Mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny

Abstract

Murine embryonic stem cells (mESCs) inoculated at passage P13 with the mycoplasma species M. hominis, M. fermentans and M. orale and cultured over 20 passages showed reduced growth rate and viability (P < 0.0001) compared to control mESCs. Spectral karyotypic analysis of mycoplasma-infected mESCs showed a number of non-clonal chromosomal aberrations which increased with the duration of infection. The differentiation status of the infected mESCs was most affected at passage P13+6 where the infection was strongest and 46.3% of the mESCs expressed both POU5F1 and SSEA-1 markers whereas 84.8% of control mESCs expressed both markers. The percentage of germline chimeras from mycoplasma-infected mESCs was examined after blastocyst injection and embryo transfer to suitable recipients at different passages and, compared to the respective control group, was most affected at passage P13+5 (50% vs. 90%; P < 0.07). Further reductions were obtained at the same passage in the percentage of litters born (50% vs. 100%; P < 0.07) and in the percentage of pups born (22% vs. 45%; P < 0.001). Thirty three chimeras (39.8%) obtained from blastocyst injection with mycoplasma-infected mESCs showed reduced body weight (P < 0.0001), nasal discharge, osteoarthropathia, and cachexia. Flow cytometric analysis of plasma from chimeras produced with mycoplasma-infected mESCs revealed statistically significant differences in the proportions of T-cells and increased levels of IgG1 (P < 0.001), IgG2a (P < 0.05) and IgM (P < 0.05), anti-DNA antibodies (P < 0.05) and rheumatoid factor (P < 0.01). The present data indicate that mycoplasma contamination of mESCs affects various cell parameters, germline transmission, and postnatal development of the resulting chimeras.

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

“…In this study, the false negative results for enzymatic method were observed in only two cell lines i.e., PEER (NCBI C511) and SH-SY5Y (NCBI C611). In the other 28 cases, the mycoalert Ò results were concordant with the real-time PCR results (Mariotti et al 2008;Markoullis et al 2009;Volokhov et al 2011;Molla Kazemiha et al 2014).…”

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

“…Because of incomplete respiration pathway in mycoplasma and the absence of tricarboxylic cycle and cytochrome, the ATP production that depends on oxidative phosphorylation (except malate dehydrogenase avtivity) is stopped. Therefore, the fermentative arginine and carbohydrate pathways are considered as ATP production survival systems in mollicultes (except in ureaplasmas for which ATP production depends on urea hydrolysis) (Markoullis et al 2009). …”

“…From more than 200 mycoplasma species, 20 have been isolated from cell cultures (Uphoff and Drexler 2013). Eight species including M. arginini, M. fermentans, M. hominis, M. orale, M. hyorhinis, M. salivarium, M. pirum and A. laidlawii are responsible for more than 95 % of cell culture contaminations (Harlin and Gajewski 2008;Markoullis et al 2009;Timenetsky et al 2006). The main sources of mycoplasma contamination in cell cultures are laboratory staff, serum, culture media, materials, reagents, instruments and previously contaminated cells.…”

Germline Transmission of a Novel Rat Embryonic Stem Cell Line Derived from Transgenic Rats

“…Pathogen status of the ES cell line also affects various properties of ES cells, including their germline transmissibility. It has been demonstrated in mouse ES cells that the presence of virus, bacteria, or Mycoplasma affects germline transmissibility [24,25]. In our study, we conducted a comprehensive test for rat pathogens, such as H1 parvovirus, Kilham's rat virus, Mycoplasma spp., rat minute virus, and rat parvovirus.…”