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Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells.

ABSTRACT: Bone-marrow-derived mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and differentiation into multiple cell types. Accumulating preclinical and clinical evidence indicates that MSCs are good candidates to use as cell therapy in many degenerative diseases. For MSC clinical applications, an adequate number of cells are necessary so an extensive expansion is required. However, spontaneous immortalization and malignant transformation of MSCs after culture expansion have been reported in human and mouse, while very few data are present for rat MSCs (rMSCs). In this study, we monitored the chromosomal status of rMSCs at several passages in vitro, also testing the influence of four different cell culture conditions. We first used the conventional traditional cytogenetic techniques, in order to have the opportunity to observe even minor structural abnormalities and to identify low-degree mosaic conditions. Then, a more detailed genomic analysis was conducted by array comparative genomic hybridization. We demonstrated that, irrespective of culture conditions, rMSCs manifested a markedly aneuploid karyotype and a progressive chromosomal instability in all the passages we analyzed and that they are anything but stable during in vitro culture. Despite the fact that the risk of neoplastic transformation associated with this genomic instability needs to be further addressed and considering the apparent genomic stability reported for in vitro cultured human MSCs (hMSCs), our findings underline the fact that rMSCs may not in fact be a good model for effectively exploring the full clinical therapeutic potential of hMSCs.

SUBMITTER: Foudah D

PROVIDER: S-EPMC2793379 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells.

Foudah Dana D Redaelli Serena S Donzelli Elisabetta E Bentivegna Angela A Miloso Mariarosaria M Dalprà Leda L Tredici Giovanni G

Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology 20091202 8

Bone-marrow-derived mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and differentiation into multiple cell types. Accumulating preclinical and clinical evidence indicates that MSCs are good candidates to use as cell therapy in many degenerative diseases. For MSC clinical applications, an adequate number of cells are necessary so an extensive expansion is required. However, spontaneous immortalization and malignant transformation of MSCs after culture expansion have be ...[more]

PMID: 19957104

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Project description:INTRODUCTION: Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into different cell lineages and have emerged as a promising tool for cell-targeted therapies and tissue engineering. Their use in a therapeutic context requires large-scale in vitro expansion, increasing the probability of genetic and epigenetic instabilities. Some evidence shows that an organized program of replicative senescence is triggered in human BM-MSCs (hBM-MSCs) on prolonged in vitro expansion that includes alterations in phenotype, differentiation potential, telomere length, proliferation rates, global gene-expression patterns, and DNA methylation profiles. METHODS: In this study, we monitored the chromosomal status, the biologic behavior, and the senescence state of hBM-MSCs derived from eight healthy donors at different passages during in vitro propagation. For a more complete picture, the telomere length was also monitored in five of eight donors, whereas the genomic profile was evaluated in three of eight donors by array-comparative genomic hybridization (array-CGH). Finally, an epigenomic profile was delineated and compared between early and late passages, by pooling DNA of hBM-MSCs from four donors. RESULTS: Our data indicate that long-term culture severely affects the characteristics of hBM-MSCs. All the observed changes (that is, enlarged morphology, decreased number of cell divisions, random loss of genomic regions, telomere shortening) might be regulated by epigenetic modifications. Gene Ontology analysis revealed that specific biologic processes of hBM-MSCs are affected by variations in DNA methylation from early to late passages. CONCLUSIONS: Because we revealed a significant decrease in DNA methylation levels in hBM-MSCs during long-term culture, it is very important to unravel how these modifications can influence the biologic features of hBM-MSCs to keep track of this organized program and also to clarify the conflicting observations on hBM-MSC malignant transformation in the literature.

Project description:Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1?h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies.

Project description:Microglia contribute to the regulation of neuroinflammation and play an important role in the pathogenesis of brain diseases. Thus, regulation of neuroinflammation triggered by activated microglia in brain diseases has become a promising curative strategy. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been shown to have therapeutic effects, resulting from the regulation of inflammatory conditions in the brain. In this study, we investigated differential gene expression in rat BM-MSCs (rBM-MSCs) that were cocultured with lipopolysaccharide- (LPS-) stimulated primary rat microglia using microarray analysis and evaluated the functional relationships through Ingenuity Pathway Analysis (IPA). We also evaluated the effects of rBM-MSC on LPS-stimulated microglia using a reverse coculture system and the same conditions of the transcriptomic analysis. In the transcriptome of rBM-MSCs, 67 genes were differentially expressed, which were highly related with migration of cells, compared to control. The prediction of the gene network using IPA and experimental validation showed that LPS-stimulated primary rat microglia increase the migration of rBM-MSCs. Reversely, expression patterns of the transcriptome in LPS-stimulated primary rat microglia were changed when cocultured with rBM-MSCs. Our results showed that 65 genes were changed, which were highly related with inflammatory response, compared to absence of rBM-MSCs. In the same way with the aforementioned, the prediction of the gene network and experimental validation showed that rBM-MSCs decrease the inflammatory response of LPS-stimulated primary rat microglia. Our data indicate that LPS-stimulated microglia increase the migration of rBM-MSCs and that rBM-MSCs reduce the inflammatory activity in LPS-stimulated microglia. The results of this study show complex mechanisms underlying the interaction between rBM-MSCs and activated microglia and may be helpful for the development of stem cell-based strategies for brain diseases.

Dataset Information

Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells.

Publications

Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells.

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