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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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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DOI: https://doi.org/10.1007/s12015-021-10263-2