Medicine" Strategies.

Excessive ethanol exposure can cause mitochondrial and cellular toxicity. In order to discover potential counteracting interventions, it is essential to develop assays capable of capturing the consequences of ethanol exposure in human neurons, and particularly dopaminergic neurons that are crucial for the development of alcohol use disorders (AUD). Here, we developed a novel high-throughput (HT) assay to quantify mitochondrial and neuronal toxicity in human dopaminergic neuron-containing cultures (DNs) from induced pluripotent stem cells (iPSCs). The assay, dubbed mitochondrial neuronal health (MNH) assay, combines live-cell measurement of mitochondrial membrane potential (MMP) with quantification of neuronal branching complexity post-fixation. Using the MNH assay, we demonstrated that chronic ethanol exposure in human iPSC-derived DNs decreases MMP and neuronal outgrowth in a dose-dependent manner. The toxic effect of ethanol on DNs was already detectable after 1 h of exposure, and occurred similarly in DNs derived from healthy individuals and from patients with AUD. We next used the MNH assay to carry out a proof-of-concept compound screening using FDA-approved drugs. We identified potential candidate compounds modulating acute ethanol toxicity in human DNs. We found that disulfiram and baclofen, which are used for AUD treatment, and lithium caused neurotoxicity also in the absence of ethanol, while the spasmolytic drug flavoxate positively influenced MNH. Altogether, we developed an HT assay to probe human MNH and used it to assess ethanol neurotoxicity and to identify modulating agents. The MNH assay represents an effective new tool for discovering modulators of MNH and toxicity in live human neurons.

Assessment of Ethanol-Induced Toxicity on iPSC-Derived Human Neurons Using a Novel High-Throughput Mitochondrial Neuronal Health (MNH) Assay.

Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.

Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome.

The high attrition rate in drug development processes calls for additional human-based model systems. However, in the context of brain disorders, sampling live neuronal cells for compound testing is not applicable. The use of human induced pluripotent stem cells (iPSCs) has revolutionized the field of neuronal disease modeling and drug discovery. Thanks to the development of iPSC-based neuronal differentiation protocols, including tridimensional cerebral organoids, it is now possible to molecularly dissect human neuronal development and human brain disease pathogenesis in a dish. These approaches may allow dissecting patient-specific treatment efficacy in a disease-relevant cellular context. For drug discovery approaches, however, a highly reproducible and cost-effective cell model is desirable. Here, we describe a step-by-step process for generating robust and expandable neural progenitor cells (NPCs) from human iPSCs. NPCs generated with this protocol are homogeneous and highly proliferative. These features make NPCs suitable for the development of high-throughput compound screenings for drug discovery. Human iPSC-derived NPCs show a metabolism dependent on mitochondrial activity and can therefore be used also to investigate neurological disorders in which mitochondrial function is affected. The protocol covers all steps necessary for the preparation, culture, and characterization of human iPSC-derived NPCs. .

Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders.

Cellular metabolism contributes to cell fate decisions. Bioenergetic profiling can therefore provide considerable insights into cellular identity and specification. Given the current importance of human pluripotent stem cells (hPSCs) for biomedical applications, assessing the bioenergetic properties of hPSCs and derivatives can unveil relevant mechanisms in the context of development biology and molecular disease modeling. Here, we describe a method to facilitate bioenergetic profiling of hPSCs in a reproducible and scalable manner. After simultaneous assessment of mitochondrial respiration and glycolytic capacity using Seahorse XFe96 Analyzer, we measure lactate concentration in the cellular media. Finally, we normalize the values based on DNA amount. We describe the procedures with specific requirements related to hPSCs . However, the same protocol can be easily adapted to other cell types, including differentiated progenies from hPSCs .

Bioenergetic Profiling of Human Pluripotent Stem Cells.

Mitochondria are organelles with recognized key roles in cellular homeostasis, including bioenergetics, redox, calcium signaling, and cell death. Mitochondria are essential for neuronal function, given the high energy demands of the human brain. Consequently, mitochondrial diseases affecting oxidative phosphorylation (OXPHOS) commonly exhibit neurological impairment. Emerging evidence suggests that mitochondria are important not only for mature postmitotic neurons but also for the regulation of neural progenitor cells (NPCs) during the process of neurogenesis. These recent findings put mitochondria as central regulator of cell fate decisions during brain development. OXPHOS mutations may disrupt the function of NPCs and thereby impair the metabolic programming required for neural fate commitment. Promoting the mitochondrial function of NPCs could therefore represent a novel interventional approach against incurable mitochondrial diseases.

Article	Year
Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes. BMC genomics\| PubMed ID: 23758669	2013
A mitochondrial strategy for safeguarding the reprogrammed genome. Cell regeneration (London, England)\| PubMed ID: 25408884	2014
Induced pluripotent stem cell-derived neuronal cells from a sporadic Alzheimer's disease donor as a model for investigating AD-associated gene regulatory networks. BMC genomics\| PubMed ID: 25765079	2015
Erratum: Induced pluripotent stem cell-derived neuronal cells from a sporadic Alzheimer's disease donor as a model for investigating AD-associated gene regulatory networks. BMC genomics\| PubMed ID: 26048372	2015
Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders. Cell stem cell\| PubMed ID: 28132834	2017
Human mesenchymal factors induce rat hippocampal- and human neural stem cell dependent oligodendrogenesis. Glia\| PubMed ID: 28940767	2018
Nuclear inclusions of pathogenic ataxin-1 induce oxidative stress and perturb the protein synthesis machinery. Redox biology\| PubMed ID: 32145456	2020
Tackling mitochondrial diversity in brain function: from animal models to human brain organoids. The international journal of biochemistry & cell biology\| PubMed ID: 32339638	2020
PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification. The Journal of experimental medicine\| PubMed ID: 32609329	2020
Biocompatibility of α-AlO Ceramic Substrates with Human Neural Precursor Cells. Journal of functional biomaterials\| PubMed ID: 32947990	2020
Therapeutic Approaches to Treat Mitochondrial Diseases: "One-Size-Fits-All" and "Precision Medicine" Strategies. Pharmaceutics\| PubMed ID: 33187380	2020
Assessment of Ethanol-Induced Toxicity on iPSC-Derived Human Neurons Using a Novel High-Throughput Mitochondrial Neuronal Health (MNH) Assay. Frontiers in cell and developmental biology\| PubMed ID: 33224955	2020
Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome. Nature communications\| PubMed ID: 33771987	2021
Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders. Bio-protocol\| PubMed ID: 33796613	2021
Bioenergetic Profiling of Human Pluripotent Stem Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34080164	2021
Mitochondria in neurogenesis: Implications for mitochondrial diseases. Stem cells (Dayton, Ohio)\| PubMed ID: 34089537	2021

Alessandro Prigione

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