Electrophysiological- and Neuropharmacological-Based Benchmarking of Human Induced Pluripotent Stem Cell-Derived and Primary Rodent Neurons

Jezierski, Anna; Baumann, Ewa; Aylsworth, Amy; Costain, Willard J.; Corluka, Slavisa; Banderali, Umberto; Sodja, Caroline; Ribecco-Lutkiewicz, Maria; Alasmar, Salma; Martina, Marzia; Tauskela, Joseph S.

doi:10.1007/s12015-021-10263-2

Cited by 8 publications

(6 citation statements)

References 73 publications

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“…The proconvulsant, 4-aminopyridine (4-AP), robustly evoked increases in action potential frequency, single cell burst firing, and synchronized network bursting in our human EC stem cell derived neuroglial cultures, emulating epileptiform activity, and consistent with primary rodent brain slice and cultured rat cortical neurons data (e.g., Heuzeroth et al, 2019;Tukker and Westerink, 2021). In contrast, two recent studies reported that 4-AP did not induce epileptiform activity in several iPSC derived neuron lines, including iNeurons, iCell Glutaneurons or CNS.4 U neuron cultures (Tukker et al, 2020;Jezierski et al, 2022) suggesting they do not fully replicate the cellular properties of mature neurons or neural circuit complexity. Several reasons for their insensitivity to 4-AP include the maturity of these neurons, the cell composition (e.g., absence or presence of glia) of the cultures (e.g., Jezierski et al, 2022) but represent critical questions when validating a neuroglial network for in vitro disease modeling and/or drug discovery.…”

Section: Discussionsupporting

confidence: 86%

“…Human ES and iPS cell derived neurons in vitro show a similar developmental chronology but require co-culture with rodent or human astrocytes and show limited levels of bursting, low synchrony and electrical activity diminishes in longer term culture ( Odawara et al, 2016 ; Kayama et al, 2018 ; Hedegaard et al, 2020 ; Kreir et al, 2022 ). Moreover, Jezierski et al (2022) in benchmarking human amniotic fluid iPSC-derived iNeurons reported that they did not create a functional neural network or exhibit properties of fully mature neurons. In contrast, the TERA2.cl.SP12 stem cell derived neuroglia cultures described here recapitulated, over 12 months, the chronological sequence and physiological maturation of neural development observed in vivo ( Khazipov and Luhmann, 2006 ; Kohler et al, 2011 ; Moore et al, 2011 ; Nicholas et al, 2013 ) and notably did not show diminished activity, even at 1 year in vitro .…”

Section: Discussionmentioning

confidence: 99%

“…Initial evaluation of neurons from human pluripotent stem cells were often limited to protein and gene expression data, along with morphological changes in the differentiated cells ( Halliwell, 2017 ). More recent studies include electrophysiological data, which is essential in exploiting such human cells in drug discovery and safety evaluation for neuropsychiatric medicines ( Halliwell, 2017 ; Sharlow et al, 2020 ; Jezierski et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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Neuropharmacology of human TERA2.cl.SP12 stem cell-derived neurons in ultra-long-term culture for antiseizure drug discovery

et al. 2023

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Modeling the complex and prolonged development of the mammalian central nervous system in vitro remains a profound challenge. Most studies of human stem cell derived neurons are conducted over days to weeks and may or may not include glia. Here we have utilized a single human pluripotent stem cell line, TERA2.cl.SP12 to derive both neurons and glial cells and determined their differentiation and functional maturation over 1 year in culture together with their ability to display epileptiform activity in response to pro-convulsant agents and to detect antiseizure drug actions. Our experiments show that these human stem cells differentiate in vitro into mature neurons and glia cells and form inhibitory and excitatory synapses and integrated neural circuits over 6–8 months, paralleling early human neurogenesis in vivo; these neuroglia cultures display complex electrochemical signaling including high frequency trains of action potentials from single neurons, neural network bursts and highly synchronized, rhythmical firing patterns. Neural activity in our 2D neuron–glia circuits is modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and these actions were consistent in both young and highly mature neuron cultures. We also show for the first time that spontaneous and epileptiform activity is modulated by first, second and third generation antiseizure agents consistent with animal and human studies. Together, our observations strongly support the value of long-term human stem cell-derived neuroglial cultures in disease modeling and neuropsychiatric drug discovery.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Neuropharmacology of human TERA2.cl.SP12 stem cell-derived neurons in ultra-long-term culture for antiseizure drug discovery

et al. 2023

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“…Although these methods allow for some degree of control over the initiation of differentiation, there are still many outstanding questions as to how to define cellular maturity and the most salient features may depend on the experimental question. Typically, the state of maturation is determined through immunohistochemical analyses of protein expression or morphology, gene expression via RNA-sequencing ( 130 ), or activity levels via electrophysiological properties and calcium transients ( 131 ). Single-cell sequencing and electrophysiological assays have also shown that co-culturing NPCs and neurons with astrocytes can enhance and accelerate the expression of transcriptional signatures associated with neuronal maturation ( 132 ).…”

Section: Two-dimensional Culturesmentioning

confidence: 99%

Using 2D and 3D pluripotent stem cell models to study neurotropic viruses

2022

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Understanding the impact of viral pathogens on the human central nervous system (CNS) has been challenging due to the lack of viable human CNS models for controlled experiments to determine the causal factors underlying pathogenesis. Human embryonic stem cells (ESCs) and, more recently, cellular reprogramming of adult somatic cells to generate human induced pluripotent stem cells (iPSCs) provide opportunities for directed differentiation to neural cells that can be used to evaluate the impact of known and emerging viruses on neural cell types. Pluripotent stem cells (PSCs) can be induced to neural lineages in either two- (2D) or three-dimensional (3D) cultures, each bearing distinct advantages and limitations for modeling viral pathogenesis and evaluating effective therapeutics. Here we review the current state of technology in stem cell-based modeling of the CNS and how these models can be used to determine viral tropism and identify cellular phenotypes to investigate virus-host interactions and facilitate drug screening. We focus on several viruses (e.g., human immunodeficiency virus (HIV), herpes simplex virus (HSV), Zika virus (ZIKV), human cytomegalovirus (HCMV), SARS-CoV-2, West Nile virus (WNV)) to illustrate key advantages, as well as challenges, of PSC-based models. We also discuss how human PSC-based models can be used to evaluate the safety and efficacy of therapeutic drugs by generating data that are complementary to existing preclinical models. Ultimately, these efforts could facilitate the movement towards personalized medicine and provide patients and physicians with an additional source of information to consider when evaluating available treatment strategies.

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“…To date, only two studies have examined a direct effect of sFlt-1 in neurons, whereby sFlt-1 reduced NPC proliferation and increased apoptosis [ 32 , 37 ], although in the 20 years since their publication, it has not been shown whether sFlt-1 directly affects neurogenesis or neuronal morphology. Furthermore, the entire literature on VEGFA and sFlt-1 in vitro and in vivo is confined to rodent neurons, and thus it is currently unclear to what extent a potential anti-neurogenic effect of sFlt-1 might be conserved in humans, owing to species differences in gene expression profiles, electrophysiology, and developmental rates [ 40 , 41 ]. The current study sought to test the hypothesis that exogenous sFlt-1 exerts anti-neurogenic, and neurite growth inhibitory effects in developing human neurons; and, secondly, that these effects are mediated through inhibition of endogenous VEGFA signalling.…”

Section: Introductionmentioning

confidence: 99%

sFlt-1 impairs neurite growth and neuronal differentiation in SH-SY5Y cells and human neurons

Barron,

Barrett,

Tuulari

et al. 2024

Bioscience Reports

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Pre-eclampsia (PE) is a hypertensive disorder of pregnancy which is associated with increased risk of neurodevelopmental disorders in exposed offspring. The pathophysiological mechanisms mediating this relationship are currently unknown, and one potential candidate is the anti-angiogenic factor soluble Fms-like tyrosine kinase 1 (sFlt-1), which is highly elevated in PE. While sFlt-1 can impair angiogenesis via inhibition of VEGFA signalling, it is unclear whether it can directly affect neuronal development independently of its effects on the vasculature. To test this hypothesis, the current study differentiated the human neural progenitor cell (NPC) line ReNcell® VM into a mixed culture of mature neurons and glia, and exposed them to sFlt-1 during development. Outcomes measured were neurite growth, cytotoxicity, mRNA expression of nestin, MBP, GFAP, and βIII-tubulin, and neurosphere differentiation. sFlt-1 induced a significant reduction in neurite growth and this effect was timing- and dose-dependent up to 100 ng/mL, with no effect on cytotoxicity. sFlt-1 (100 ng/mL) also reduced βIII-tubulin mRNA and neuronal differentiation of neurospheres. Undifferentiated NPCs and mature neurons/glia expressed VEGFA and VEGFR-2, required for endogenous autocrine and paracrine VEGFA signalling, while sFlt-1 treatment prevented the neurogenic effects of exogenous VEGFA. Overall, these data provide the first experimental evidence for a direct effect of sFlt-1 on neurite growth and neuronal differentiation in human neurons through inhibition of VEGFA signalling, clarifying our understanding of the potential role of sFlt-1 as a mechanism by which PE can affect neuronal development.

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Electrophysiological- and Neuropharmacological-Based Benchmarking of Human Induced Pluripotent Stem Cell-Derived and Primary Rodent Neurons

Cited by 8 publications

References 73 publications

Neuropharmacology of human TERA2.cl.SP12 stem cell-derived neurons in ultra-long-term culture for antiseizure drug discovery

Neuropharmacology of human TERA2.cl.SP12 stem cell-derived neurons in ultra-long-term culture for antiseizure drug discovery

Using 2D and 3D pluripotent stem cell models to study neurotropic viruses

sFlt-1 impairs neurite growth and neuronal differentiation in SH-SY5Y cells and human neurons

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