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Wagner W.,RWTH Aachen | Bork S.,University of Heidelberg | Lepperdinger G.,Institute for Biomedical Aging Research | Joussen S.,RWTH Aachen | And 3 more authors.
Aging | Year: 2010

Mesenchymal stromal cells (MSC) are currently tested in a large number of clinical trials and raise high hope inregenerative medicine. These cells have to be expanded in vitro before transplantation and several studies demonstratedthat long-term culture evokes continuous changes in MSC: proliferation rate decays, the cell size increases, differentiationpotential is affected, chromosomal instabilities may arise and molecular changes are acquired. Long-term culture of cellpreparations might also have therapeutic consequences, although this has hardly been addressed in ongoing trials so far. Reliable therapeutic regimens necessitate quality control of cellular products. This research perspective summarizesavailable methods to track cellular aging of MSC. We have demonstrated that gene expression changes [1] and epigeneticmodifications [2] are continuously acquired during replicative senescence. Molecular analysis of a suitable panel of genesmight provide a robust tool to assess efficiency and safety of long-term expansion. © Wagner et al. Source


Greussing R.,Institute for Biomedical Aging Research
Journal of visualized experiments : JoVE | Year: 2012

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins (1, 2). Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response (3), cell cycle regulation and cellular differentiation (4) or in immune system response (5). The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases (4, 6). Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging (7, 8, 9, 10). Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)-dgn fusion protein (GFP-dgn, unstable) and a variant carrying a frameshift mutation (GFP-dgnFS, stable (11)) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP-dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry. Source


Boraschi D.,CNR Institute of Biomedical Technologies | Del Giudice G.,Novartis | Dutel C.,Fondation Merieux | Ivanoff B.,Institute for Biomedical Aging Research | And 2 more authors.
Vaccine | Year: 2010

Among the greatest achievements of the 20th century, prolongation of life expectancy has been the result of improved health conditions, decreased childhood mortality, lower incidence of infectious diseases. The consequence is the rapid ageing of the world population, with the elderly representing over 25% of the entire population by the year 2030, of which 75% living in less developed countries. Ageing thus represents one of the major public health challenges of the 21st century. Indeed, unhealthy ageing and frailty of the aged population has an important impact on the economic development and social costs of a country, a problem even more acute in less developed countries. A better knowledge of immune senescence and the design of customised vaccination strategies for the elderly are the immediate challenges posed to scientists and physicians. The conference "Ageing and immunity", recently held in Siena (Italy), has addressed these issues and defined the global strategic priorities for research and health policies aimed at ensuring healthy ageing. Source


Greussing R.,Institute for Biomedical Aging Research | Unterluggauer H.,Institute for Biomedical Aging Research | Koziel R.,Institute for Biomedical Aging Research | Maier A.B.,Leiden University | Jansen-Durr P.,Institute for Biomedical Aging Research
Journal of Visualized Experiments | Year: 2012

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)-dgn fusion protein (GFP-dgn, unstable) and a variant carrying a frameshift mutation (GFP-dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP-dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry. Source

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