Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues

Limone, Francesco; Juan, Irune Guerra San; Mitchell, Jana M.; Smith, Janell L.M.; Raghunathan, Kavya; Meyer, David G.; Ghosh, Sulagna; Couto, Alexander Benavides; Klim, Joseph R.; Joseph, Brian; Gold, John; Mello, Curtis J.; Nemesh, James; Curtiss, Brittany M.; Verhage, Matthijs; McCarroll, Steven A.; Pietiläinen, Olli; Nehme, Ralda; Eggan, Kevin

doi:10.1016/j.celrep.2022.111896

Cited by 18 publications

(17 citation statements)

References 75 publications

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“…6a). To recapitulate proteostatic stress we applied a proteasome inhibitor to human Pluripotent Stem Cells (hPSC)-derived neurons 58,59 and induced nuclear loss of TDP-43 (Extended Data Fig. 6b,c).…”

Section: Resultsmentioning

confidence: 99%

“…Given the stress signature identified in neurons, we wondered whether these transcriptomic changes might be driven by neuronal distress. We differentiated microglia-like cells (iMGLs) 68 and neurons (piNs) 59 from hPSCs, triggered neuronal apoptosis and then introduced apoptotic neurons to iMGLs in vitro (Extended Data Fig. 10b-c).…”

Section: Resultsmentioning

confidence: 99%

“…For feeding assays, neurons were generated from human iPSCs using an NGN2 overexpression system as described previously 59,96,97 . Day30 hiPSC-neurons “piNs” were treated with 2 μ M H 2 O 2 for 24 hours to induce apoptosis.…”

Section: Methodsmentioning

confidence: 99%

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Single-nucleus sequencing reveals enriched expression of genetic risk factors in Extratelencephalic Neurons sensitive to degeneration in ALS

Limone

Mordes

Couto

et al. 2021

Preprint

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Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterised by a progressive loss of motor function. While it is known that the eponymous spinal sclerosis observed upon autopsy is the result of Cortico-Spinal Motor Neuron (CSMN) degeneration, it remains unclear why this neuronal subtype is selectively affected. To understand the unique molecular properties that sensitise deep-layer CSMNs to ALS, we performed RNA sequencing of 79,169 single nuclei from the frontal cortex of patients and controls. In unaffected individuals, we found that expression of ALS risk genes was most significantly enriched only in THY1+ presumptive CSMNs and not in other cortical cell types. In patients, these genetic risk factors, as well as additional genes involved in protein homeostasis and stress responses, were significantly induced in THY1+ CSMNs and a wider collection of deep layer neurons, but not in neurons with more superficial identities. Examination of oligodendroglial and microglial nuclei also revealed patient-specific gene expression changes We show microglial alterations can in part be explained by interactions with degenerating neurons. Overall, our findings suggest the selective vulnerability of CSMNs is due to a "first over the line" mechanism by which their intrinsic molecular properties sensitise them to genetic and mechanistic contributors to degeneration.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Single-nucleus sequencing reveals enriched expression of genetic risk factors in Extratelencephalic Neurons sensitive to degeneration in ALS

Limone

Mordes

Couto

et al. 2021

Preprint

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show abstract

“…In future studies, efforts could be made to accelerate both maturation (Hergenreder et al, 2022) and aging (Miller et al, 2013; Vera et al, 2016) “in the dish” and to combine neurons and glia in co-culture (we purposely showed proof-of-principle that our tractable pB system could be used for glia just as easily; Figures 1D-1E; S1A-S1B ). An increasing number of CNS cell types are being generated through transdifferentiation (Chen et al, 2021; Dräger et al, 2022; Holzer et al, 2022; Limone et al, 2022; Sonn et al, 2022). Recent studies(Guttikonda et al, 2021) show that the combination of glia in the right proportions are important for triggering appropriate neuroinflammatory responses.…”

Section: Discussionmentioning

confidence: 99%

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of α-synuclein inclusions

Lam

Ndayisaba

Lewis

et al. 2022

Preprint

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In neurodegenerative proteinopathies, intracellular inclusions are histopathologically and ultrastructurally heterogeneous but the significance of this heterogeneity is unclear. Patient- derived iPSC models, while promising for disease modeling, do not form analogous inclusions in a reasonable timeframe and suffer from limited tractability and scalability. Here, we developed an iPSC toolbox that utilizes piggyBac-based or targeted transgenes to rapidly induce CNS cells with concomitant expression of misfolding-prone proteins. The system is scalable and amenable to screening and longitudinal tracking at single-cell and single-inclusion resolution. For proof-of- principle, cortical neuron alpha-synuclein inclusionopathy models were engineered to form inclusions spontaneously or through exogenous seeding by alpha-synuclein fibrils. These models recapitulated known fibril- and lipid-rich inclusion subtypes in human brain, shedding light on their formation and consequences. Genetic-modifier and protein-interaction screens identified sequestered proteins in these inclusions, including RhoA, that were deleterious to cells when lost. This new iPSC platform should facilitate biological and drug discovery for neurodegenerative proteinopathies.

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“…In the brain, pioneering studies have adapted pluripotent stem cell (PSC)-derived 2-dimensional (2D) neuronal cultures to produce population-scale models comprising multiple donor cell lines of different genetic backgrounds (Cederquist et al, 2020; Cuomo et al, 2020; Jerber et al, 2021; Limone et al, 2023; Mitchell et al, 2020; Wells et al, 2023). However, 2D cultures typically produce a limited spectrum of cell types that does not reflect the diversity of the endogenous brain.…”

Section: Introductionmentioning

confidence: 99%

Multi-donor human cortical Chimeroids reveal individual susceptibility to neurotoxic triggers

Bolaños,

Faravelli,

Faits

et al. 2023

Preprint

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Inter-individual genetic variation affects the susceptibility to and progression of many diseases. Efforts to study the molecular mechanisms mediating the impact of human genetic variation on normal development and disease phenotypes are limited, however, by the paucity of faithful cellular human models, and the difficulty of scaling current systems to represent multiple individuals. Here, we present human brain “Chimeroids”, a highly reproducible, multi-donor human brain cortical organoid model generated by the co-development of cells from a panel of individual donors in a single organoid, while maintaining fidelity to endogenous tissue. By reaggregating cells from multiple single-donor organoids at the neural stem or committed progenitor cell stage, we generate Chimeroids in which each donor produces all cell lineages of the cerebral cortex, even when using pluripotent stem cell lines with notable growth biases. We leveraged Chimeroids to investigate inter-individual variation in susceptibility to neurotoxic stressors that exhibit high clinical phenotypic variability: ethanol and the anti-epileptic drug valproic acid. Individual donors varied in both the penetrance of the effect on target cell types, and the molecular phenotype within each affected cell type. Our results show that human genetic background may be an important mediator of neurotoxin susceptibility and introduce Chimeroids as a scalable system for high-throughput investigation of the contribution of human genetic variation to brain development and disease.

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Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues

Cited by 18 publications

References 75 publications

Single-nucleus sequencing reveals enriched expression of genetic risk factors in Extratelencephalic Neurons sensitive to degeneration in ALS

Single-nucleus sequencing reveals enriched expression of genetic risk factors in Extratelencephalic Neurons sensitive to degeneration in ALS

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of α-synuclein inclusions

Multi-donor human cortical Chimeroids reveal individual susceptibility to neurotoxic triggers

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