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Papers by Rainer Saffrich

Experimental Cell Research - EXP CELL RES, 2006

Over the past several years, it has become clear that the Rho family of GTPases plays an importan... more Over the past several years, it has become clear that the Rho family of GTPases plays an important role in various aspects of neuronal development including cytoskeleton dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated Rac1 and Cdc42 can rescue the formation of these structures. We have also established quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes.

Experimental Hematology, 2006

Journal of Magnetic Resonance (1969), 1990

... Recording and processing parameters as described under Materials and Methods. The Ha Ho regio... more ... Recording and processing parameters as described under Materials and Methods. The Ha Ho region of the spectrum is shown, and the multiplets found are indicated. ... 17. JC HOCH, S. HENGYI, M. KJAER, S. LUDVIGSEN, AND FM POULSEN, Carlherg Res. Commun. ...

Journal of Magnetic Resonance, Series B, 1993

Cytotherapy, 2015

Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regene... more Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Strahlentherapie und Onkologie, 2014

Mesenchymal stem cells (MSCs) can regenerate damaged tissues and may therefore be of importance f... more Mesenchymal stem cells (MSCs) can regenerate damaged tissues and may therefore be of importance for normal tissue repair after cancer treatment. Small molecule receptor kinase inhibitors (RKIs) have recently been introduced into cancer treatment. However, the influence of these drugs-particularly in combination with radiotherapy-on the survival of MSCs is largely unknown. The sensitivity of human primary MSCs from healthy volunteers and primary human fibroblast cells to small molecule kinase inhibitors of the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ) receptors, as well to inhibitors of c-Kit, was examined in combination with ionizing radiation (IR); cell survival and proliferation were assessed. Expression patterns of different kinase receptors and ligands were investigated using gene arrays. MSCs were highly sensitive to the tyrosine kinase inhibitors SU14816 (imatinib) and SU11657 (sunitinib), but showed only moderate sensitivity to the selective TGFβ receptor 1 inhibitor LY2109761. Primary adult human fibroblasts were comparably resistant to all three inhibitors. The addition of IR had an additive or supra-additive effect in the MSCs, but this was not the case for differentiated fibroblasts. Proliferation was markedly reduced in MSCs following kinase inhibition, both with and without IR. Gene expression analysis revealed high levels of the PDGF α and β receptors, and lower levels of the TGFβ receptor 2 and Abl kinase. IR did not alter the expression of kinase receptors or their respective ligands in either MSCs or adult fibroblasts. These data show that MSCs are highly sensitive to RKIs and combination treatments incorporating IR. Expression analyses suggest that high levels of PDGF receptors may contribute to this effect.

Computational Aspects of the Study of Biological Macromolecules by Nuclear Magnetic Resonance Spectroscopy, 1991

Microinjection and Transgenesis, 1998

British Journal of Cancer, 2008

Journal of Cellular Physiology - J CELL PHYSIOL, 2000

Neuronal differentiation of PC12 cells is achieved by stimulation with nerve growth factor (NGF) ... more Neuronal differentiation of PC12 cells is achieved by stimulation with nerve growth factor (NGF) but not by epidermal growth factor (EGF). However, features of differentiation such as neurite outgrowth are observable at the earliest after several hours. Using actin staining of the cells, we show here that NGF stimulation leads to lamellipodia formation within only 3 min at the periphery of the PC12 cells. EGF stimulation or microinjection of differentiation-inducing c-Crk I protein does not cause lamellipodia. The actin reorganization after NGF stimulation is blocked by microinjecting dominant negative Rac protein. The lamellipodia formation is also abolished by inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY 294002 in a concentration-dependent manner. Phase-contrast time-lapse microscopy was used to analyze membrane dynamics in real time and to confirm the induction of lamellipodia by NGF and their inhibition by pretreatment with both wortmannin and LY 294002. The results indicate that NGF, but not EGF, leads to rapid lamellipodia formation in PC12 cells via phosphatidylinositol 3-kinase and the small GTPase Rac, thereby defining a novel role for these factors in early NGF signaling.

Transfusion Medicine and Hemotherapy, 2008

Experimental Cell Research - EXP CELL RES, 2006

Over the past several years, it has become clear that the Rho family of GTPases plays an importan... more Over the past several years, it has become clear that the Rho family of GTPases plays an important role in various aspects of neuronal development including cytoskeleton dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated Rac1 and Cdc42 can rescue the formation of these structures. We have also established quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes.

Experimental Hematology, 2006

Journal of Magnetic Resonance (1969), 1990

... Recording and processing parameters as described under Materials and Methods. The Ha Ho regio... more ... Recording and processing parameters as described under Materials and Methods. The Ha Ho region of the spectrum is shown, and the multiplets found are indicated. ... 17. JC HOCH, S. HENGYI, M. KJAER, S. LUDVIGSEN, AND FM POULSEN, Carlherg Res. Commun. ...

Journal of Magnetic Resonance, Series B, 1993

Cytotherapy, 2015

Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regene... more Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Strahlentherapie und Onkologie, 2014

Mesenchymal stem cells (MSCs) can regenerate damaged tissues and may therefore be of importance f... more Mesenchymal stem cells (MSCs) can regenerate damaged tissues and may therefore be of importance for normal tissue repair after cancer treatment. Small molecule receptor kinase inhibitors (RKIs) have recently been introduced into cancer treatment. However, the influence of these drugs-particularly in combination with radiotherapy-on the survival of MSCs is largely unknown. The sensitivity of human primary MSCs from healthy volunteers and primary human fibroblast cells to small molecule kinase inhibitors of the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ) receptors, as well to inhibitors of c-Kit, was examined in combination with ionizing radiation (IR); cell survival and proliferation were assessed. Expression patterns of different kinase receptors and ligands were investigated using gene arrays. MSCs were highly sensitive to the tyrosine kinase inhibitors SU14816 (imatinib) and SU11657 (sunitinib), but showed only moderate sensitivity to the selective TGFβ receptor 1 inhibitor LY2109761. Primary adult human fibroblasts were comparably resistant to all three inhibitors. The addition of IR had an additive or supra-additive effect in the MSCs, but this was not the case for differentiated fibroblasts. Proliferation was markedly reduced in MSCs following kinase inhibition, both with and without IR. Gene expression analysis revealed high levels of the PDGF α and β receptors, and lower levels of the TGFβ receptor 2 and Abl kinase. IR did not alter the expression of kinase receptors or their respective ligands in either MSCs or adult fibroblasts. These data show that MSCs are highly sensitive to RKIs and combination treatments incorporating IR. Expression analyses suggest that high levels of PDGF receptors may contribute to this effect.

Computational Aspects of the Study of Biological Macromolecules by Nuclear Magnetic Resonance Spectroscopy, 1991

Microinjection and Transgenesis, 1998

British Journal of Cancer, 2008

Journal of Cellular Physiology - J CELL PHYSIOL, 2000

Neuronal differentiation of PC12 cells is achieved by stimulation with nerve growth factor (NGF) ... more Neuronal differentiation of PC12 cells is achieved by stimulation with nerve growth factor (NGF) but not by epidermal growth factor (EGF). However, features of differentiation such as neurite outgrowth are observable at the earliest after several hours. Using actin staining of the cells, we show here that NGF stimulation leads to lamellipodia formation within only 3 min at the periphery of the PC12 cells. EGF stimulation or microinjection of differentiation-inducing c-Crk I protein does not cause lamellipodia. The actin reorganization after NGF stimulation is blocked by microinjecting dominant negative Rac protein. The lamellipodia formation is also abolished by inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY 294002 in a concentration-dependent manner. Phase-contrast time-lapse microscopy was used to analyze membrane dynamics in real time and to confirm the induction of lamellipodia by NGF and their inhibition by pretreatment with both wortmannin and LY 294002. The results indicate that NGF, but not EGF, leads to rapid lamellipodia formation in PC12 cells via phosphatidylinositol 3-kinase and the small GTPase Rac, thereby defining a novel role for these factors in early NGF signaling.

Transfusion Medicine and Hemotherapy, 2008