Furthermore, expression analysis of Sox17, Foxa2, and HNF4at protein level by Western blotting from samples at day 5 and day 9 of differentiation showed similar results as observed by qRT-PCR (Physique 5(b))
Furthermore, expression analysis of Sox17, Foxa2, and HNF4at protein level by Western blotting from samples at day 5 and day 9 of differentiation showed similar results as observed by qRT-PCR (Physique 5(b)). 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 Nonivamide effect was seen around the HOXA2 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. 1. Introduction 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. However, 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 . Cytotoxic effects of gentamicin have been reported in animal models (for a review, see ). Gentamicin is also widely used for the treatment of infections caused by gram-negative bacteria. In animal and human models, the use of gentamicin is usually reported to cause ototoxicity and nephrotoxicity [3, 4]. Animals treated with high therapeutic doses of gentamicin show extensive necrosis of proximal kidney tubular cells Nonivamide  while low doses of gentamicin induced programmed cell death through the activation of caspase cascade . In addition, therapeutic doses of gentamicin have been shown to cause hearing loss and nephrotoxicity in neonates [6, 7]. Although Nonivamide 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. Human embryonic stem cells (hESCs) are pluripotent cells which can be differentiated into all three germ layers, the ectoderm, mesoderm, and endoderm, and the protocols for the directed differentiation of hESCs towards specific cell lineages have been published [8C11]. The availability of hESC-derived cell lines had opened up the possibility to detect cytotoxicity of various drugs as well as the possibility to use them as a developmental model to understand the effect of different toxins or teratogens on early human embryogenesis which is usually otherwise possible only in animal models. Since gentamicin can cross the placenta during pregnancy, it may cause adverse effects around the developing organs of the fetus. This study was therefore designed to understand the effect of routinely used antibiotics such as pen-strep and gentamicin on hESC proliferation and their differentiation towards neural and hepatic fate keeping in mind that, this might also help to understand the side effects of these aminoglycosides in early human embryogenesis in vivo. 2. Materials and Methods 2.1. Cell Culture, Differentiation, and Antibiotic Treatment hESCs (H9, WiCell Institute) were maintained in feeder-free condition on Matrigel- (Corning, cat. number 354227) coated plates in mTeSR1 medium (Stem Cell Technologies, cat. number 05850) and were between passages 37 to 46 in all of the experiments. Neural induction protocol was replicated as published previously [11, 12]. Briefly, 50,000 cells/cm2 were plated on a 24 well plate coated with Matrigel and maintained in mTeSR1 medium until fully confluent. The medium was then replaced with neural induction medium containing KSR media (15% Knockout Serum Replacement (KO-SR Gibco, cat. number 10828028), 1% L-glutamine (100x-Gibco, cat. number 25030081), 1% MEM (Hyclone, cat. number SH40003.01), and 0.1% beta-mercaptoethanol (Gibco, cat. number 31350010) in knockout DMEM (Gibco, cat. number 10829018) supplemented with LDN193189 (Stem Cell Technologies, cat. number 72142-1?mg lot number SCO4565), inhibitor BMP type 1 receptors (100?nM) and SB431542 (Milipore, cat. number 616461-5?mg Lot number D00165595), and activin receptor inhibitor (10? 0.05, ?? 0.01, and ??? 0.001). 3. Results 3.1. Effect of Gentamicin and Penicillin-Streptomycin on hESC Proliferation In order to understand the effect of the antibiotics, gentamicin and pen-strep around the growth and viability of hESCs, the most widely used hESC line, H9 cells were produced in feeder-free conditions on mTeSR1 medium and treated with different concentrations of gentamicin ranging from 0, 10, 25, 50, and 200.