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

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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.

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Acknowledgments

This study was supported by the NGFN grant 01GS0852 to T.A. and D.H.B. We thank the animal caretakers and Ruth Schmöller for technical assistance.

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Authors and Affiliations

  1. Department of Comparative Medicine, Helmholtz Center Munich, German Research Center for Environmental Health, Ingolstädter Landstrasse 1, 85764, Neuherberg, Germany

    Kyriaki Markoullis, Diana Bulian, Jörg Schmidt & Esther Mahabir

  2. Institute of Pathology, Helmholtz Center Munich, German Research Center for Environmental Health, 85764, Neuherberg, Germany

    Gabriele Hölzlwimmer & Leticia Quintanilla-Martinez

  3. Institute of Molecular Radiobiology, Helmholtz Center Munich, German Research Center for Environmental Health, 85764, Neuherberg, Germany

    Katrin-Janine Heiliger & Horst Zitzelsberger

  4. Institute of Biomathematics and Biometry, Helmholtz Center Munich, German Research Center for Environmental Health, 85764, Neuherberg, Germany

    Hagen Scherb

  5. Institute of Molecular Immunology, Helmholtz Center Munich, German Research Center for Environmental Health, 81337, Munich, Germany

    Josef Mysliwietz

  6. Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms and Cell Cultures, 38124, Braunschweig, Germany

    Cord C. Uphoff & Hans G. Drexler

  7. Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, 81675, Munich, Germany

    Thure Adler & Dirk H. Busch

  8. Clinical Cooperation Group ‘Immune-Monitoring’, Helmholtz Center Munich, German Research Center for Environmental Health, 85764, Neuherberg, Germany

    Dirk H. Busch

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  1. Kyriaki Markoullis
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  2. Diana Bulian
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  3. Gabriele Hölzlwimmer
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  9. Cord C. Uphoff
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  14. Esther Mahabir
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Correspondence to Esther Mahabir.

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Markoullis, K., Bulian, D., Hölzlwimmer, G. et al. Mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny. Transgenic Res 18, 71–87 (2009). https://doi.org/10.1007/s11248-008-9218-z

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  • DOI: https://doi.org/10.1007/s11248-008-9218-z

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