The percentage of CD133+ cells in human colorectal cancer cell lines is influenced by Mycoplasma hyorhinis infection

<p>Abstract</p> <p>Background</p> <p><it>Mollicutes </it>contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of <it>Mycoplasma hyorhinis </it>contamination on CD133 expression in human colon cancer cell lines.</p> <p>Methods</p> <p>MycoAlert^®and mycoplasma agar culture were used to detect mycoplasma contamination on GEO, SW480 and HT-29 cell lines. Restriction fragment length polymorphism assay was used to determine mycoplasma species. All cellular models were decontaminated by the use of a specific antibiotic panel (Enrofloxacin, Ciprofloxacin, BM Cyclin 1 and 2, Mycoplasma Removal Agent and MycoZap^®). The percentage of CD133 positive cells was analyzed by flow cytometry on GEO, SW480 and HT-29 cell lines, before and after <it>Mycoplasma hyorhinis </it>eradication.</p> <p>Results</p> <p><it>Mycoplasma hyorhinis </it>infected colon cancer cell lines showed an increased percentage of CD133+ cells as compared to the same cell lines rendered mycoplasma-free by effective exposure to antibiotic treatment. The percentage of CD133 positive cells increased again when mycoplasma negative cells were re-infected by <it>Mycoplasma hyorhinis</it>.</p> <p>Conclusions</p> <p><it>Mycoplasma hyorhinis </it>infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression. Possible explanations are (i) the direct involvement of Mycoplasma on CD133 expression or (ii) the selective pressure on a subpopulation of cells characterized by constitutive CD133 expression.</p> <p>In keeping with United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines, the present data indicate the mandatory prerequisite, for investigators involved in human colon cancer research area, of employing mycoplasma-free cell lines in order to avoid the production of non-reproducible or even false data.</p

Impairment of germline transmission after blastocyst injection with murine embryonic stem cells cultured with mouse hepatitis virus and mouse minute virus

The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 100 TCID50 MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10−5 TCID50 MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs

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

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 &lt; 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 &lt; 0.07). Further reductions were obtained at the same passage in the percentage of litters born (50% vs. 100%; P &lt; 0.07) and in the percentage of pups born (22% vs. 45%; P &lt; 0.001). Thirty three chimeras (39.8%) obtained from blastocyst injection with mycoplasma-infected mESCs showed reduced body weight (P &lt; 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 &lt; 0.001), IgG2a (P &lt; 0.05) and IgM (P &lt; 0.05), anti-DNA antibodies (P &lt; 0.05) and rheumatoid factor (P &lt; 0.01). The present data indicate that mycoplasma contamination of mESCs affects various cell parameters, germline transmission, and postnatal development of the resulting chimeras

Evaluation of polymerase chain reaction methods for detection of murine Helicobacter in nine diagnostic laboratories.

Helicobacter infections of laboratory animals may influence the results of in vivo experiments, necessitating diagnostic methods that are specific, sensitive and rapid. Polymerase chain reaction (PCR) is currently the preferred diagnostic tool for detecting Helicobacter infections in mice; however, detection ability may vary considerably among laboratories. Nine commercial and academic European labs participated in a 3-year ring study that was designed to explore this problem. The authors sought to identify which PCR methods were used for detection of murine Helicobacter spp. in fecal pellets and to compare the sensitivity, specificity and reproducibility of these methods. The study consisted of four rounds in which labs tested mouse fecal samples spiked with H. bilis, H. hepaticus or H. muridarum. The first round showed differences of up to 3 logs in detection sensitivity. Over the course of the study, sensitivity, specificity and reproducibility of PCR results in all labs improved substantially. By the study&#39;s conclusion, diagnostic ability in all labs was sufficient to reliably detect Helicobacter in naturally infected mice