The effect of rho kinase inhibition on morphological and electrophysiological maturity in iPSC-derived neurons
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.
KeywordsRho kinase Induced pluripotent stem cells Excitability Neuronal maturation Y-27632
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.
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.
- Belinsky GS, Rich MT, Sirois CL, Short SM, Pedrosa E, Lachman HM, Antic SD (2014) Patch-clamp recordings and calcium imaging followed by single-cell PCR reveal the developmental profile of 13 genes in iPSC-derived human neurons. Stem Cell Res 12:101–118. https://doi.org/10.1016/j.scr.2013.09.014 CrossRefPubMedGoogle Scholar
- Brennand KJ, Simone A, Jou J, Gelboin-Burkhart C, Tran N, Sangar S, Li Y, Mu Y, Chen G, Yu D, McCarthy S, Sebat J, Gage FH (2011) Modelling schizophrenia using human induced pluripotent stem cells. Nature 473(7346):221–225Google Scholar
- Chan CC, Khodarahmi K, Liu J, Sutherland D, Oschipok LW, Steeves JD, Tetzlaff W (2005) Dose-dependent beneficial and detrimental effects of ROCK inhibitor Y27632 on axonal sprouting and functional recovery after rat spinal cord injury. Exp Neurol 196:352–364. https://doi.org/10.1016/j.expneurol.2005.08.011 CrossRefPubMedGoogle Scholar
- Lingor P, Teusch N, Schwarz K, Mueller R, Mack H, Bahr M, Mueller BK (2007) Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo. J Neurochem 103:181–189. https://doi.org/10.1111/j.1471-4159.2007.04756.x PubMedGoogle Scholar
- Song M, Mohamad O, Chen D, Yu SP (2013) Coordinated development of voltage-gated Na+ and K+ currents regulates functional maturation of forebrain neurons derived from human induced pluripotent stem cells. Stem Cells Dev 22:1551–1563. https://doi.org/10.1089/scd.2012.0556 CrossRefPubMedGoogle Scholar
- Tan HB, Zhong YS, Cheng Y, Shen X (2011) Rho/ROCK pathway and neural regeneration: a potential therapeutic target for central nervous system and optic nerve damage. Int J Ophthalmol 4:652–657. https://doi.org/10.3980/j.issn.2222-3959.2011.06.16 PubMedPubMedCentralGoogle Scholar
- Tang X, Zhou L, Wagner AM, Marchetto MC, Muotri AR, Gage FH, Chen G (2013) Astroglial cells regulate the developmental timeline of human neurons differentiated from induced pluripotent stem cells. Stem Cell Res 11:743–757. https://doi.org/10.1016/j.scr.2013.05.002 CrossRefPubMedPubMedCentralGoogle Scholar