4018 Research Article Introduction Marrow stoma cells or mesenchymal stem cells are multipotent, and therefore an important source for the regeneration of damaged tissues and for homeostasis maintenance (Gerson, 1999; Le Blanc and Pittenger, 2005). The relative ease by which they can be isolated and their subsequent manipulation to differentiate into several cell lineages (Pittenger et al., 1999) is a significant advantage for therapeutic use. When grown ex vivo, the division rate of human mesenchymal stem cells (hMSCs) reduces after a few passages, corresponding to cellular senescence and aging (Baxter et al., 2004; Bonab et al., 2006). As the hMSCs exhibit fast cellular senescence without the need for additional manipulations, they can be used as a model system to study the process of cellular senescence. The process of cellular senescence refers to irreversible growth arrest, which is characterized by quantitative changes in nuclear factors that record the proliferative history of cells, known as Hayflick factors. In human cells, the three best known Hayflick senescence-associated factors are telomere shortening, which results from limited activity and reduced expression of the telomere reverse transcriptase (TERT), accumulation of DNA damage in senescence-associated DNA foci and derepression of the ARF/INK4a locus (reviewed by Collado et al., 2007). Although the general molecular mechanisms underlying the initiation and maintenance of cellular senescence are increasingly recognized, qualitative and quantitative differences are found between organisms and cell types (Ben Porath and Weinberg, 2005; Shay and Wright, 2007; Collado et al., 2007). Nuclei of senescent cells show significant structural changes, including the formation of the senescence-associated heterochromatic foci (SAHF), which are enriched for several heterochromatin-binding proteins (Narita et al., 2003), the aggregation of the promyelocytic leukemia-nuclear bodies (PML- NBs), which serve as storage hubs for many nuclear proteins (Narita et al., 2003) and the appearance of γH2AX-foci, which are centers for DNA double-strand breaks (Sedelnikova et al., 2004; Cowell et al., 2007). In addition, distorted organization of the nuclear lamina was found in senescent cells (reviewed by Gruenbaum et al., 2005) and in fibroblasts derived from patients carrying mutations in the lamin A gene (Broers et al., 2006; Capell and Collins, 2006). Furthermore, these cells revealed an accumulation of DNA damage and altered histone modifications (Scaffidi and Misteli, 2006), and telomere shortening (Huang et al., 2008), suggesting that changes in lamina organization trigger changes in nuclear function. Ex vivo, human mesenchymal stem cells (hMSCs) undergo spontaneous cellular senescence after a limited number of cell divisions. Intranuclear structures of the nuclear lamina were formed in senescent hMSCs, which are identified by the presence of Hayflick-senescence-associated factors. Notably, spatial changes in lamina shape were observed before the Hayflick senescence-associated factors, suggesting that the lamina morphology can be used as an early marker to identify senescent cells. Here, we applied quantitative image-processing tools to study the changes in nuclear architecture during cell senescence. We found that centromeres and telomeres colocalised with lamina intranuclear structures, which resulted in a preferred peripheral distribution in senescent cells. In addition, telomere aggregates were progressively formed during cell senescence. Once formed, telomere aggregates showed colocalization with γ-H2AX but not with TERT, suggesting that telomere aggregates are sites of DNA damage. We also show that telomere aggregation is associated with lamina intranuclear structures, and increased telomere binding to lamina proteins is found in cells expressing lamina mutants that lead to increases in lamina intranuclear structures. Moreover, three-dimensional image processing revealed spatial overlap between telomere aggregates and lamina intranuclear structures. Altogether, our data suggest a mechanical link between changes in lamina spatial organization and the formation of telomere aggregates during senescence of hMSCs, which can possibly contribute to changes in nuclear activity during cell senescence. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/24/4018/DC1 Key words: Cell senescence, Telomere aggregates, Nuclear lamina, Spatial organization Summary The nuclear lamina promotes telomere aggregation and centromere peripheral localization during senescence of human mesenchymal stem cells Vered Raz 1,2, *, Bart J. Vermolen 3,4 , Yuval Garini 4,5 , Jos J. M. Onderwater 1 , Mieke A. Mommaas-Kienhuis 1 , Abraham J. Koster 1 , Ian T. Young 4 , Hans Tanke 1 and Roeland W. Dirks 1 1 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands 3 Biophysical Engineering Group, Faculty of Sciences and Technology, Twente University, 7500 AE Enschede, The Netherlands 4 Quantitative Imaging Group, Department of Applied Sciences Delft University of Technology, Delft, The Netherlands 5 Physics Department and Institute of Nanotechnology, Bar-Ilan University, Ramat-Gan 52900, Israel *Author for correspondence (e-mail: v.raz@lumc.nl) Accepted 3 September 2008 Journal of Cell Science 121, 4018-4028 Published by The Company of Biologists 2008 doi:10.1242/jcs.034876 Journal of Cell Science