Abstract
Human embryonic stem cells (hESCs) are being used extensively in array of studies to understand different mechanisms such as early human embryogenesis, drug toxicity testing, disease modeling, and cell replacement therapy. The protocols for the directed differentiation of hESCs towards specific cell types often require long-term cell cultures. To avoid bacterial contamination, these protocols include addition of antibiotics such as pen-strep and gentamicin. Although aminoglycosides, streptomycin, and gentamicin have been shown to cause cytotoxicity in various animal models, the effect of these antibiotics on hESCs is not clear. In this study, we found that antibiotics, pen-strep, and gentamicin did not affect hESC cell viability or expression of pluripotency markers. However, during directed differentiation towards neural and hepatic fate, significant cell death was noted through the activation of caspase cascade. Also, the expression of neural progenitor markers Pax6, Emx2, Otx2, and Pou3f2 was significantly reduced suggesting that gentamicin may adversely affect early embryonic neurogenesis whereas no effect was seen on the expression of endoderm or hepatic markers during differentiation. Our results suggest that the use of antibiotics in cell culture media for the maintenance and differentiation of hESCs needs thorough investigation before use to avoid erroneous results.
Highlights
Antibiotics are routinely used in long-term stem cell cultures in the laboratories to avoid general bacterial contamination
In order to understand the effect of the antibiotics, gentamicin and pen-strep on the growth and viability of Human embryonic stem cells (hESCs), the most widely used hESC line, H9 cells were grown in feeder-free conditions on mTeSR1 medium and treated with different concentrations of gentamicin ranging from 0, 10, 25, 50, and 200 μg/ml combined without or with pen-strep (100 U/ml-100 μg/ml) for 2 and 5 days to test the effect on cell viability
No difference in cell viability was observed with the treatment of different concentrations of gentamicin alone or in combination with pen-strep including those treated with a high concentration of gentamicin, that is, 200 μg/ml alone or combined with pen-strep (Figure 1(a))
Summary
Antibiotics are routinely used in long-term stem cell cultures in the laboratories to avoid general bacterial contamination. Penicillin-streptomycin (pen-strep) is one of the most commonly used antibiotics in the cell culture media to control bacterial contamination. Many strains of bacteria are found to be resistant to pen-strep. In these situations, other broad spectrum antibiotics such as normocin and gentamicin are used [1]. Cytotoxic effects of gentamicin have been reported in animal models (for a review, see [2]). In animal and human models, the use of gentamicin is reported to cause ototoxicity and nephrotoxicity [3, 4]. It is known that aminoglycosides can cross placenta, the effect of maternal use of these antibiotics on early embryonic development if any is still not well known
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