Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) represent a promising cell-based therapy for a number of degenerative conditions. Many applications require cell expansion and involve the treatment of diseases and conditions found in an aging population. Therefore, the effects of donor age and long-term passage must be clarified. In this study, the effects of donor age and long-term passage on the morphology, proliferation potential, characteristics, mesodermal differentiation ability, and transdifferentiation potential of hMSCs towards neurogenic lineage were evaluated. Cells from child donors (0-12 years, n = 6) maintained their fibroblast-like morphology up to higher passages and proliferated in a greater number than those from adult (25-50 years, n = 6) and old (over 60 years, n = 6) donors. Adipogenic, osteogenic, and neurogenic differentiation potential decreased with age, while chondrogenic potential did not change. Long-term passage affected the morphology and proliferation of hMSCs from all ages. With increasing passage number, proliferation rate decreased and cells lost their typical morphology. Expression levels of neural markers (β III tubulin and NSE) and topo II isoforms in populations of nondifferentiated hMSCs were investigated by reverse transcription polymerase chain reaction analysis. While neural marker and topo IIβ expression levels increased due to increasing passage number in adult hMSCs compared to child hMSCs, topo IIα decreased in both. These results indicated that, even under highly standardized culture conditions, donor age and long-term passage have effects on hMSC characteristics, which should be taken into account prior to stem cell-based therapies.

DNA topoisomerase I and II have been shown to be modi¢ed with a ubiquitin-like protein SUMO in response to their speci¢c inhibitors called 'poisons'. These drugs also damage DNA by stabilizing the enzyme^DNA cleavable complex and induce a degradation of the enzymes through the 26S proteasome system. A plausible link between sumoylation and degradation has not yet been elucidated. We demonstrate here that topoisomerase IIL L, but not its isoform IIK K, is selectively degraded through proteasome by exposure to the catalytic inhibitor ICRF-193 which does not damage DNA. The L L isoform immunoprecipitated from ICRF-treated cells was modi¢ed by multiple modi¢ers, SUMO-2/3, SUMO-1, and polyubiquitin. When the SUMO conjugating enzyme Ubc9 was conditionally knocked out, the ICRF-induced degradation of topoisomerase IIL L did not occur, suggesting that the SUMO modi¢cation pathway is essential for the degradation.

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Two isoforms of DNA topoisomerase II (topo II) have been identified in mammalian cells, named topo IIα and topo IIβ. Topo IIα plays an essential role in segregation of daughter chromosomes and thus for cell proliferation in mammalian cells. Unlike its isozyme topo IIα, topo IIβ is greatly expressed upon terminal differentiation of neuronal cells. Although there have been accumulating evidence about the crucial role of topo IIβ in neural development through activation or repression of developmentally regulated genes at late stages of neuronal differentiation, there have been no reports that analyzed the roles of topo IIβ in the neural trans differentiation process of multipotent stem cells. Terminal differentiation of neurons and transdifferentiation of Mesenchymal Stem Cells (MSCs) are two distinct processes. Therefore, the functional significance of topo IIβ may also be different in these differentiation systems. MSC transdifferentiation into neuron-like cells represents an useful model to further validate the role of topo IIβ in neuronal differentiation. The aim of this study is to evaluate the subset of genes that are regulated in neural transdifferentiation of bone marrow-derived human MSCs (BM-hMSCs) in vitro and find genes related with topo IIβ. For this purpose, topo IIβ was silenced by specific small interfering RNAs in hMSCs and cells were induced to differentiate into neuron-like cells. Differentiation and silencing of topo IIβ were monitored by real-time cell analysis and also expressions of topo II isoforms were analyzed. Change in transcription patterns of genes upon topo IIβ silencing was identified by DNA microarray analysis, and apparently genes involved in regulation of several ion channels and transporters, vesicle function, and cell calcium metabolism were particularly affected by topo IIβ silencing suggesting that topoIIβ silencing can significantly alter the gene expression pattern of genes involved in variety of biological processes and signal transduction pathways including transcription, translation, cell trafficking, vesicle function, transport, cell morphology, neuron guidance, growth, polarity, and axonal growth. It appears that the deregulation of these pathways may contribute to clarify the further role of topo IIβ in neural differentiation.

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Our findings suggest that topo IIβ could be a down-stream target of signaling pathways contributing to AD-like pathology. However, further studies must be carried out in vivo to elucidate the precise association topo IIβ with AD.