An Autaptic Culture System for Standardized Analyses of iPSC-Derived Human Neurons

Rhee, Hong Jun; Shaib, Ali; Rehbach, Kristina; Lee, Choong-Ku; Seif, Peter; Thomas, Carolina; Gideons, Erinn S.; Guenther, Anja; Krutenko, Tamara; Hebisch, Matthias; Peitz, Michael; Brose, Nils; Brüstle, Oliver; Rhee, Jeong Seop

doi:10.1016/j.celrep.2019.04.059

Cited by 49 publications

(58 citation statements)

References 42 publications

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“…We next developed a sparse labeling approach that enables the tracing of individual cells in the dense connected neuronal cultures. This consisted of transfecting iNs with a GFP-encoding plasmid four days prior to fixation followed by staining with an axonal marker (Pan-Neu).The majority of iNs (25/26 cells) had a single axon, which is in line with previous findings [25]. In addition, it was always the longest neurite which was found to be positive for the axonal marker (n=26 cells, Supplementary Figure 4).…”

Section: Introductionsupporting

confidence: 84%

“…We removed cells with less than 500 or more than 6000 genes detected and cells in which 23 more than 5% of the transcripts accounted for mitochondrial genes were. After filtering, 25,707 cells on culturing day 5, day14, day 28 and day 35 were retained, and 1,993 genes and 7,051 UMIs were detected in each single cell on average. For human and chimpanzee iPSCs, 3,329 cells were retained after filtering, and 2,140 genes and 7,651 raw reads were detected in each single cell on average.…”

Section: Data Processingmentioning

confidence: 99%

“…The binomial test was performed using the "binom.test" function in the R package. The significantly enriched GO categories were selected based25 on the p values being less than 0.05. To visualize how DEGs (neuron-expressed genes) regulate synaptic functions at each time point, we perform GO enrichment analysis with the online SynGO annotations including 179 synaptic processes based on published, expert-curated evidence[36].…”

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confidence: 99%

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Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes

Schoernig

Fast

et al. 2020

Preprint

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We generated induced excitatory sensory neurons (iNeurons, iNs) from chimpanzee, bonobo and human stem cells by expressing the transcription factor neurogenin-2 (NGN2). Single cell-RNA sequencing showed that genes involved in dendrite and synapse development are expressed earlier during iNs maturation in the chimpanzee than the human cells. In accordance, during the first two weeks of differentiation, chimpanzee and bonobo iNs showed repetitive action potentials and more spontaneous excitatory activity than human iNs, and extended neurites of higher total length. However, the axons of human iNs were slightly longer at 5 weeks of differentiation. The timing of the establishment of neuronal polarity did not differ between the species. Chimpanzee, bonobo and human neurites eventually reached the same level of structural complexity.Thus, human iNs develop slower than chimpanzee and bonobo iNs and this difference in timing likely depends on functions downstream of NGN2. 4Here, we use a direct conversion system in which chimpanzee, bonobo and human iPSCs are converted into neurons using an NGN2 overexpression system [23,24].Unlike what has been previously reported, we obtain a neuronal cell population containing a majority of sensory neurons. We describe their temporal differentiation in terms of gene expression, electrophysiology and dendritic arborization. 5 RESULTS Maturation of human and ape induced neurons in vitroWe generated iNeurons from three chimpanzee (SandraA, JoC, ciPS01), one bonobo (BmRNA) and three human (409B2, SC102A, HmRNA) iPSC lines and from one human ESC line (H9) using forced expression of the pan-neurogenic transcription factor neurogenin 2 (NGN 2; Figure 1A) [23,24]. We followed their differentiation using molecular, electrophysiological and morphological approaches for up to 8 weeks, as indicated in the ( Figure 1A, B). Single cell RNA sequencing (scRNAseq) was performed for one chimpanzee (SandraA) and three human (409B2, SC102A1 and H9) cell lines, electrophysiology was performed using all eight cell lines and morphological analyses were done with two chimpanzee (SandraA and JoC), the bonobo (BmRNA) and three human (409B2, SC102A1 and H9) cell lines. We will refer to ape iNs for results where we combined chimpanzee and bonobo iNs for analysis.Differentiation was characterized by a downregulation of the stem cell markers NANOG, OCT4 and SOX2 and of the neural progenitor's markers ID1 and ID3 in both chimpanzee and human cells ( Supplementary Figure 1), by a change in cellular morphology and by the extension of neurites ( Figure 1C). Chimpanzee, bonobo and human iNs showed a neuron-like morphology at day 7 (d7) of differentiation and formed a dense network by d14. Neurites were positive for TUJI (beta-III-tubulin, a neuronal marker) starting from d3 of differentiation in apes and humans ( Supplementary Figure 2).By the end of the differentiation at d35, both ape and human cells formed networks that were positive for MAP2 (microtubule associated protein-2, marker for mature

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Section: Introductionsupporting

confidence: 84%

Section: Data Processingmentioning

confidence: 99%

mentioning

confidence: 99%

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Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes

Schoernig

Fast

et al. 2020

Preprint

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Section: Improvements In 2d Culture: Single Cell Models To Study Synamentioning

confidence: 99%

“…Recently, three independent research groups developed efficient protocols to improve the survival rate of human single-cell cultures and obtain human autaptic neurons (Figure 1) [67][68][69]. [68,69] or random integration/two phase protocol [67]) for generating mature human neurons from iPSCs to study the morphological and functional parameters underlying human synaptic transmission in specific neuronal subtypes.…”

Section: Improvements In 2d Culture: Single Cell Models To Study Synamentioning

confidence: 99%

Progress of Induced Pluripotent Stem Cell Technologies to Understand Genetic Epilepsy

Sterlini

Fruscione

Baldassari

et al. 2020

IJMS

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The study of the pathomechanisms by which gene mutations lead to neurological diseases has benefit from several cellular and animal models. Recently, induced Pluripotent Stem Cell (iPSC) technologies have made possible the access to human neurons to study nervous system disease-related mechanisms, and are at the forefront of the research into neurological diseases. In this review, we will focalize upon genetic epilepsy, and summarize the most recent studies in which iPSC-based technologies were used to gain insight on the molecular bases of epilepsies. Moreover, we discuss the latest advancements in epilepsy cell modeling. At the two dimensional (2D) level, single-cell models of iPSC-derived neurons lead to a mature neuronal phenotype, and now allow a reliable investigation of synaptic transmission and plasticity. In addition, functional characterization of cerebral organoids enlightens neuronal network dynamics in a three-dimensional (3D) structure. Finally, we discuss the use of iPSCs as the cutting-edge technology for cell therapy in epilepsy.

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Generation of Human iPSC‐Derived Neurons on Nanowire Arrays Featuring Varying Lengths, Pitches, and Diameters

Harberts

Siegmund

Hedrich

et al. 2022

Adv Materials Inter

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In this context, vertically aligned high aspect ratio nanostructures-so-called nanowire (NW) arrays-used as cell culture substrates play an increasingly important role in establishing novel tools for interrogating and stimulating cells on molecular and cellular levels. [4][5][6][7] In recent years, studies testing the unique capabilities of NW arrays have been conducted with a variety of cell types, for instance, basic cell lines such as GPE86, HEK293, and HeLa cells, primary rodent neurons, and (mesenchymal) stem cells (MSCs), to name a few. [8,9] The impact of such studies on medical applications such as drug screenings and neurodegenerative disease studies might, however, be significantly improved by employing more sophisticated cells, namely, cells derived from human induced pluripotent stem cells (iPSCs). [10] Human iPSC technologies have changed the way of preclinical research and application by enabling human (patientspecific) in vitro models without restrictions in cell availability. [11] Not only political and ethical controversies raised by using embryonic stem cells (ESCs) are avoided, but also all major cell types including blood-brain barrier models and brain organoids can be generated. [12,13] For studying neurodegenerative diseases such as Alzheimer's or Parkinson's, neurons derived from human iPSCs are of particular interest since adequate models are otherwise scarcely available. [14] Apart from ESCs,

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An Autaptic Culture System for Standardized Analyses of iPSC-Derived Human Neurons

Cited by 49 publications

References 42 publications

Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes

Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes

Progress of Induced Pluripotent Stem Cell Technologies to Understand Genetic Epilepsy

Generation of Human iPSC‐Derived Neurons on Nanowire Arrays Featuring Varying Lengths, Pitches, and Diameters

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