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The effect of rho kinase inhibition on morphological and electrophysiological maturity in iPSC-derived neurons

Abstract

Induced pluripotent stem cell (iPSC)-derived neurons permit the study of neurogenesis and neurological disease in a human setting. However, the electrophysiological properties of iPSC-derived neurons are consistent with those observed in immature cortical neurons, including a high membrane resistance depolarized resting membrane potential and immature firing properties, limiting their use in modeling neuronal activity in adult cells. Based on the proven association between inhibiting rho kinase (ROCK) and increased neurite complexity, we seek to determine if short-term ROCK inhibition during the first 1–2 weeks of differentiation would increase morphological complexity and electrophysiological maturity after several weeks of differentiation. While inhibiting ROCK resulted in increased neurite formation after 24 h, this effect did not persist at 3 and 6 weeks of age. Additionally, there was no effect of ROCK inhibition on electrophysiological properties at 2–3, 6, or 12 weeks of age, despite an increase in evoked and spontaneous firing and a more hyperpolarized resting membrane potential over time. These results indicate that while there is a clear effect of time on electrophysiological maturity, ROCK inhibition did not accelerate maturity.

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Acknowledgments

We wish to thank Kristen Brennand (Icahn School of Medicine at Mount Sinai) for providing the neurotypic iPSC line used in this study. We also thank Keena Thomas and Amy Bouton for assistance with the pMLC Western blot. Additionally, we would like to thank Peter Klein and Adam Lu for help with figure generation and statistics, Ruth Stornetta for help in the neurite tracing experiments and Neurolucida software and Stefan Bekiranov for valuable conversations on statistics.

Funding

LJH and NM received support from a neuroscience training grant (NIH/NIGM T32GM008328-24). MPB is supported by NIH Grant R01NS099586-01. MJM is supported by NIMH U01 MH106882 and the Owens Philanthropic Fund. KJL was supported by a Hartwell Post-doctoral Fellowship.

Author information

Correspondence to Mark P. Beenhakker or Michael J. McConnell.

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Harbom, L.J., Rudisill, T.L., Michel, N. et al. The effect of rho kinase inhibition on morphological and electrophysiological maturity in iPSC-derived neurons. Cell Tissue Res 375, 641–654 (2019). https://doi.org/10.1007/s00441-018-2942-7

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Keywords

  • Rho kinase
  • Induced pluripotent stem cells
  • Excitability
  • Neuronal maturation
  • Y-27632