scholarly journals Human iPSC-derived cerebral organoids model features of Leigh Syndrome and reveal abnormal corticogenesis

2020 ◽  
Author(s):  
Alejandra I. Romero-Morales ◽  
Anuj Rastogi ◽  
Hoor Temuri ◽  
Megan L. Rasmussen ◽  
Gregory Scott McElroy ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neural Progenitor Cells ◽  
Three Dimensional ◽  
Leigh Syndrome ◽  
Mitochondrial Respiratory Chain ◽  
Model Systems ◽  
Phenotypic Characterization ◽  
Mitochondrial Morphology ◽  
Induced Pluripotent ◽  
Model Features

SummaryLeigh syndrome (LS) is a rare, inherited neuro-metabolic disorder that presents with bilateral brain lesions. This disease is caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded proteins. We generated induced pluripotent stem cells (iPSCs) from three widely available LS fibroblast lines and identified, through whole exome and mitochondrial sequencing, unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). LS derived cell lines were viable and able to differentiate into key progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. The DLD-mutant line showed decreased neural rosette (NR) formation, and there were differences in NR lumen area in all three LS lines compared to control. LS-derived cerebral organoids showed defects in neural epithelial bud generation and reduced size when grown for 100 days. Loss of cortical architecture and markers were detected at days 30 and 100. The MT-ATP6/PDH line produced organoid neural progenitor cells with an abnormal mitochondrial morphology characterized by fragmentation and disorganization, and demonstrated increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that could be used as LS model systems.

scholarly journals Neurosphere Based Differentiation of Human iPSC Improves Astrocyte Differentiation

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Shuling Zhou ◽  
Karolina Szczesna ◽  
Anna Ochalek ◽  
Julianna Kobolák ◽  
Eszter Varga ◽  
...  
Keyword(s):  
Neural Progenitor Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Neural Progenitor Cell ◽  
Neural Progenitor ◽  
Culture Methods ◽  
Differentiation Potential ◽  
Differentiated Cells ◽  
Astrocyte Differentiation ◽  
Induced Pluripotent

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers,PAX6andNESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAPandaquaporin 4(AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.

scholarly journals SURF1 mutations causative of Leigh syndrome impair human neurogenesis

2019 ◽  
Author(s):  
Gizem Inak ◽  
Agnieszka Rybak-Wolf ◽  
Pawel Lisowski ◽  
René Jüttner ◽  
Annika Zink ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Neural Progenitor Cells ◽  
Leigh Syndrome ◽  
Mitochondrial Complex ◽  
Complex Iv ◽  
Disease Mechanisms ◽  
Assembly Factor ◽  
Potential Strategy ◽  
Induced Pluripotent

AbstractMutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a rare fatal neurological disorder. SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering our understanding of the disease mechanisms. We generated induced pluripotent stem cells from LS patients carrying homozygous SURF1 mutations (SURF1 iPS) and performed biallelic correction via CRISPR/Cas9. In contrast to corrected cells, SURF1 iPS showed impaired neuronal differentiation. Aberrant bioenergetics in SURF1 iPS occurred already in neural progenitor cells (NPCs), disrupting their neurogenic potency. Cerebral organoids from SURF1 iPS were smaller and recapitulated the neurogenesis defects. Our data imply that SURF1 mutations cause a failure in the development of maturing neurons. Using NPC function as an interventional target, we identified SURF1 gene augmentation as a potential strategy for restoring neurogenesis in LS patients carrying SURF1 mutations.

scholarly journals In Vitro and In Vivo Models to Study Nephropathic Cystinosis

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 6
Author(s):  
Pang Yuk Cheung ◽  
Patrick T. Harrison ◽  
Alan J. Davidson ◽  
Jennifer A. Hollywood
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Primary Cultures ◽  
Model Systems ◽  
In Vivo Model ◽  
Nephropathic Cystinosis ◽  
In Vivo Models ◽  
Induced Pluripotent

The development over the past 50 years of a variety of cell lines and animal models has provided valuable tools to understand the pathophysiology of nephropathic cystinosis. Primary cultures from patient biopsies have been instrumental in determining the primary cause of cystine accumulation in the lysosomes. Immortalised cell lines have been established using different gene constructs and have revealed a wealth of knowledge concerning the molecular mechanisms that underlie cystinosis. More recently, the generation of induced pluripotent stem cells, kidney organoids and tubuloids have helped bridge the gap between in vitro and in vivo model systems. The development of genetically modified mice and rats have made it possible to explore the cystinotic phenotype in an in vivo setting. All of these models have helped shape our understanding of cystinosis and have led to the conclusion that cystine accumulation is not the only pathology that needs targeting in this multisystemic disease. This review provides an overview of the in vitro and in vivo models available to study cystinosis, how well they recapitulate the disease phenotype, and their limitations.

scholarly journals Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage

2018 ◽  
Vol 19 (9) ◽  
pp. 2788 ◽  
Author(s):  
Guang Yang ◽  
Hyenjong Hong ◽  
April Torres ◽  
Kristen Malloy ◽  
Gourav Choudhury ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
In Vitro Models ◽  
The Body ◽  
Model Systems ◽  
Induced Pluripotent

Humans and nonhuman primates (NHP) are similar in behavior and in physiology, specifically the structure, function, and complexity of the immune system. Thus, NHP models are desirable for pathophysiology and pharmacology/toxicology studies. Furthermore, NHP-derived induced pluripotent stem cells (iPSCs) may enable transformative developmental, translational, or evolutionary studies in a field of inquiry currently hampered by the limited availability of research specimens. NHP-iPSCs may address specific questions that can be studied back and forth between in vitro cellular assays and in vivo experimentations, an investigational process that in most cases cannot be performed on humans because of safety and ethical issues. The use of NHP model systems and cell specific in vitro models is evolving with iPSC-based three-dimensional (3D) cell culture systems and organoids, which may offer reliable in vitro models and reduce the number of animals used in experimental research. IPSCs have the potential to give rise to defined cell types of any organ of the body. However, standards for deriving defined and validated NHP iPSCs are missing. Standards for deriving high-quality iPSC cell lines promote rigorous and replicable scientific research and likewise, validated cell lines reduce variability and discrepancies in results between laboratories. We have derived and validated NHP iPSC lines by confirming their pluripotency and propensity to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) according to standards and measurable limits for a set of marker genes. The iPSC lines were characterized for their potential to generate neural stem cells and to differentiate into dopaminergic neurons. These iPSC lines are available to the scientific community. NHP-iPSCs fulfill a unique niche in comparative genomics to understand gene regulatory principles underlying emergence of human traits, in infectious disease pathogenesis, in vaccine development, and in immunological barriers in regenerative medicine.

scholarly journals Cancer Cell Lines Are Useful Model Systems for Medical Research

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1098 ◽  
Author(s):  
Mirabelli ◽  
Coppola ◽  
Salvatore
Keyword(s):  
Medical Research ◽  
Cell Lines ◽  
Cancer Cell ◽  
Three Dimensional ◽  
Cancer Cell Lines ◽  
Culture Conditions ◽  
The Cancer Genome Atlas ◽  
Model Systems

Cell lines are in vitro model systems that are widely used in different fields of medical research, especially basic cancer research and drug discovery. Their usefulness is primarily linked to their ability to provide an indefinite source of biological material for experimental purposes. Under the right conditions and with appropriate controls, authenticated cancer cell lines retain most of the genetic properties of the cancer of origin. During the last few years, comparing genomic data of most cancer cell lines has corroborated this statement and those that were observed studying the tumoral tissue equivalents included in the The Cancer Genome Atlas (TCGA) database. We are at the disposal of comprehensive open access cell line datasets describing their molecular and cellular alterations at an unprecedented level of accuracy. This aspect, in association with the possibility of setting up accurate culture conditions that mimic the in vivo microenvironment (e.g., three-dimensional (3D) coculture), has strengthened the importance of cancer cell lines for continuing to sustain medical research fields. However, it is important to consider that the appropriate use of cell lines needs to follow established guidelines for guaranteed data reproducibility and quality, and to prevent the occurrence of detrimental events (i.e., those that are linked to cross-contamination and mycoplasma contamination)

scholarly journals 2518. Development And Characterization Of Human Microglial Models To Elucidate HIV Transmission Events And Pathogenesis

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S874-S875
Author(s):  
Mohammad Ali Rai ◽  
Jason E Hammonds ◽  
Paul Spearman ◽  
Paul Spearman
Keyword(s):  
Flow Cytometry ◽  
Hiv Infection ◽  
Cell Lines ◽  
Microglial Cell ◽  
Hiv Transmission ◽  
Cell Types ◽  
Model Systems ◽  
Cellular Events ◽  
The Central Nervous System ◽  
Induced Pluripotent

Abstract Background HIV-associated neurocognitive disorders cause significant morbidity and mortality despite the advent of antiretroviral therapy. An understanding of fundamental mechanisms underlying HIV infection and transmission events in the central nervous system (CNS) is needed. Microglia are resident myeloid cells that are readily infected by HIV and may constitute a CNS reservoir. We evaluated and compared existing microglial cell lines and primary cell-derived microglia as potential model systems for studying HIV-microglia interactions. Methods We cultured two immortalized human microglial lines (HMC3, C20) and developed two primary microglial models: induced microglia (iMG) derived from primary human monocytes; and microglial-like cells (iMGL) differentiated from induced pluripotent stem cells (iPSCs). We compared these four microglial cell types to commercially available fetal microglia (PM) for a microglial comparator, and monocyte-derived macrophages as a non-microglial comparator cell. Each cell type was evaluated for the presence of typical myeloid and microglia-specific markers by flow cytometry and immunofluorescence microscopy. HIV infection was performed using macrophage-tropic HIV or VSV-G-pseudotyped HIV. Results After differentiation, the iMG and iMGL displayed characteristic microglial morphology: a spindle shape and a reduction in the central body, along with ramified cell processes. Flow cytometry revealed significant differences in surface markers among the cell types. iMG and iMGL displayed CD11b, CD45, CXCR4, CCR5 and lack of expression of CD4 and CX3CR1. In contrast, HMC3, C20, and PM were negative for CD11b, CD45, CX3CR1, CD4, CXCR4. Immunostaining showed that iMG and iMGL were positive for microglial markers TMEM119 and P2RY12. RNA Seq analysis is currently underway to determine gene expression differences between the microglial cell lines and our microglia models. In preliminary results, iMG and iMGL were both readily infected with HIV, and comparison with other lines is ongoing. Conclusion There is no standard model available for defining the molecular and cellular events involved in HIV infection of microglia. Significant differences in microgial markers and in HIV receptor and coreceptor levels were noted in this study. iMG and iMGL appear to be viable microglial models susceptible to HIV infection. Disclosures All authors: No reported disclosures.

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

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gizem Inak ◽  
Agnieszka Rybak-Wolf ◽  
Pawel Lisowski ◽  
Tancredi M. Pentimalli ◽  
René Jüttner ◽  
...  
Keyword(s):  
Mitochondrial Disease ◽  
Neural Progenitor Cells ◽  
Leigh Syndrome ◽  
Metabolic Programming ◽  
Human Model ◽  
Neuronal Morphogenesis ◽  
Severe Manifestation ◽  
Neural Cultures ◽  
Induced Pluripotent ◽  
Effective Models

AbstractLeigh 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.

scholarly journals Patterns of Herpes Simplex Virus 1 Infection in Neural Progenitor Cells

2020 ◽  
Vol 94 (16) ◽  
Author(s):  
Wenxiao Zheng ◽  
Alissa M. Klammer ◽  
Jennifer N. Naciri ◽  
Jason Yeung ◽  
Matthew Demers ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Neural Progenitor Cells ◽  
Three Dimensional ◽  
Two Dimensional ◽  
Herpes Simplex Virus 1 ◽  
3D Cultures ◽  
Potential Value ◽  
Induced Pluripotent ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) can induce damage in brain regions that include the hippocampus and associated limbic structures. These neurogenic niches are important because they are associated with memory formation and are highly enriched with neural progenitor cells (NPCs). The susceptibility and fate of HSV-1-infected NPCs are largely unexplored. We differentiated human induced pluripotent stem cells (hiPSCs) into NPCs to generate two-dimensional (2D) and three-dimensional (3D) culture models to examine the interaction of HSV-1 with NPCs. Here, we show that (i) NPCs can be efficiently infected by HSV-1, but infection does not result in cell death of most NPCs, even at high multiplicities of infection (MOIs); (ii) limited HSV-1 replication and gene expression can be detected in the infected NPCs; (iii) a viral silencing mechanism is established in NPCs exposed to the antivirals (E)-5-(2-bromovinyl)-2′-deoxyuridine (5BVdU) and alpha interferon (IFN-α) and when the antivirals are removed, spontaneous reactivation can occur at low frequency; (iv) HSV-1 impairs the ability of NPCs to migrate in a dose-dependent fashion in the presence of 5BVdU plus IFN-α; and (v) 3D cultures of NPCs are less susceptible to HSV-1 infection than 2D cultures. These results suggest that NPC pools could be sites of HSV-1 reactivation in the central nervous system (CNS). Finally, our results highlight the potential value of hiPSC-derived 3D cultures to model HSV-1–NPC interaction. IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model the interaction of HSV-1 with NPCs, which reside in the neurogenic niches of the CNS and play a fundamental role in adult neurogenesis. Herein, we provide evidence that in NPCs infected at an MOI as low as 0.001, HSV-1 can establish a latent state, suggesting that (i) a variant of classical HSV-1 latency can be established during earlier stages of neuronal differentiation and (ii) neurogenic niches in the brain may constitute additional sites of viral reactivation. Lytic HSV-1 infections impaired NPC migration, which represents a critical step in neurogenesis. A difference in susceptibility to HSV-1 infection between two-dimensional (2D) and three-dimensional (3D) NPC cultures was observed, highlighting the potential value of 3D cultures for modeling host-pathogen interactions. Together, our results are relevant in light of observations relating HSV-1 infection to postencephalitic cognitive dysfunction.

scholarly journals 16p11.2 microdeletion imparts transcriptional alterations in human iPSC-derived models of early neural development

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Julien G Roth ◽  
Kristin L Muench ◽  
Aditya Asokan ◽  
Victoria M Mallett ◽  
Hui Gai ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neural Development ◽  
Developmental Disorders ◽  
Neural Progenitor Cells ◽  
Expression Patterns ◽  
Clinical Information ◽  
Copy Number Variations ◽  
Chromosomal Locus ◽  
Transcriptional Alterations ◽  
Induced Pluripotent

Microdeletions and microduplications of the 16p11.2 chromosomal locus are associated with syndromic neurodevelopmental disorders and reciprocal physiological conditions such as macro/microcephaly and high/low body mass index. To facilitate cellular and molecular investigations into these phenotypes, 65 clones of human induced pluripotent stem cells (hiPSCs) were generated from 13 individuals with 16p11.2 copy number variations (CNVs). To ensure these cell lines were suitable for downstream mechanistic investigations, a customizable bioinformatic strategy for the detection of random integration and expression of reprogramming vectors was developed and leveraged towards identifying a subset of ‘footprint’-free hiPSC clones. Transcriptomic profiling of cortical neural progenitor cells derived from these hiPSCs identified alterations in gene expression patterns which precede morphological abnormalities reported at later neurodevelopmental stages. Interpreting clinical information—available with the cell lines by request from the Simons Foundation Autism Research Initiative—with this transcriptional data revealed disruptions in gene programs related to both nervous system function and cellular metabolism. As demonstrated by these analyses, this publicly available resource has the potential to serve as a powerful medium for probing the etiology of developmental disorders associated with 16p11.2 CNVs.

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