Cytokinesis-block micronucleus assay adapted for analyzing genomic instability of human mesenchymal stem cells.
Stem Cells Dev
; 23(8): 823-38, 2014 Apr 15.
Article
en En
| MEDLINE
| ID: mdl-24328548
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Inestabilidad Genómica
/
Citocinesis
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Células Madre Mesenquimatosas
Tipo de estudio:
Guideline
Límite:
Female
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Humans
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Male
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Middle aged
/
Newborn
Idioma:
En
Revista:
Stem Cells Dev
Asunto de la revista:
HEMATOLOGIA
Año:
2014
Tipo del documento:
Article
País de afiliación:
Brasil
Pais de publicación:
Estados Unidos