Effect of Antibiotics against Mycoplasma sp. on Human Embryonic Stem Cells Undifferentiated Status, Pluripotency, Cell Viability and Growth

Abstract

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of PlasmocinTM and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days PlasmocinTM 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with PlasmocinTM 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that PlasmocinTM and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.

An evaluation of a human stem cell line to identify risk of developmental neurotoxicity with antiepileptic drugs

“…Notably, ciprofloxacin increased the proportion of cells in the S and G 2 /M phase of the cell cycle in non-differentiating and differentiating conditions but this did not impact neurogenesis. Consistent with this observation, ciprofloxacin can induce cell cycle arrest at the S and G 2 /M checkpoint in mamamlian cells (Seo et al, 2012) but it has no effect on human embryonic stem cell pluripotency (Romorini et al, 2013). Since neither ciprofloxacin nor perfluorooctanoic acid are associated with developmental neurotoxicity, these data suggest that an in vitro stem cell model of neurogenesis may be able to differentiate between non-neurotoxic and developmental neurotoxic agents.…”

Effects of ciprofloxacin-containing antimicrobial scaffolds on dental pulp stem cell viability—In vitro studies

“…30 Taken together, mammalian cells are therefore susceptible to CIP toxic effects, particularly cells with high proliferation rates, i.e. , human hematopoietic stem cells 31,32 , embryonic stem cells (hESCs) 33 , and dental-related cells. 15,16 Worth mentioning, one should note that the cytotoxic effects associated with CIP also depend on the dosage and time of exposure.…”

“…15,16 Worth mentioning, one should note that the cytotoxic effects associated with CIP also depend on the dosage and time of exposure. In a recent in vitro study, embryonic stem cells were able to regain baseline metabolic activity after CIP withdrawal 33 ; thus, decreasing the drug concentration or/and time of exposure may significantly improve stem cells viability. Based on the aforementioned, here we investigated whether synthesis, via electrospinning, of the CIP-containing scaffolds with reduced CIP concentrations would enhance dental pulp stem cell viability, while preserving antimicrobial properties.…”

Regulation of cyclin E1 expression in human pluripotent stem cells and derived neural progeny

“…All cell lines were free of Mycoplasma sp. infection, which was tested as previously described [66].…”