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    • Multi electrode array approaches have been proposed as

    2018-10-24

    Multi-electrode array approaches have been proposed as a tool for detecting functional changes in electrically excitable cells, including neurons, exposed to drugs or toxins and allow use in high throughput studies (McConnell et al., 2012). Although there are a number of electrophysiological measures to evaluate, mean firing rate has been shown to be sensitive, robust and accurate for the identification of the effect of compounds on neural network function (McConnell et al., 2012; Novellino et al., 2011; Vassallo et al., 2016; Defranchi et al., 2011). Recent investigations with human iPSC-derived neuronal cultures appear to be useful for high throughput screening studies (Rosenkopf, 1989). In our studies, we were able to measure significant decreases in mean firing rate in iCell® Neurons indicative of neurotoxicity for paclitaxel and, as expected, increases following treatment with bicuculline, a GABA antagonist used as a positive control (McConnell et al., 2012). Mean firing rate could be another phenotype to evaluate potential neuroprotectants. There is a great need for discovery of agents to prevent peripheral neuropathy in patients at risk. One such pharmacological herbal mixture, GJG, has been shown in animal studies and small clinical studies to prevent CIPN (Schroder et al., 2013; Tawata et al., 1994). GJG alleviates paclitaxel induced hyperalgesia by preventing degeneration of the ganglion ganciclovir and suppressing TRPV4 expression (Matsumura et al., 2014), bortezomib-induced mechanical allodynia through the kappa opioid receptor (Higuchi et al., 2015) and oxaliplatin through attenuation of the generation of oxaliplatin-induced reactive oxygen species (Kono et al., 2015). Our research has demonstrated the potentiality of GJG to protect human iPSC-derived cortical neurons against paclitaxel-induced neuropathy without causing decreased sensitivity of particular cancer cells (i.e. A549, SKOV3) to paclitaxel. However, we did not observe neuroprotection of oxaliplatin with GJG consistent with lack of clinical evidence from a randomized phase III study of GJG combined with oxaliplatin (Oki et al., 2015). The following are the supplementary data related to this article.
    Funding source This work is supported, in part, by the NIH/NIGMS Pharmacogenomics of Anticancer Agents Research Grant U01 GM61393NIH/NCIRO1 CA136765 (M.E.D.), NIH/NCI RO1 CA157823 (M.E.D.), the University of Chicago Comprehensive Cancer Center (CA14599) and Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust.
    Acknowledgements
    Resource table Resource details Epidermal fibroblasts were obtained from skin biopsies of a 50years old healthy male subject (EPS1133). Reprogramming was performed on patient fibroblasts using non-integrating episomal vectors to deliver OCT4, SOX2, KLF4, L-MYC, LIN28 and shRNA to p53 (Piao et al., 2014; Wang et al., 2016). A clonal hiPSC line (CERAi001-A-6 hiPSC) was established and further characterized for pluripotency. Characterization of CERAi001-A-6 hiPSCs demonstrated that the cells were positive for the pluripotent markers TRA-1-60 and OCT4 (Fig. 1A and B, respectively). Upon embryoid body formation, CERAi001-A-6 hiPSCs differentiated into cells representative of ectoderm (NESTIN positive cells, Fig. 1C), mesoderm (SMA positive cells, Fig. 1D) and endoderm (AFP positive cells, Fig. 1E). RT-PCR analysis demonstrated gene expression of NANOG and endogenous OCT4 and SOX2, suggesting successful reprogramming of CERAi001-A-6 hiPSCs (Fig. 1F). Altogether, these results demonstrate that the CERAi001-A-6 hiPSCs retain the potential to differentiate in vitro into the three germ layers. To ensure the genetic integrity of the CERAi001-A-6 hiPSCs generated, we performed genome-wide copy number variation (CNV) profiling using Illumina HumanCore Beadchip. Our results demonstrate that the parental fibroblasts and CERAi001-A-6 hiPSCs were both male (XY) and exhibited a normal diploid chromosomal content (Fig. 1G). Furthermore, microsatellite analysis confirmed that the CERAi001-A-6 hiPSCs originated from the parental patient fibroblasts (Fig. 1H). The established CERAi001-A-6 is negative for mycoplasma testing (Fig. 1I).