Identification of new genes associated to senescent and tumorigenic phenotypes in mesenchymal stem cells.

Although human mesenchymal stem cells (hMSCs) are a powerful tool for cell therapy, prolonged culture times result in replicative senescence or acquisition of tumorigenic features. To identify a molecular signature for senescence, we compared the transcriptome of senescent and young hMSCs with normal karyotype (hMSCs/n) and with a constitutional inversion of chromosome 3 (hMSC/inv). Senescent and young cells from both lineages showed differentially expressed genes (DEGs), with higher levels in senescent hMSCs/inv. Among the 30 DEGs in senescent hMSC/inv, 11 are new candidates for biomarkers of cellular senescence. The functional categories most represented in senescent hMSCs were related to cellular development, cell growth/proliferation, cell death, cell signaling/interaction, and cell movement. Mapping of DEGs onto biological networks revealed matrix metalloproteinase-1, thrombospondin 1, and epidermal growth factor acting as topological bottlenecks. In the comparison between senescent hMSCs/n and senescent hMSCs/inv, other functional annotations such as segregation of chromosomes, mitotic spindle formation, and mitosis and proliferation of tumor lines were most represented. We found that many genes categorized into functional annotations related to tumors in both comparisons, with relation to tumors being highest in senescent hMSCs/inv. The data presented here improves our understanding of the molecular mechanisms underlying the onset of cellular senescence as well as tumorigenesis.

Cytokinesis-block micronucleus assay adapted for analyzing genomic instability of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are multipotent cells used in cell therapy research. One of the problems involving hMSCs is the possibility of genetic instability during in vitro expansion required to obtain a suitable number of cells for clinical applications. The cytokinesis-block micronucleus (CBMN) assay measures genetic instability by analyzing the presence of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) in binucleated cells. The present study describes modifications in the CBMN assay methodology to analyze genetic instability in hMSCs isolated from the umbilical vein and in vitro expanded. The best protocol to achieve binucleated hMSCs with preserved cytoplasm was as follows: cytochalasin B concentration (4.0 µg/mL), use of hypotonic treatment (3 min), and the fixative solution (9 methanol:1 acetic acid). These adaptations were reproduced in three hMSC primary cell cultures and also in XP4PA and A549 cell lines. The frequency of hMSCs treated with mitomycin-C presenting MN was lower than that with other nuclear alterations, indicating that the hMSCs contain mechanisms to avoid a high level of chromosomal breaks. However, a high frequency of cells with NPBs was detected and spontaneous anaphase bridges under normal hMSC in vitro culture were observed. Considering that anaphase bridges are characteristic alterations in tumor cells, the CBMN assay is indicated as an important tool associated with other genetic analyses in order to ensure the safe clinical use of hMSCs in cell therapy.

Cytogenetic findings in 14 benign cartilaginous neoplasms.

Benign cartilaginous tumors represent a spectrum of neoplastic processes with variable clinical and pathologic presentations. These tumors are histologically characterized by the presence of chondrocytes surrounded by a cartilaginous matrix. Few studies describe karyotypic abnormalities in these benign lesions. We report a series of 14 chondromas from a single institution. Conventional cytogenetics was performed on short term cultures from all cases. Clonal chromosome aberrations were found in nine tumors. One soft tissue chondroma contained three clones with t(6;12)(q12;p11.2), t(3;7)(q13;p12), and der(2)t(2;18)(p11.2;q11.2). Three periosteal chondromas displayed random structural aberrations of chromosomes 2, 3, 6, 7, and 11 and loss of chromosome 13. Among the enchondromas, three tumors displayed chromosome losses, one contained a complex translocation involving chromosomes 12, 15, and 21 as well as an inv(2)(p21q31),t(12;15;21)(q13;q14;q22) and a separate enchondroma showed a translocation involving chromosomes 12 and 22. Our data suggest that considerable cytogenetic heterogeneity exists among benign chondromatous tumors.

Effect of titanium surface modified by plasma energy source on genotoxic response in vitro.

Titanium (Ti) is currently the most widely used material for the manufacture of orthopedic and dental implants. Changes in the surface of commercial pure Ti (cp Ti) can determine the functional response of cells, and is therefore a critical factor for the success of the implant. However, the genotoxicity of titanium surfaces has been poorly studied. Thus, the purpose of this study was to evaluate the genotoxic potential of a new titanium surface developed by plasma treatment using argon-ion bombardment and compare it with an untreated titanium surface. Accordingly, comet assay, analysis of chromosomal aberrations (CAs), and Cytokinesis Block Micronucleus (CBMN) assay were carried out, using CHO-K1 (Chinese hamster ovary) cells grown on both titanium surfaces. Our results show that the untreated titanium surface caused a significant increase in % tail moment, in the number of cells with CAs, tetraploidy, micronucleus frequency, and other nuclear alterations when compared with the negative control and with the plasma-treated titanium surface. This difference may be attributed to increased surface roughness and changes in titanium oxide layer thickness.

Chromosome alterations associated with positive and negative lymph node involvement in breast cancer.

Genetic heterogeneity is high in breast cancer, and hence it is difficult to link a specific chromosome alteration to a specific clinicopathologic feature. We examined clonal chromosome alterations in 45 breast carcinomas and statistically correlated the findings with clinical-histopathological parameters of the patients. The most common abnormalities were losses of chromosomes 19, 22, 21, X, and 17 and gains of chromosomes 9 and 18. A statistically significant correlation was found between clonal aberrations in chromosomes 17, 20, and 21 and positive lymph node involvement (LN+) and between clonal aberrations in chromosomes X and 6 and negative involvement (LN-). The average number of chromosome abnormalities was the same for both LN- and LN+ groups, and numerical and structural alterations were equally distributed. The mean number of chromosome aberrations did not differ significantly among tumor grades, but when aberrations were analyzed as monosomies, trisomies, and structural aberrations, a heterogeneous distribution was observed. Further cytogenetic investigation of breast tumors and their variable pathological features is undoubtedly necessary. The recognition and ultimately the molecular understanding of these abnormalities may improve breast cancer taxonomy and provide important prognostic information for both the patient and clinician.

Comprehensive cytogenetic evaluation of a mature ovarian teratoma case.

Mature ovarian teratomas are benign ovarian germ cell tumors that usually present with a normal karyotype. There are very few reports describing chromosomal abnormalities in these tumors, none of which are recurrent. In this study we report on a mature teratoma case with clonal chromosomal alterations which include monosomies of chromosomes 6, 14, 16, and 21; trisomies of chromosomes 14 and 21; and deletions of Xq, 5p, 16p, and 17p. Comparative genomic hybridization evaluation of the sample revealed a normal profile. These findings are discussed together with the cytogenetic reports on other cases of ovarian teratomas described in the literature.