Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Calabrese, Barbara; Powers, Regina M; Slepian, Alexandria J.; Halpain, Shelley

doi:10.3791/59305

Cited by 7 publications

(18 citation statements)

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“…Replating of cultured neurons has been reported for various neuron subtypes including primary dorsal root ganglia (DRG) neurons, primary cortical neurons or stem cell (SC)-derived neurons ( Caviedes et al, 1990a , b ; Koechling et al, 2011 ; Saijilafu et al, 2013 ; Frey et al, 2015 ; Biswas and Kalil, 2018 ; Calabrese et al, 2019 ; Lee et al, 2020a ). Neuron replating has been implemented to reduce neuron complexity and cell membrane surface area, thereby improving accessibility for electrophysiological recordings, because passive membrane properties such as membrane capacitance or resistance were altered ( Caviedes et al, 1990a , b ).…”

Section: Discussionmentioning

confidence: 99%

Neuron Replating, a Powerful and Versatile Approach to Study Early Aspects of Neuron Differentiation

Schneider

Duong

Rust

2021

eNeuro

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Neuron differentiation includes formation and outgrowth of neurites that differentiate into axons or dendrites. Directed neurite outgrowth is controlled by growth cones that protrude and retract actin-rich structures to sense environmental cues. These cues Significance statementUnraveling the molecular mechanisms that control neuron differentiation requires reporter gene expression or gene inactivation. In mouse primary hippocampal neurons, a widely used cellular system to study neuron differentiation, classical transfection methods are restricted to later stages of differentiation. Instead, electroporation allows genetic manipulation prior to seeding. However, time course of reporter gene expression or gene inactivation frequently hinders a full characterization of neuron differentiation, specifically of early stages. To circumvent this limitation, we combined electroporation-based genetic manipulation prior to initial seeding with a replating step after two days in vitro, which reset neurons into an undifferentiated stage. We show that replated neurons differentiated similar to nonreplated neurons. We provide a detailed protocol that allows to comprehensively characterize the molecular mechanisms underlying neuron differentiation.

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Section: Discussionmentioning

confidence: 99%

Neuron Replating, a Powerful and Versatile Approach to Study Early Aspects of Neuron Differentiation

Schneider

Duong

Rust

2021

eNeuro

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“…Achieving monolayer cultures with hiPSCs was noticeably more difficult compared to primary cultures due to a well-recognized lengthy process of transition from neural progenitors cells (NPC) to neuronal networks requiring careful control of growth and maturation (Calabrese et al 2019). Specifically, neurons growing for a long period of time in the same dish commonly form clustered, interconnected networks producing robust patterns of network level activity (Segev et al 2001, Wagenaar et al 2006.…”

Section: Dmentioning

confidence: 99%

“…To mitigate these problems, we modified a published cell culture protocol (Calabrese et al 2019) resulting in well-adhered, relatively sparse and uniform cultures with limited cell clustering. Our protocol included the following critical steps:…”

Section: Dmentioning

confidence: 99%

“…In prior studies, the voltage sensor and OG actuator were co-expressed using viral transduction (Bando et al 2019, Hochbaum et al 2014, Kiskinis et al 2018. However, growing hiPSC-derived neuronal networks is a challenging procedure (Calabrese et al 2019). One specific problem is that introduction of large or multiple expression vectors can often lead to high rate of cell death (Detrait et al 2002, Ding & Kilpatrick 2013, Johnston et al 2020.…”

Section: Introductionmentioning

confidence: 99%

“…(iii)They contained NPCs that remained undifferentiated, continued to divide and, in many cases, dominated over differentiated neurons competing with them for the growth substrate. Under these conditions, NPCs could also deprive differentiated neurons of their nutrients.To mitigate these problems, we modified a published cell culture protocol(Calabrese et al 2019) resulting in well-adhered, relatively sparse and uniform cultures with limited cell clustering. Our protocol included the following critical steps:(i)The initial 4-5 weeks of NPC differentiation in 10-cm Petri dishes.…”

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confidence: 99%

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All-optical electrophysiology in hiPSC-derived neurons with synthetic voltage sensors

Puppo

Sadegh

Trujillo

et al. 2021

Preprint

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Voltage imaging and “all-optical electrophysiology” in human induced pluripotent stem cell (hiPSC)-derived neurons have opened unprecedented opportunities for high-throughput phenotyping of activity in neurons possessing unique genetic backgrounds of individual patients. While prior all-optical electrophysiology studies relied on genetically encoded voltage indicators, viral transduction of human neurons with large or multiple expression vectors can impact cell function and often lead to massive cell death. Here, we demonstrate an alternative protocol using a synthetic voltage sensor and genetically encoded optogenetic actuator that generate robust and reproducible results. We demonstrate the functionality of this method by measuring spontaneous and evoked activity in three independent hiPSC-derived neuronal cell lines with distinct genetic backgrounds.

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Dopaminergic induction of human dental pulp stem cells by photobiomodulation: comparison of 660nm laser light and polychromatic light in the nir

Yurtsever

Kiremitçi

Gümüşderelı́oğlu

2020

Journal of Photochemistry and Photobiology B: Biology

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Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Cited by 7 publications

References 0 publications

Neuron Replating, a Powerful and Versatile Approach to Study Early Aspects of Neuron Differentiation

Neuron Replating, a Powerful and Versatile Approach to Study Early Aspects of Neuron Differentiation

All-optical electrophysiology in hiPSC-derived neurons with synthetic voltage sensors

Dopaminergic induction of human dental pulp stem cells by photobiomodulation: comparison of 660nm laser light and polychromatic light in the nir

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