Cell cycle reactivation of cochlear progenitor cells in neonatal FUCCI mice by a GSK3 small molecule inhibitor

Abstract

Due to the lack of regenerative capacity of the mammalian auditory epithelium, sensory hair cell loss results in permanent hearing deficit. Nevertheless, a population of tissue resident stem/progenitor cells has been recently described. Identification of methods to trigger their activity could lead to exploitation of their potential therapeutically. Here we validate the use of transgenic mice reporting cell cycle progression (FUCCI), and stemness (Lgr5-GFP), as a valuable tool to identify regulators of cell cycle re-entry of supporting cells within the auditory epithelium. The small molecule compound CHIR99021 was used to inhibit GSK3 activity. This led to a significant increase in the fraction of proliferating sphere-forming cells, labeled by the FUCCI markers and in the percentage of Lgr5-GFP + cells, as well as a selective increase in the fraction of S-G2-M cells in the Lgr5 + population. Using whole mount cultures of the organ of Corti we detected a statistically significant increment in the fraction of proliferating Sox2 supporting cells after CHIR99021 treatment, but only rarely appearance of novel MyoVIIa+/Edu + hair cells. In conclusion, these tools provide a robust mean to identify novel regulators of auditory organ regeneration and to clarify the contribution of stem cell activity.

A novel polysaccharide derived from algae extract induces apoptosis and cell cycle arrest in human gastric carcinoma MKN45 cells via ROS/JNK signaling pathway

“…MKN45 cells were exposed to 100 µg/ml polysaccharide for 48 h. MKN45 cells expressing two Fucci probes: cells emit red fluorescence (SCF Skp2 ) in G1/G0 phase and green fluorescence (APC Cdh1 ) in S/G2/M phases (18). As described (19), fluorescence and phase contrast images were observed using an FV10i-DOC confocal laser scanning microscope (Olympus, Tokyo, Japan) with a UPLSAPO 60x Wobjective lens.…”

Inner ear organoids: new tools to understand neurosensory cell development, degeneration and regeneration

“…Additional cell sorting experiments have isolated supporting cells based on the expression of Sox2, p27, p75 (Ngfr), Lgr5 and others (Chai et al, 2012;Roccio et al, 2015;Shi et al, 2012;Sinkkonen et al, 2011;White et al, 2006). These experiments have revealed that, although several cell types in the cochlear epithelium can respond to mitogenic stimulation, the capacity to give rise to hair cells is limited to a subset of the supporting cells.…”

“…The Lgr5positive cells show a spontaneous response to damage in the newborn cochlea, where they proliferate and give rise to hair cells (Bramhall et al, 2014). This can be further enhanced by stimulation of Wnt signaling, Sox2 manipulation or Notch manipulation (Bramhall et al, 2014;Chai et al, 2012;Roccio et al, 2015;Shi et al, 2013Shi et al, , 2012. However, these properties are not exclusive to cells that express high levels of Lgr5 in the native cochlea, and recruitment of supporting cells that do not express Lgr5 has been observed after damage.…”

“…We have demonstrated that the addition of Matrigel to cultures of cochlear sensory epitheliumderived dissociated cells gives rise to epithelial cysts, rather than spheres. In addition to growth factor stimulation (Oshima et al, 2007) and activation of Wnt signaling using CHIR99021 (Roccio et al, 2015), the modulation of chromatin remodeling by the histone deacetylase inhibitor valproic acid (Stockhausen et al, 2005) allows for robust expansion of Lgr5-positive supporting cells in these 3D cultures. Although the starting cell population contains only a small percentage of Lgr5-positive cells, exposure to these treatments upregulates Lgr5 and drives their expansion.…”

“…Moreover, responsiveness to GSI-treatment in the adult inner ear may depend on damage; expression of Notch downstream effectors increases after trauma, which causes overt hair cell loss (Batts et al, 2009;Du et al, 2018;Mizutari et al, 2013), but not after exposure to noise that induces less extensive hair cell death (Maass et al, 2015). Activation of canonical Wnt signaling though β-catenin stabilization also results in cell cycle re-entry of supporting cells (Li et al, 2015;Roccio et al, 2015;Samarajeewa et al, 2018) and prolongs the time window for GSI-induced hair cell differentiation. However, the effects decline after the first postnatal week.…”