Dataset Information

Evaluation of genotoxicity and mutagenic effects of vector/DNA nanocomplexes in transfected mesenchymal stem cells by flow cytometry.

ABSTRACT: In recent years, there has been a great deal of interest in ex-vivo genetic modification of mesenchymal stem cells (MSCs) to meet various biomedical needs. Considering the self-renewal potential of MSCs, it is critically important to ensure that transfection vectors (gene carriers) do not induce genotoxicity because they could theoretically turn a single stem cell into a cancer-initiating cell. Unfortunately, there is currently no reliable, unbiased, and quantitative method to measure genotoxicity (micronuclei formation) of gene carriers directly in transfected MSCs. Consequently, it has not been possible to study the correlation of vectors' physicochemical characteristics with their impact on stem cell genome stability. To address this deficiency, a flow cytometry-based method with a specialized gating protocol was developed that not only measures micronuclei formation, but also determines the mechanism of mutagenesis (i.e., clastogenic vs. aneugenic) of each vector in transfected MSCs. This gating protocol effectively eliminates all interfering signals associated with aggregated nanoparticles (viral and non-viral), exogenous DNA, and apoptotic/necrotic bodies from the micronuclei measurement process. The presented gating protocol for flow cytometry, which is provided as a template, enables investigators in academia, industry and regulatory bodies to rapidly and reliably evaluate the genosafety profiles of gene carriers. The findings of this study also indicate that highly positively charged lipid- and polymeric-based vectors can induce genotoxicity even without manifesting substantial somatic toxicity. Thus, extreme care must be taken before implanting ex-vivo-modified MSCs back into a patient's body.

Statement of significance

There is a great interest in genetic modification of stem cells (SCs) by using vectors for various biomedical needs. Considering the self-renewal potential of SCs, it is essential to ensure that such vectors do not induce genetic aberrations (genotoxicity) because they could theoretically turn a single stem cell into a cancer-initiating cell. Unfortunately, there is currently no reliable method to measure genotoxicity of vectors directly in transfected SCs. To address this deficiency, a specialized flow cytometry-based method was developed that quantitatively analyzed genotoxicity and determined the mechanism of mutagenesis that occurred in transfected SCs during the transfection process. The developed technique will enable scientists to design safer vectors for genetic modification of stem cells.

SUBMITTER: Nomani A

PROVIDER: S-EPMC6004338 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Publications

Evaluation of genotoxicity and mutagenic effects of vector/DNA nanocomplexes in transfected mesenchymal stem cells by flow cytometry.

Nomani Alireza A Chen Xuguang X Hatefi Arash A

Acta biomaterialia 20180519

In recent years, there has been a great deal of interest in ex-vivo genetic modification of mesenchymal stem cells (MSCs) to meet various biomedical needs. Considering the self-renewal potential of MSCs, it is critically important to ensure that transfection vectors (gene carriers) do not induce genotoxicity because they could theoretically turn a single stem cell into a cancer-initiating cell. Unfortunately, there is currently no reliable, unbiased, and quantitative method to measure genotoxici ...[more]

PMID: 29783088

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Project description:Acoustic imaging is affordable and accessible without ionizing radiation. Photoacoustic imaging increases the contrast of traditional ultrasound and can offer good spatial resolution when used at high frequencies with excellent temporal resolution. Prussian blue nanoparticles (PBNPs) are an emerging photoacoustic contrast agent with strong optical absorption in the near-infrared region. In this study, we developed a simple and efficient method to label human mesenchymal stem cells (hMSCs) with PBNPs and imaged them with photoacoustic imaging. First, PBNPs were synthesized by the reaction of FeCl3 with K4[Fe(CN)6] in the presence of citric acid and complexed with the cationic transfection agent poly-l-lysine (PLL). The PLL-coated PBNPs (PB-PLL nanocomplexes) have a maximum absorption peak at 715 nm and could efficiently label hMSCs. Cellular uptake of these nanocomplexes was studied using bright field, fluorescence, and transmission electron microscopy. The labeled stem cells were successfully differentiated into two downstream lineages of adipocytes and osteocytes, and they showed positive expression for surface markers of CD73, CD90, and CD105. No changes in viability or proliferation of the labeled cells were observed, and the secretome cytokine analysis indicated that the expression levels of 12 different proteins were not dysregulated by PBNP labeling. The optical properties of PBNPs were preserved postlabeling, suitable for the sensitive and quantitative detection of implanted cells. Labeled hMSCs exhibited strong photoacoustic contrast in vitro and in vivo when imaged at 730 nm, and the detection limit was 200 cells/μL in vivo. The photoacoustic signal increased as a function of cell concentration, indicating that the number of labeled cells can be quantified during and after cell transplantations. In hybrid ultrasound/photoacoustic imaging, this approach offers real-time and image-guided cellular injection even through an intact skull for brain intraparenchymal injections. Our labeling and imaging technique allowed the detection and monitoring of 5 × 104 mesenchymal stem cells in living mice over a period of 14 days.

Project description:BackgroundMesenchymal stem/stromal cells (MSC) have immunomodulatory properties, studied in a wide range of diseases. Validated quality controls must confirm this activity in the context of clinical trials. This study presents a method's validation, assessing MSC's ability to inhibit lymphocyte proliferation, according to the ICH Q2 standard.MethodsMSC were co-cultured with CellTrace™ Violet-labeled Peripheral blood mononuclear cells (PBMC) coming from a bank of ten donors, at seven different ratios for 7 days. Cell trace violet PBMC bank was validated in parallel. Flow cytometry analysis was used to obtain the division percentage of T cells. The percentage of inhibition of lymphocyte proliferation by MSC, for each ratio X, was calculated using the formula: Ratio × percentage of inhibition = (control percentage of division-ratio × percentage of division)/control percentage of division. The inhibition percentage of lymphocyte proliferation function of co-culture ratios was represented in a line graph. The corresponding area under the curve was calculated, representing MSC's ability to inhibit lymphocyte proliferation.ResultsTwo cell trace violet PBMC banks were compared for bank validation. When compared using four different MSC samples coming each from a different donor, their area under the curve did not show any statistical differences and were correlated. Moreover, the stability of one cell trace violet PBMC bank was confirmed up to 509 days of storage. Analytical parameters were investigated for method validation. Analysis of repeatability and reproducibility respectively showed a standard deviation of 6.1% and 4.6%. The assay was robust regarding PBMC, as no statistical differences were found between inhibitory activities when testing three adjacent concentrations of PBMC. Still, attention is needed on MSC quantity as it can influence results. Linearity was evaluated: the percentage of inhibition of lymphocyte proliferation function of co-culture ratios was linear on the exploited range. Finally, the assay measurement range allowed to differentiate MSC presenting different inhibition activities.ConclusionThis quantification method displayed low analytical variability and no inter-bank variability of PBMC. However, MSC quantification should be checked before co-culture to reduce variability. Therefore, it could be used for the qualification of MSC batches' immunomodulatory activity.

Dataset Information

Evaluation of genotoxicity and mutagenic effects of vector/DNA nanocomplexes in transfected mesenchymal stem cells by flow cytometry.

Statement of significance

Publications

Evaluation of genotoxicity and mutagenic effects of vector/DNA nanocomplexes in transfected mesenchymal stem cells by flow cytometry.

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