Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders

Sauter, Evelyn J.; Kutsche, Lisa K.; Klapper, Simon D.; Busskamp, Volker

doi:10.1007/978-1-4939-9080-1_9

Cited by 9 publications

(6 citation statements)

References 19 publications

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“…Previous studies on hiPSC-derived networks were either limited to short-term evaluation of less than a month ( Ronchi et al, 2021 ; Akarca et al, 2022 ) or probing the activity with conventional MEAs with limited number of electrodes ( Odawara et al, 2014 , 2016 ; Hyvärinen et al, 2019 ; Lu et al, 2019 ; Schmieder et al, 2022 ). Here we exploited the advantages of HD-MEAs and our established long-term culturing protocol for hiPSC-derived networks ( Klapper et al, 2017 ; Sauter et al, 2019 ; Schmieder et al, 2022 ) to extract functional and morphological data of developing hiPSC-derived networks for 3 months. We constantly applied astrocyte-conditioned medium to the neuronal cultures to improve their functional maturation ( Klapper et al, 2017 ; Taga et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

Long-term morphological and functional dynamics of human stem cell-derived neuronal networks on high-density micro-electrode arrays

et al. 2022

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Comprehensive electrophysiological characterizations of human induced pluripotent stem cell (hiPSC)-derived neuronal networks are essential to determine to what extent these in vitro models recapitulate the functional features of in vivo neuronal circuits. High-density micro-electrode arrays (HD-MEAs) offer non-invasive recording with the best spatial and temporal resolution possible to date. For 3 months, we tracked the morphology and activity features of developing networks derived from a transgenic hiPSC line in which neurogenesis is inducible by neurogenic transcription factor overexpression. Our morphological data revealed large-scale structural changes from homogeneously distributed neurons in the first month to the formation of neuronal clusters over time. This led to a constant shift in position of neuronal cells and clusters on HD-MEAs and corresponding changes in spatial distribution of the network activity maps. Network activity appeared as scarce action potentials (APs), evolved as local bursts with longer duration and changed to network-wide synchronized bursts with higher frequencies but shorter duration over time, resembling the emerging burst features found in the developing human brain. Instantaneous firing rate data indicated that the fraction of fast spiking neurons (150–600 Hz) increases sharply after 63 days post induction (dpi). Inhibition of glutamatergic synapses erased burst features from network activity profiles and confirmed the presence of mature excitatory neurotransmission. The application of GABAergic receptor antagonists profoundly changed the bursting profile of the network at 120 dpi. This indicated a GABAergic switch from excitatory to inhibitory neurotransmission during circuit development and maturation. Our results suggested that an emerging GABAergic system at older culture ages is involved in regulating spontaneous network bursts. In conclusion, our data showed that long-term and continuous microscopy and electrophysiology readouts are crucial for a meaningful characterization of morphological and functional maturation in stem cell-derived human networks. Most importantly, assessing the level and duration of functional maturation is key to subject these human neuronal circuits on HD-MEAs for basic and biomedical applications.

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Section: Discussionmentioning

confidence: 99%

Long-term morphological and functional dynamics of human stem cell-derived neuronal networks on high-density micro-electrode arrays

et al. 2022

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“…Human embryonic stem cells (hESCs) ( Ilic & Ogilvie, 2017 ) and human-induced pluripotent stem cells (hiPSCs) ( Hockemeyer & Jaenisch, 2016 ; Shi et al, 2017 ) offer an almost unlimited cell source for neuronal cell and circuit engineering. Many protocols to drive stem cells into neurons require multiple steps and long time periods using soluble factors and specific culturing techniques ( Sauter et al, 2019 ). There exist also single-step differentiation protocols by inducible neurogenic transcription factor expression that result in rapid neurogenesis ( Pang et al, 2011 ; Zhang et al, 2013 ; Busskamp et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

Tracking connectivity maps in human stem cell–derived neuronal networks by holographic optogenetics

et al. 2022

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Neuronal networks derived from human induced pluripotent stem cells have been exploited widely for modeling neuronal circuits, neurological diseases, and drug screening. As these networks require extended culturing periods to functionally mature in vitro, most studies are based on immature networks. To obtain insights on long-term functional features, we improved a glia–neuron co-culture protocol within multi-electrode arrays, facilitating continuous assessment of electrical features in weekly intervals. By full-field optogenetic stimulation, we detected an earlier onset of neuronal firing and burst activity compared with spontaneous activity. Full-field stimulation enhanced the number of active neurons and their firing rates. Compared with full-field stimulation, which evoked synchronized activity across all neurons, holographic stimulation of individual neurons resulted in local activity. Single-cell holographic stimulation facilitated to trace propagating evoked activities of 400 individually stimulated neurons per multi-electrode array. Thereby, we revealed precise functional neuronal connectivity motifs. Holographic stimulation data over time showed increasing connection numbers and strength with culture age. This holographic stimulation setup has the potential to establish a profound functional testbed for in-depth analysis of human-induced pluripotent stem cell-derived neuronal networks.

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“…[75][76][77][78] Disease-causing mutations may also be introduced in iPSCs from healthy individuals by genomic modifications using clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems, for instance, to derive isogenic backgrounds. [79,80] The identification of genetic variation that predisposes to disease is of tremendous importance when attempting to identify the molecular and cellular underpinnings of a pathological process; [73] in particular when studying genetic variants that only mildly increase disease risk. Hereby, correcting a disease mutation in a patient-derived cell line to the wildtype genotype may be more beneficial for disease modeling since the genetic background is already permissive to disease progression.…”

Section: Modeling Pch2a With Patient-derived Cellsmentioning

confidence: 99%

What connects splicing of transfer RNA precursor molecules with pontocerebellar hypoplasia?

Sekulovski

Trowitzsch

2022

BioEssays

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Transfer RNAs (tRNAs) represent the most abundant class of RNA molecules in the cell and are key players during protein synthesis and cellular homeostasis. Aberrations in the extensive tRNA biogenesis pathways lead to severe neurological disorders in humans. Mutations in the tRNA splicing endonuclease (TSEN) and its associated RNA kinase cleavage factor polyribonucleotide kinase subunit 1 (CLP1) cause pontocerebellar hypoplasia (PCH), a heterogeneous group of neurodegenerative disorders, that manifest as underdevelopment of specific brain regions typically accompanied by microcephaly, profound motor impairments, and child mortality. Recently, we demonstrated that mutations leading to specific PCH subtypes destabilize TSEN in vitro and cause imbalances of immature to mature tRNA ratios in patient-derived cells. However, how tRNA processing defects translate to disease on a systems level has not been understood. Recent findings suggested that other cellular processes may be affected by mutations in TSEN/CLP1 and obscure the molecular mechanisms of PCH emergence. Here, we review PCH disease models linked to the TSEN/CLP1 machinery and discuss future directions to study neuropathogenesis.

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Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders

Cited by 9 publications

References 19 publications

Long-term morphological and functional dynamics of human stem cell-derived neuronal networks on high-density micro-electrode arrays

Long-term morphological and functional dynamics of human stem cell-derived neuronal networks on high-density micro-electrode arrays

Tracking connectivity maps in human stem cell–derived neuronal networks by holographic optogenetics

What connects splicing of transfer RNA precursor molecules with pontocerebellar hypoplasia?

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