Wnt signaling regulates the invasion capacity of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) exhibit the potential to contribute to a wide variety of endogenous organ tissue repair. However, the signals governing hMSC mobilization out of the bone marrow, release into the bloodstream, and migration/invasion into the target tissue are largely unknown. Since canonical Wnt signaling regulates not only tumor but also various stem cell attributes, we hypothesized that this signal transduction pathway might also be involved in governing the transmigration of hMSCs through human extracellular matrix (ECM). Stimulation of hMSCs with recombinant Wnt3a or LiCl resulted in the accumulation of the transcriptional activator beta-catenin, its translocation into the nucleus, and the upregulation of typical Wnt target genes such as cyclin D1 and membrane-type matrix metalloproteinase-1 (MT1-MMP). Moreover, both stimuli significantly enhanced hMSC proliferation up to 40%. In addition, an increase of more than twofold in the ability of hMSCs to transmigrate through Transwell filters coated with human ECM was observed. In a reverse approach, Wnt signaling in hMSCs was inhibited by knocking down the expression of either beta-catenin or low-density lipoprotein receptor-related protein 5 using RNA interference technology. These inhibition strategies resulted in downregulation of the Wnt target genes cyclin D1 and MT1-MMP, in a reduced proliferation rate, and in a strikingly diminished invasion capacity (64% and 52%). Taken together, this study provides for the first time decisive evidence that canonical Wnt signaling is critically involved in the regulation of the proliferation, as well as of the migration/invasion capacity of hMSCs, representing essential stem cell features indispensable during tissue regeneration processes.

Nonviral genetic modification mediates effective transgene expression and functional RNA interference in human mesenchymal stem cells.

BACKGROUND: Human mesenchymal stem cells (hMSC) are increasingly the focus of both basic and clinical research due to their ability to strike a balance between self-renewal and commitment to mesodermal differentiation. However, the promising therapeutic utility of hMSC in regenerative medical approaches requires detailed knowledge about their molecular characteristics. Therefore, genetic modification of hMSC provides a powerful tool to understand their complex molecular regulation mechanisms. METHODS: Here we describe a proof of concept approach of separate and combined gene transfer and gene silencing by nonviral DNA transfection of enhanced green fluorescent protein (EGFP) and EGFP-targeted small interfering RNAs (siRNAs) in hMSC. For optimization of nonviral DNA and siRNA transfer different liposomal-based transfection strategies were validated. RESULTS: The highest fraction of EGFP-expressing hMSC was obtained using Lipofectamine 2000 (50%) which also mediated the highest transfection rates of siRNAs into hMSC (>or=92%). Stably EGFP-expressing hMSC maintained their proliferation capacity paired with the ability to differentiate into different mesodermal lineages (bone, cartilage, and fat) without loss of transgene expression. Based on our nonviral nucleic acid delivery technique we showed efficient, functional, and long-term RNA interference (RNAi) in hMSC by gene specific knock-down of transiently and stably expressed EGFP (88-98%). CONCLUSIONS: This is the first demonstration of efficient nonviral transfer of both nucleic acids (DNA and siRNA) into hMSC, exhibiting the potential of targeted modification of hMSC. In particular, the combination of these techniques represents a powerful gene transfer/silencing strategy, thus facilitating detailed genetic approaches to study regulatory networks in stem cell differentiation processes.