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Effect of F68 on cryopreservation of mesenchymal stem cells derived from human tooth germ
New Biotechnology · Volume 29S · September 2012
Oral 2.2.02 Generating cartilage from mesenchymal stem cells Anthony P. Hollander ∗ , Sally C. Dickinson, W.Z. Kafienah School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK Methods for deriving chondrocytes from mesenchymal stem cells (MSCs) have been developed in a number of laboratories, but turning these methods into clinical therapies has been particularly challenging. We have used specific cartilage marker assays to monitor the quality of generated tissue in our in vitro cartilage tissue engineering assays. On this basis we have been able to develop an optimised protocol for cartilage tissue engineering from MSCs. Whilst the original purpose of our work was to develop treatments for cartilage degradation in osteoarthritis, we were able to adapt our method in order to engineer a functional airway that was successfully implanted into the left bronchus of a patient with bronchomalacia. In order now to progress with the development of a treatment for osteoarthritis, we need to understand how to reduce the variability of chondrogenesis between different MSC preparations. We have used MSC cloning and gene array analysis to begin the process of identifying cell surface markers that may be used to reduce the variability of outcome. This approach is likely to lead to a translatable chondrogenic differentiation method for treatment of a range of cartilage diseases. http://dx.doi.org/10.1016/j.nbt.2012.08.036 Oral 2.2.03 Therapeutic effect of genetically modified mesenchymal stem cells with improved VEGF production on spinal cord injury in rats Gökhan Duruksu 1,∗ , Serdar Kabatas 2 , Alparslan Okcu 1 , Cansu Subasi 1 , Gizem Turac 1 , Irem Yilmaz 1 , Erdinc Civelek 2 , Erdal Karaoz 1 1
Kocaeli University, Centre for Stem Cell and Gene Therapies Research and Practice, Izmit, Kocaeli, Turkey 2 Baskent University, Faculty of Medicine, Department of Neurosurgery, Istanbul, Turkey Mesenchymal stem cells (MSCs) are multipotent cells, found in almost all tissue, and involve in tissue regeneration by differentiation into various cell types. Tropism to tumors and migration to the damaged site makes them candidates for drug carrier in therapies. In the previous study, bone marrow derived (BM-) MSCs were injected into lesion site after spinal cord injury in rats, and partial regaining of mobility was observed. To improve the regeneration capacity of MSCs, they were transfected with vascular endothelial growth factor (VEGF) gene, which is well known for its function in angiogenesis and neurogenesis as neurotrophic factor. Before the transplantation of BM-MSC in rat laminectomy and trauma models, GFP gene was co-transfected with VEGF gene into cells by electroporation to distinguish the delivered MSCs from the rest of the tissue. The motility of rats was scored regularly. After 5weeks, rats were sacrificed, and their spinal cords were examined by immunostainings. For almost full recovery was observed in the S16
www.elsevier.com/locate/nbt
animal group treated with GFP+ /VEGF+ BM-MSCs. The immunestaining of tissue sections with appropriate antibodies revealed the genetically improved MSCs were extensively participated in the regeneration, despite the in vitro neural differentiation was failed. The GFP+ cells in the tissue sections were widely expressing astrocyte markers GFAP and S100 besides the neurotrophin BDNF, whereas the expression of neuron and oligodentrocyte markers, 3 -tubulin and CNPase, was induced slightly. The gene expression analysis of VEGF+ MSCs showed that not only the VEGF, but also the expression of other growth and transcription factors was upregulated. http://dx.doi.org/10.1016/j.nbt.2012.08.037 Oral 2.2.04 Effect of F68 on cryopreservation of mesenchymal stem cells derived from human tooth germ Ays¸egül Do˘ gan 1,∗ , Mehmet Emir Yalvac¸ 2 , Albert Rizvanov 1,3 , Fikrettin S¸ahin 1 1
Department of Genetics and BioEngineering, College of Engineering and Architecture, Yeditepe University, 26 A˘gustos Campus, Kayisdagi cad., Kayisdagi, TR-34755 Istanbul, Turkey 2 The Research Institute at Nationwide Children’s Hospital, 43205, Columbus, OH, USA 3 Department of Genetics, Faculty of Biology and Soil Sciences, Kazan State University, ul. Kremlevskaya 18, R-420008 Kazan, Russia The use of stem cell based therapies in regenerative medicine and in the treatment of disorders such as Parkinson, Alzheimer’s Disease, Diabetes, Spinal Cord Injuries and Cancer has been shown to be promising. Among all stem cells, mesenchymal stem cells (MSCs) were reported to have anti-apoptotic, immunomodulatory and angiogenic effects which are attributed to restorative capacity of these cells. Human tooth germ stem cells (HTGSCs) having mesenchymal stem cell characteristics have been proven to exert high proliferation and differentiation capacity. Regardless of their sources, the stem cells are exposed to physical and chemical stresses during cryopreservation hindering their therapeutic capacity. Amelioration of side effects of cryopreservation on MSCs seems to be a priority in order to maximizing the therapeutic efficacy of these cells. In this study, we tested the effect of Pluronic 188 (F-68) on HTGSCs during long term cryopreservation and repeated freezing and defrosting cycles. Our data revealed that F68 has a protective role on survival and differentiation of HTGSCs in long term cryopreservation. Pluronic F68 does not change the mesenchymal stem cell characteristics and fatty acid profile of the cells after long term cryopreservation. Instead F68 increased the osteogenic, chondrogenic and adipogenic differentiation potential of cells. Moreover F68 reduces the expression of apoptotic genes. Keywords: Pluronic; Mesenchymal stem cells; Cryopreservation http://dx.doi.org/10.1016/j.nbt.2012.08.038
Oral 2.2.02 Generating cartilage from mesenchymal stem cells Anthony P. Hollander ∗ , Sally C. Dickinson, W.Z. Kafienah School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK Methods for deriving chondrocytes from mesenchymal stem cells (MSCs) have been developed in a number of laboratories, but turning these methods into clinical therapies has been particularly challenging. We have used specific cartilage marker assays to monitor the quality of generated tissue in our in vitro cartilage tissue engineering assays. On this basis we have been able to develop an optimised protocol for cartilage tissue engineering from MSCs. Whilst the original purpose of our work was to develop treatments for cartilage degradation in osteoarthritis, we were able to adapt our method in order to engineer a functional airway that was successfully implanted into the left bronchus of a patient with bronchomalacia. In order now to progress with the development of a treatment for osteoarthritis, we need to understand how to reduce the variability of chondrogenesis between different MSC preparations. We have used MSC cloning and gene array analysis to begin the process of identifying cell surface markers that may be used to reduce the variability of outcome. This approach is likely to lead to a translatable chondrogenic differentiation method for treatment of a range of cartilage diseases. http://dx.doi.org/10.1016/j.nbt.2012.08.036 Oral 2.2.03 Therapeutic effect of genetically modified mesenchymal stem cells with improved VEGF production on spinal cord injury in rats Gökhan Duruksu 1,∗ , Serdar Kabatas 2 , Alparslan Okcu 1 , Cansu Subasi 1 , Gizem Turac 1 , Irem Yilmaz 1 , Erdinc Civelek 2 , Erdal Karaoz 1 1
Kocaeli University, Centre for Stem Cell and Gene Therapies Research and Practice, Izmit, Kocaeli, Turkey 2 Baskent University, Faculty of Medicine, Department of Neurosurgery, Istanbul, Turkey Mesenchymal stem cells (MSCs) are multipotent cells, found in almost all tissue, and involve in tissue regeneration by differentiation into various cell types. Tropism to tumors and migration to the damaged site makes them candidates for drug carrier in therapies. In the previous study, bone marrow derived (BM-) MSCs were injected into lesion site after spinal cord injury in rats, and partial regaining of mobility was observed. To improve the regeneration capacity of MSCs, they were transfected with vascular endothelial growth factor (VEGF) gene, which is well known for its function in angiogenesis and neurogenesis as neurotrophic factor. Before the transplantation of BM-MSC in rat laminectomy and trauma models, GFP gene was co-transfected with VEGF gene into cells by electroporation to distinguish the delivered MSCs from the rest of the tissue. The motility of rats was scored regularly. After 5weeks, rats were sacrificed, and their spinal cords were examined by immunostainings. For almost full recovery was observed in the S16
www.elsevier.com/locate/nbt
animal group treated with GFP+ /VEGF+ BM-MSCs. The immunestaining of tissue sections with appropriate antibodies revealed the genetically improved MSCs were extensively participated in the regeneration, despite the in vitro neural differentiation was failed. The GFP+ cells in the tissue sections were widely expressing astrocyte markers GFAP and S100 besides the neurotrophin BDNF, whereas the expression of neuron and oligodentrocyte markers, 3 -tubulin and CNPase, was induced slightly. The gene expression analysis of VEGF+ MSCs showed that not only the VEGF, but also the expression of other growth and transcription factors was upregulated. http://dx.doi.org/10.1016/j.nbt.2012.08.037 Oral 2.2.04 Effect of F68 on cryopreservation of mesenchymal stem cells derived from human tooth germ Ays¸egül Do˘ gan 1,∗ , Mehmet Emir Yalvac¸ 2 , Albert Rizvanov 1,3 , Fikrettin S¸ahin 1 1
Department of Genetics and BioEngineering, College of Engineering and Architecture, Yeditepe University, 26 A˘gustos Campus, Kayisdagi cad., Kayisdagi, TR-34755 Istanbul, Turkey 2 The Research Institute at Nationwide Children’s Hospital, 43205, Columbus, OH, USA 3 Department of Genetics, Faculty of Biology and Soil Sciences, Kazan State University, ul. Kremlevskaya 18, R-420008 Kazan, Russia The use of stem cell based therapies in regenerative medicine and in the treatment of disorders such as Parkinson, Alzheimer’s Disease, Diabetes, Spinal Cord Injuries and Cancer has been shown to be promising. Among all stem cells, mesenchymal stem cells (MSCs) were reported to have anti-apoptotic, immunomodulatory and angiogenic effects which are attributed to restorative capacity of these cells. Human tooth germ stem cells (HTGSCs) having mesenchymal stem cell characteristics have been proven to exert high proliferation and differentiation capacity. Regardless of their sources, the stem cells are exposed to physical and chemical stresses during cryopreservation hindering their therapeutic capacity. Amelioration of side effects of cryopreservation on MSCs seems to be a priority in order to maximizing the therapeutic efficacy of these cells. In this study, we tested the effect of Pluronic 188 (F-68) on HTGSCs during long term cryopreservation and repeated freezing and defrosting cycles. Our data revealed that F68 has a protective role on survival and differentiation of HTGSCs in long term cryopreservation. Pluronic F68 does not change the mesenchymal stem cell characteristics and fatty acid profile of the cells after long term cryopreservation. Instead F68 increased the osteogenic, chondrogenic and adipogenic differentiation potential of cells. Moreover F68 reduces the expression of apoptotic genes. Keywords: Pluronic; Mesenchymal stem cells; Cryopreservation http://dx.doi.org/10.1016/j.nbt.2012.08.038