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Adenoviral vector mediated ferritin over-expression in mesenchymal stem cells detected by 7T MRI in vitro.

ABSTRACT: The aim of the present work was to verify whether adenoviral vector mediated ferritin over-expression in mesenchymal stem cells could be detected by 7T MRI device, and to explore the relationship between ferritin content and MRI signal intensities.A recombined adenoviral vector (rAdV) encoding ferritin heavy chain (FTH1) subunit was specially designed for the aim of infecting bone marrow mesenchymal stem cells (BMSCs). Ferritin over-expression in BMSCs was determined by cell immunocytochemistry and the ferritin content in cells was determined by ELISA assay. BMSCs were subjected to cell viability, proliferation and multi-differentiation analyses as well as 7T MRI test using fast spin-echo pulse sequence. The R2 value and?R2 were calculated according to T2 mapping images.As was confirmed by cell immunocytochemistry and ELISA assay, rAdV mediated ferritin was over-expressed in BMSCs. Ferritin over-expression did not interfere with stem cell viability or pluripotent differentiation but slowed cell proliferation. The R2 value of BMSCs-FTH1 vs control BMSCs from 1-4 weeks was16.65±1.28 s-1 vs 13.99±0.80 s-1, (t = 3.94, p = 0.004), 15.63±1.37 s-1 vs 13.87±0.83 s-1 (t = 2.47, p = 0.039), 15.53±0.88 s-1 vs 14.25±0.53 s-1 (t = 2.80, p = 0.023) and 14.61±1.28 s-1 vs 13.69±1.03 s-1 (t = 1.25, p = 0.24), respectively. ?R2 gradually decreased from 1-4 weeks and the difference between the groups had statistical significance (F = 12.45, p<0.01).?R2 was positively correlated with OD value (r = 0.876, p<0.01) and ferritin concentration (r = 0.899, p<0.01) as determined by Pearson correlation.Our study confirms that ferritin could be over-expressed in BMSCs as a result of rAdV mediated infection and could be quantitatively detected by 7T MRI device. The differences in T2 signal intensities and R2 values stem from internal contrast generated by endogenous ferritin over-expression. The correlation between ?R2, OD and ferritin concentration suggests that MRI can detect ferritin signal change accurately.

SUBMITTER: Dai HY

PROVIDER: S-EPMC5612726 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Adenoviral vector mediated ferritin over-expression in mesenchymal stem cells detected by 7T MRI in vitro.

Dai Hai-Yang HY He Rong R Zhang Ying Y Wu Ren-Hua RH Xiao Ye-Yu YY

PloS one 20170925 9

<h4>Objective</h4>The aim of the present work was to verify whether adenoviral vector mediated ferritin over-expression in mesenchymal stem cells could be detected by 7T MRI device, and to explore the relationship between ferritin content and MRI signal intensities.<h4>Methods</h4>A recombined adenoviral vector (rAdV) encoding ferritin heavy chain (FTH1) subunit was specially designed for the aim of infecting bone marrow mesenchymal stem cells (BMSCs). Ferritin over-expression in BMSCs was deter ...[more]

PMID: 28945778

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Project description:IntroductionA combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.MethodsTransduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-?-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.ResultsThe second generation lentiviral vector rHIV-pWPT-EF1-?-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.ConclusionsCollectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.

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Adenoviral vector mediated ferritin over-expression in mesenchymal stem cells detected by 7T MRI in vitro.

Publications

Adenoviral vector mediated ferritin over-expression in mesenchymal stem cells detected by 7T MRI in vitro.

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