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

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Abstract

Human induced pluripotent stem cell (iPSC)-derived neurons are of interest for studying neurological disease mechanisms, developing potential therapies and deepening our understanding of the human nervous system. However, compared to an extensive history of practice with primary rodent neuron cultures, human iPSC-neurons still require more robust characterization of expression of neuronal receptors and ion channels and functional and predictive pharmacological responses. In this study, we differentiated human amniotic fluid-derived iPSCs into a mixed population of neurons (AF-iNs). Functional assessments were performed by evaluating electrophysiological (patch-clamp) properties and the effect of a panel of neuropharmacological agents on spontaneous activity (multi-electrode arrays; MEAs). These electrophysiological data were benchmarked relative to commercially sourced human iPSC-derived neurons (CNS.4U from Ncardia), primary human neurons (ScienCell™) and primary rodent cortical/hippocampal neurons. Patch-clamp whole-cell recordings showed that mature AF-iNs generated repetitive firing of action potentials in response to depolarizations, similar to that of primary rodent cortical/hippocampal neurons, with nearly half of the neurons displaying spontaneous post-synaptic currents. Immunochemical and MEA-based analyses indicated that AF-iNs were composed of functional glutamatergic excitatory and inhibitory GABAergic neurons. Principal component analysis of MEA data indicated that human AF-iN and rat neurons exhibited distinct pharmacological and electrophysiological properties. Collectively, this study establishes a necessary prerequisite for AF-iNs as a human neuron culture model suitable for pharmacological studies.

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Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon request.

Abbreviations

ACh:

Acetylcholine

AF:

Amniotic fluid

AF-iPSC:

Amniotic fluid derived induced pluripotent stem cells

AF-iNs:

AF-iPSC derived neurons

AF-iPSC-NP:

AF-iPSC derived neural progenitors

AP:

Action potential

ATP:

Adenosine triphosphate

BDNF:

Brain-derived neurotrophic factor

ChAT:

Choline acetyltransferase

CNS:

Central nervous system

DHPG:

R,S-3,5-dihydroxyphenylglycine

dl-TBOA:

dl-threo-benzyloxyaspartic acid

DMSO:

Dimethyl sulfoxide

GABA:

Gamma-Aminobutyric acid

GAD:

Glutamic acid decarboxylase

GDNF:

Glial cell line-derived neurotrophic factor

GFAP:

Glial fibrillary acidic protein

hiN:

Induced neurons (direct reprogramming)

iPSC:

Induced pluripotent stem cells

iPSC-iN:

Induced pluripotent stem cell derived neurons

K+ :

Potassium

Na+ :

Sodium

NBQX:

2,3-Dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline

NCAM:

Neural cell adhesion molecule

NeuN:

Neuron-specific nuclear protein

NMDAR1:

N-Methyl-d-aspartate receptor 1

Map2:

Microtubule-associated protein 2

MEA:

Multi-electrode array

MFR:

Mean firing rate

MPEP:

2-Methyl-6-(phenylethynyl) pyridine

VGLUT-2:

Vesicular glutamate transporter 2

PBS:

Phosphate buffered solution

PEI:

Polyethyleneimine

PLO:

Poly-l-ornithine

PLL:

Poly-l-lysine

SDHA:

Spike duration at half amplitude

sEPSC:

Spontaneous excitatory postsynaptic currents

sIPSCs:

Spontaneous inhibitory postsynaptic currents

SMADi:

SMAD inhibitor

sPSCs:

Spontaneous post-synaptic currents

TH:

Tyrosine hydroxylase

TTX:

Tetrodotoxin

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Acknowledgements

We would like to acknowledge Ncardia SA for provision of cells and supplements, and for advice provided by Drs. G Luerman, D Hess and C Fleming. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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AJ: Conceptualization, Methodology, Supervision, Writing. EB: Methodology, Experimentation, Validation, Investigation, Visualization. SC: Investigation, Formal Analysis. AA: Investigation, Formal Analysis. UB: Investigation, Formal Analysis. CS: Investigation. MR-L: Investigation. SA: Investigation. WJC: Formal Analysis, Writing. MM: Methodology, Investigation, Formal Analysis, Writing. JST: Conceptualization, Methodology, Supervision, Writing.

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Correspondence to Anna Jezierski.

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Jezierski, A., Baumann, E., Aylsworth, A. et al. Electrophysiological- and Neuropharmacological-Based Benchmarking of Human Induced Pluripotent Stem Cell-Derived and Primary Rodent Neurons. Stem Cell Rev and Rep 18, 259–277 (2022). https://doi.org/10.1007/s12015-021-10263-2

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