In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells

Wind, Matt; Tsakiridis, Anestis

doi:10.1002/cpz1.244

Cited by 6 publications

(2 citation statements)

References 33 publications

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“…Furthermore, as the cardiomyocyte response to neuronal stimulation was observed, we have shown here that the cell types used form a functional connection. There are several protocols for differentiating specific types of neurons [ 15 , 36 , 37 ], which can be utilized in our future studies of the neuron–CM interaction and further evaluate the CM response to specific peripheral types of neurons. Moreover, we used neuronal high K + exposure and video microscopy to evaluate the functionality of the neuron–CM interactions, although previous studies [ 10 , 11 , 12 ] have used MEAs to electrically stimulate neurons and record the CM response, allowing better spatiotemporal induction of the stimulation and the measurement of the response in both neurons and CMs.…”

Section: Discussionmentioning

confidence: 99%

Human Neurons Form Axon-Mediated Functional Connections with Human Cardiomyocytes in Compartmentalized Microfluidic Chip

Häkli

Jäntti

Joki

et al. 2022

IJMS

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The cardiac autonomic nervous system (cANS) regulates cardiac function by innervating cardiac tissue with axons, and cardiomyocytes (CMs) and neurons undergo comaturation during the heart innervation in embryogenesis. As cANS is essential for cardiac function, its dysfunctions might be fatal; therefore, cardiac innervation models for studying embryogenesis, cardiac diseases, and drug screening are needed. However, previously reported neuron-cardiomyocyte (CM) coculture chips lack studies of functional neuron–CM interactions with completely human-based cell models. Here, we present a novel completely human cell-based and electrophysiologically functional cardiac innervation on a chip in which a compartmentalized microfluidic device, a 3D3C chip, was used to coculture human induced pluripotent stem cell (hiPSC)-derived neurons and CMs. The 3D3C chip enabled the coculture of both cell types with their respective culture media in their own compartments while allowing the neuronal axons to traverse between the compartments via microtunnels connecting the compartments. Furthermore, the 3D3C chip allowed the use of diverse analysis methods, including immunocytochemistry, RT-qPCR and video microscopy. This system resembled the in vivo axon-mediated neuron–CM interaction. In this study, the evaluation of the CM beating response during chemical stimulation of neurons showed that hiPSC-neurons and hiPSC-CMs formed electrophysiologically functional axon-mediated interactions.

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Section: Discussionmentioning

confidence: 99%

Human Neurons Form Axon-Mediated Functional Connections with Human Cardiomyocytes in Compartmentalized Microfluidic Chip

Häkli

Jäntti

Joki

et al. 2022

IJMS

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“…In recent years, several groups have utilized in vitro PSC‐derived organoid models of neural tube development to model environmental neurotoxicity and disease (Wu, Peng, Finnell, & Zheng, 2021; Zheng, Zhang, Xu, & Wu, 2021). A previous Current Protocols procedure documents the derivation of posterior motor neurons from neuromesodermal progenitors in a 2D monolayer format (Wind & Tsakiridis, 2021). The protocol presented here follows the framework of a previously established 3D method to derive motor neurons in vitro (Maury et al., 2015).…”

Section: Commentarymentioning

confidence: 99%

Dynamic 3D Combinatorial Generation of hPSC‐Derived Neuromesodermal Organoids With Diverse Regional and Cellular Identities

et al. 2022

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Neuromesodermal progenitors represent a unique, bipotent population of progenitors residing in the tail bud of the developing embryo, which give rise to the caudal spinal cord cell types of neuroectodermal lineage as well as the adjacent paraxial somite cell types of mesodermal origin. With the advent of stem cell technologies, including induced pluripotent stem cells (iPSCs), the modeling of rare genetic disorders can be accomplished in vitro to interrogate cell‐type specific pathological mechanisms in human patient conditions. Stem cell‐derived models of neuromesodermal progenitors have been accomplished by several developmental biology groups; however, most employ a 2D monolayer format that does not fully reflect the complexity of cellular differentiation in the developing embryo. This article presents a dynamic 3D combinatorial method to generate robust populations of human pluripotent stem cell‐derived neuromesodermal organoids with multi‐cellular fates and regional identities. By utilizing a dynamic 3D suspension format for the differentiation process, the organoids differentiated by following this protocol display a hallmark of embryonic development that involves a morphological elongation known as axial extension. Furthermore, by employing a combinatorial screening assay, we dissect essential pathways for optimally directing the patterning of pluripotent stem cells into neuromesodermal organoids. This protocol highlights the influence of timing, duration, and concentration of WNT and fibroblast growth factor (FGF) signaling pathways on enhancing early neuromesodermal identity, and later, downstream cell fate specification through combined synergies of retinoid signaling and sonic hedgehog activation. Finally, through robust inhibition of the Notch signaling pathway, this protocol accelerates the acquisition of terminal cell identities. This enhanced organoid model can serve as a powerful tool for studying normal developmental processes as well as investigating complex neurodevelopmental disorders, such as neural tube defects. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Robust generation of 3D hPSC‐derived spheroid populations in dynamic motion settings Support Protocol 1: Pluronic F‐127 reagent preparation and coating to generate low‐attachment suspension culture dishes Basic Protocol 2: Enhanced specification of hPSCs into NMP organoids Support Protocol 2: Combinatorial pathway assay for NMP organoid protocol optimization Basic Protocol 3: Differentiation of NMP organoids along diverse cellular trajectories and accelerated terminal fate specification into neurons, neural crest, and sclerotome derivatives

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Organophosphate flame retardants tris (2-butoxyethyl) phosphate (TBEP) and tris (2-chloroethyl) phosphate (TCEP) disrupt human motor neuron development by differentially affecting their survival and differentiation

Zheng,

Li,

Nie

et al. 2024

Science of The Total Environment

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In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells

Cited by 6 publications

References 33 publications

Human Neurons Form Axon-Mediated Functional Connections with Human Cardiomyocytes in Compartmentalized Microfluidic Chip

Human Neurons Form Axon-Mediated Functional Connections with Human Cardiomyocytes in Compartmentalized Microfluidic Chip

Dynamic 3D Combinatorial Generation of hPSC‐Derived Neuromesodermal Organoids With Diverse Regional and Cellular Identities

Organophosphate flame retardants tris (2-butoxyethyl) phosphate (TBEP) and tris (2-chloroethyl) phosphate (TCEP) disrupt human motor neuron development by differentially affecting their survival and differentiation

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