title: Cytokinesis-block micronucleus assay adapted for analyzing genomic instability of human mesenchymal stem cells.
abstract: 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.
authors: 1 Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte , Natal, Brazil .
authors: Déborah Afonso
authors: DA
authors: Cornélio
authors: Joana Cristina Medeiros
authors: JC
authors: Tavares
authors: Thais Valéria Costa de Andrade
authors: TV
authors: Pimentel
authors: Geraldo Barroso
authors: GB
authors: Cavalcanti
authors: Silvia Regina
authors: SR
authors: Batistuzzo de Medeiros
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002467:Cell Nucleus; D048430:Cell Shape; D003571:Cytochalasin B / Q000494:pharmacology; D048749:Cytokinesis / Q000187:drug effects*; D005260:Female; D042822:Genomic Instability; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D015162:Micronucleus Tests / Q000379:methods; D008875:Middle Aged; D061251:Primary Cell Culture
chemical_list: D015415:Biomarkers; D003571:Cytochalasin B