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Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates
Published on: May 29, 2018
Mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric
Kyriaki Markoullis1, Diana Bulian, Gabriele Hölzlwimmer
1Department of Comparative Medicine, Helmholtz Center Munich, German Research Center for Environmental Health, Ingolstädter Landstrasse 1, 85764, Neuherberg, Germany.
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.
Insights
Mycoplasma contamination in murine embryonic stem cells (mESCs) impairs cell growth, chromosomal stability, and differentiation. This contamination also negatively impacts germline transmission and the health of resulting chimeras.
Area of Science:
- Stem Cell Biology
- Microbiology
- Developmental Biology
Background:
- Murine embryonic stem cells (mESCs) are crucial for developmental studies and regenerative medicine.
- Mycoplasma contamination is a common issue in cell culture, potentially affecting experimental outcomes.
- The impact of specific Mycoplasma species on mESC behavior and subsequent chimera development requires detailed investigation.
Purpose of the Study:
- To investigate the effects of Mycoplasma contamination on mESC growth, chromosomal integrity, and differentiation potential.
- To assess the impact of contaminated mESCs on germline transmission efficiency and the health of resulting chimeras.
Main Methods:
- Inoculation of mESCs with Mycoplasma species (M. hominis, M. fermentans, M. orale).
- Monitoring of cell growth, viability, and chromosomal aberrations via spectral karyotyping.
- Assessment of differentiation markers (POU5F1, SSEA-1) and chimera formation post-blastocyst injection and embryo transfer.
- Flow cytometry analysis of plasma from chimeras.
Main Results:
- Mycoplasma-infected mESCs exhibited reduced growth rate, viability, and increased chromosomal aberrations.
- Differentiation potential was significantly impaired, with fewer cells expressing key stem cell markers.
- Germline transmission, litter size, and pup survival were reduced in chimeras derived from contaminated mESCs.
- Chimeras showed developmental issues (reduced body weight, nasal discharge, osteoarthropathia, cachexia) and altered immune profiles (T-cell proportions, elevated immunoglobulins, anti-DNA antibodies, rheumatoid factor).
Conclusions:
- Mycoplasma contamination adversely affects critical mESC parameters, including proliferation, genetic stability, and differentiation capacity.
- Contaminated mESCs lead to compromised germline transmission and significant developmental and immunological deficits in chimeras.
- These findings underscore the importance of mycoplasma-free cell cultures for reliable stem cell research and therapeutic applications.
