Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Frega, Monica; Gestel, Sebastianus H. C. van; Linda, Katrin; Raadt, Jori van der; Keller, Jason M.; Rhijn, Jon-Ruben van; Schubert, Dirk W.; Albers, Cornelis A.; Kasri, Nael Nadif

doi:10.3791/54900-v

Cited by 12 publications

(23 citation statements)

References 12 publications

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“…We used a characterized rtTA/Ngn2 positive h-iPSC line generated from fibroblast of an healthy subject (30 year-old female) kindly provided in frozen vials by Frega et al . (Frega et al, 2017). This line was reprogrammed via episomal reprogramming (Coriell Institute for medical research, GM25256).…”

Section: Methodsmentioning

confidence: 99%

“…Neuronal differentiation and neurospheroids generation h-iPSCs were directly derived into excitatory cortical Layer 2/3 neurons by overexpressing the neuronal determinant Neurogenin 2 (Ngn2) upon doxycycline treatment as described previously (Frega et al, 2017). In particular, h-iPSCs were detached from a well after reaching confluence (6 × 10 6 cells) using ReleSR and a single-cell solution was obtained by collecting the colonies in 2 ml of "E8F+dox" medium (i.e.…”

Section: Human Induced Pluripotent Stem Cells Generation and Maintenancementioning

confidence: 99%

“…In this work, we introduce a new methodology allowing the generation of 3D neuronal networks from h-iPSCs in a relatively short time, with control over cell composition and suitable for chronical electrophysiological recordings. The proposed novel methodology is based on the combination of state-of-the art techniques to generate 3D functional neurospheroids and is based on rapid differentiation protocol for the generation of 2D population of human cortical excitatory neurons presented in Frega et al, 2017. We show the capability to grow and maintain such neurospheorids on the surface of different MEA devices commercially available for weeks. In particular we selected two planar MEAs (one based on a glass substrate providing tens of electrodes and one based on CMOS technology providing thousands of electrodes) and a 3D MEA.…”

Section: Introductionmentioning

confidence: 99%

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Human-derived cortical neurospheroids coupled to passive, high-density and 3D MEAs: a valid platform for functional tests

Muzzi

Lisa

Falappa

et al. 2023

Preprint

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With the advent of human-induced pluripotent stem cells (hiPSCs) differentiation protocols, different methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality would make them more representative of an in-vivo environment. Thus, human-derived neurospheroids and brain-organoids are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapid generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by presenting for the first-time results from the main micro-electrodes arrays (MEAs) types available on the market (passive electrode, C-MOS electrodes, 3D electrodes). Neurospheroids grown in free culture and transferred on MEA exhibited functional activity that can be chemically and electrically modulated. Our results indicates that this model holds great potential for in-depth study of signal transmission to drug screening, disease modeling and offers a reproducible and stable platform for in-vitro functional testing.

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Section: Methodsmentioning

confidence: 99%

Section: Human Induced Pluripotent Stem Cells Generation and Maintenancementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Human-derived cortical neurospheroids coupled to passive, high-density and 3D MEAs: a valid platform for functional tests

Muzzi

Lisa

Falappa

et al. 2023

Preprint

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“…We developed a protocol for the rapid generation of miniaturized and homogeneous 3D neuron/astrocyte co-cultures (Supplementary Document 1 ). Briefly, we employed a clonal hiPSC-line with doxycyclin-inducible Neurogenin 2 (Ngn2) [ 14 ], that was previously shown to facilitate the efficient generation of glutamatergic neurons from hiPSCs [ 15 , 16 ]. Ngn2-hiPSCs were pre-differentiated to neural progenitors for two days prior to 3D co-culturing with human primary astrocytes.…”

Section: Resultsmentioning

confidence: 99%

“…A previously generated hiPSC line stably expressing a doxycyclin-inducible rtTA/Ngn2 [ 14 ] was maintained feeder-free on Vitronectin (Stem Cell Technologies) coated plates in TeSR-E8 medium (Stem Cell Technologies; 1X TeSR-E8 supplement (Stem Cell Technologies), 50 U/mL Pen/strep (Gibco)) supplemented with G418 (50 µg/mL; Sigma) and puromycin (0.5 µg/mL; Sigma) at 37 °C/5% CO 2 under hypoxic conditions. Colonies were fed daily and double-volume feeding allowed for weekend-free culturing.…”

Section: Methodsmentioning

confidence: 99%

A 3D human co-culture to model neuron-astrocyte interactions in tauopathies

et al. 2023

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Background Intraneuronal tau aggregation is the major pathological hallmark of neurodegenerative tauopathies. It is now generally acknowledged that tau aggregation also affects astrocytes in a cell non-autonomous manner. However, mechanisms involved are unclear, partly because of the lack of models that reflect the situation in the human tauopathy brain. To accurately model neuron-astrocyte interaction in tauopathies, there is a need for a model that contains both human neurons and human astrocytes, intraneuronal tau pathology and mimics the three-dimensional architecture of the brain. Results Here we established a novel 100–200 µm thick 3D human neuron/astrocyte co-culture model of tau pathology, comprising homogenous populations of hiPSC-derived neurons and primary human astrocytes in microwell format. Using confocal, electron and live microscopy, we validate the procedures by showing that neurons in the 3D co-culture form pre- and postsynapses and display spontaneous calcium transients within 4 weeks. Astrocytes in the 3D co-culture display bipolar and stellate morphologies with extensive processes that ensheath neuronal somas, spatially align with axons and dendrites and can be found perisynaptically. The complex morphology of astrocytes and the interaction with neurons in the 3D co-culture mirrors that in the human brain, indicating the model’s potential to study physiological and pathological neuron-astrocyte interaction in vitro. Finally, we successfully implemented a methodology to introduce seed-independent intraneuronal tau aggregation in the 3D co-culture, enabling study of neuron-astrocyte interaction in early tau pathogenesis. Conclusions Altogether, these data provide proof-of-concept for the utility of this rapid, miniaturized, and standardized 3D model for cell type-specific manipulations, such as the intraneuronal pathology that is associated with neurodegenerative disorders.

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Changing Fate: Reprogramming Cells via Engineered Nanoscale Delivery Materials

Dehnavi¹,

Zadeh

Harvey

et al. 2022

Advanced Materials

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The incorporation of nanotechnology in regenerative medicine is at the nexus of fundamental innovations and early‐stage breakthroughs, enabling exciting biomedical advances. One of the most exciting recent developments is the use of nanoscale constructs to influence the fate of cells, which are the basic building blocks of healthy function. Appropriate cell types can be effectively manipulated by direct cell reprogramming; a robust technique to manipulate cellular function and fate, underpinning burgeoning advances in drug delivery systems, regenerative medicine, and disease remodeling. Individual transcription factors, or combinations thereof, can be introduced into cells using both viral and nonviral delivery systems. Existing approaches have inherent limitations. Viral‐based tools include issues of viral integration into the genome of the cells, the propensity for uncontrollable silencing, reduced copy potential and cell specificity, and neutralization via the immune response. Current nonviral cell reprogramming tools generally suffer from inferior expression efficiency. Nanomaterials are increasingly being explored to address these challenges and improve the efficacy of both viral and nonviral delivery because of their unique properties such as small size and high surface area. This review presents the state‐of‐the‐art research in cell reprogramming, focused on recent breakthroughs in the deployment of nanomaterials as cell reprogramming delivery tools.

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Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Cited by 12 publications

References 12 publications

Human-derived cortical neurospheroids coupled to passive, high-density and 3D MEAs: a valid platform for functional tests

Human-derived cortical neurospheroids coupled to passive, high-density and 3D MEAs: a valid platform for functional tests

A 3D human co-culture to model neuron-astrocyte interactions in tauopathies

Changing Fate: Reprogramming Cells via Engineered Nanoscale Delivery Materials

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