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.

In vitro comparative study of human mesenchymal stromal cells from dermis and adipose tissue for application in skin wound healing

“…As the prolonged cell culture could induce potential tumorigenic mutations by cumulative replicative stress (Kim et al, 2017), the genetic stability of DSCs and ASCs was evaluated by the cytokinesis-block micronucleus assay. Despite the increased value of binucleated ASCs with nuclei alterations, low frequency of errors (micronuclei, nuclear buds, and nuclear bridges) according to the literature (Cornélio et al, 2014;Fenech, 2007) were observed for DSCs and ASCs (from P2 to P8), suggesting that both are safe for future cell therapy applications in these passages. The methodology used here is a simplified method to assess nuclear alterations and predict risk factors; however, it does not identify specific chromosomic defects (Cornélio et al, 2014).…”

“…Despite the increased value of binucleated ASCs with nuclei alterations, low frequency of errors (micronuclei, nuclear buds, and nuclear bridges) according to the literature (Cornélio et al, 2014;Fenech, 2007) were observed for DSCs and ASCs (from P2 to P8), suggesting that both are safe for future cell therapy applications in these passages. The methodology used here is a simplified method to assess nuclear alterations and predict risk factors; however, it does not identify specific chromosomic defects (Cornélio et al, 2014). Thus, further genetic analysis, such as karyotyping, and the effect of the population FIGURE 6 Suggested clinical applications of dermal derived mesenchymal stromal cells (DSCs) and adipose mesenchymal stromal cells (ASCs).…”

“…Nuclear stability of DSC and ASC (DNA damage) over the time in culture was evaluated by the cytokinesis‐block micronucleus assay as described by Fenech () and Cornélio, Tavares, Pimentel, Cavalcanti‐Junior, and Medeiros (). Cells in P2 or P8 and 80% confluent monolayers were treated with 5‐mg/ml cytochalasin‐B (48 hr, Sigma Aldrich), detached by trypsinization, and centrifuged.…”

Non-proliferative neurogenesis in human periodontal ligament stem cells

“…These findings call into question that micronuclei, nucleoplasmic bridge and nuclear bud does necessarily generated during mitosis 96 . Moreover, a high frequency of human mesenquimal stem cells with nuclear bud, micronuclei and nucleoplasmic bridge was detected under normal in vitro culture 97 . Therefore, the mechanisms that lead to extra-nuclear bodies formation and their biological relevance are still far from been understood 96,98,99 . www.nature.com/scientificreports www.nature.com/scientificreports/ In this study, we show that there can be a relationship in the formation of the nuclear bud, micronuclei, nucleoplasmic bridge and nuclear envelope-limited chromatin sheets.…”

Non-proliferative adult neurogenesis in neural crest-derived stem cells isolated from human periodontal ligament

“…Although these nuclear anomalies have been associated with chromosomal instability events [76][77][78][79], recent reports showed generation of micronuclei during interphase [80][81][82]. Moreover, a high frequency of human mesenquimal stem cells with nuclear bud, micronuclei and nucleoplasmic bridge was detected under normal in vitro culture were observed [83]. Therefore, the mechanisms that lead to extra-nuclear bodies formation and their biological relevance are still far from been understood [84,85].…”