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272. Is Intravascular Transplantation of Mesenchymal Stem Cells Safe?
Mesenchymal, Muscle and Bone Stem Cells when compared to the normal MSC. Transformed cells maintained osteogenic and adipogenic capacity and formed tubular structure in ECM. Transplantation of these cells to the infarcted myocardium did not enhance myocardial functions in contrary to normal strain MSC and did not have an influence on infarction size although they enhanced new capillaries formation. Encapsulated structures with characteristics of immature bone and cartilage were found in the infarcted areas of a large fraction of injected hearts. Moreover fat like formation were observed in vitro and in vivo in colocalization with injected cells. Further studies to investigate transfection ability of these cells yielded higher results than normal phenotype MSC. In conclusion accurate investigations are needed to ensure the safe usage of MSC in transplantation-tissue regeneration models. In our study, rat MSC positive to common stem cell markers concealed occurrence of abnormal cells even at very early passages. These cells could maintain differentiation potential and have an angiogenic effect; however, they also lead to tumor-like structure formations. The effectiveness of MSC transplantation for myocardial infarction treatment needs further investigations. Moreover, deeper quality control as: tumorigenicity test, chromosome aberration analysis and prolonged culturing, are highly recommended before MSC transplantation.
270. Angiogenesis in Response to Local Delivery of Human Embryonic Stem Cell Derived Mesenchymal Stem Cells into Ischemic Rat Hind Limb
Juha P. Laurila,1,2 Lilja Laatikainen,1 Peiman Hematti,3 Mikko O. Laukkanen.1 1 Medicity Research Laboratory, University of Turku, Turku, Finland; 2Turku Graduate School of Biomedical Sciences, University of Turku, Turku, Finland; 3Department of Medicine, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI. Mesenchymal stem cells (MSCs) have caused a lot of excitement among stem cell researchers owing to their multipotential differentiation capacity and apparent lack of immunogenecity. In addition, MSCs have been found to enhance tissue regeneration and angiogenesis upon transplantation, although the mechanism remains poorly defined. MSCs derived from embryonic stem cells (hESCderived MSCs) would provide an appealing source for therapeutic material if they were to function like adult MSCs. The aim of our study was to characterize the in vivo potential of hESC-derived MSCs, and provide insight into how MSCs enhance regeneration and angiogenesis. The study was executed utilizing rat hind limb ischemia model, wherein the distribution of hESC-derived MSCs was studied by fluorescence microcopy and bioluminescent imaging. We found 70% of the transplanted cells to be lost within 6 hours of local delivery, while after three days only a few cells remained, scattered throughout the tissue. However, no cells were detected in the lungs or other tissues. Interestingly, analysis of capillary density by immunohistochemistry showed significantly enhanced angiogenesis three days after transplantation. Quantitative RT-PCR suggests this effect to be due to increased endogeneous VEGF-D expression whereas VEGF-A appears to play a lesser role. While the transplanted cells did express low level of VEGF-A 6 hours after transplantation, it was overwhelmed by recipients own VEGF-A expression. Furthermore, no graft-derived VEGF expression could be detected three days post-transplantation. The study shows hESCderived MSCs to be able to enhance angiogenesis by stimulating endogeneous VEGF-D expression although only a fraction of transplanted cells remain in the area of injury after 24 hours. The current study shows hESC-derived MSCs to harbor similar functional properties as compared to adult MSCs, thus highlighting the great promise hESC-derived MSCs hold as a potential source of allogeneic material for therapeutic applications. S102
271. Adverse Effects of Mesenchymal Stem Cell Transplantation in a Denuded Rabbit Carotid Artery
Ciara O’Shea,1 Sean O. Hynes,1 Georgina Shaw,1 Barbara A. Coen,2 Mary Murphy,1 Ailish C. Hynes,2 Frank Barry,1 Timothy O’Brien.1 1 Regenerative Medicine Institute, National University of Ireland, Galway, Ireland; 2Department of Physiology, National University of Ireland, Galway, Ireland. Endothelial dysfunction is an important factor in cardiovascular pathology. Endothelial progenitor cells have been shown to improve endothelial repair following vessel injury. It has been suggested that pluripotent mesenchymal stem cells(MSCs) may also differentiate to endothelial cells and thereby potentially contribute to endothelial repair. More rapid re-endothelialisation could be associated with a better outcome following percutaneous angioplasty procedures. The present investigation examined the delivery of MSCs to a denuded vessel in vivo. The right carotid arteries of male New Zealand white rabbits were denuded by passing an uninflated 3 French Fogarty balloon catheter along the artery three times. 1x105 MSCs in a bolus of 200 µl was then delivered intraluminally and allowed to dwell for twenty minutes. MSC engraftment was assessed using PKH26 labelling and transduction with adenoviral reporter genes. Vessels were examined at two weeks for levels of endothelialisation, as well as for neointimal hyperplasia and vasomotor function. Labelled MSCs and those transduced with adenoviral lacZ as a reporter gene were noted to be engrafted in the vessel wall following local arterial delivery. Endothelialisation was not improved following MSC delivery at two weeks. Furthermore, an increase in adverse events occurred in MSC-treated vessels with an increased number of occlusions due to thrombosis(75%) and neointimal hyperplasia (25%). Histological sections revealed an increased neointima in MSC-treated vessels. Vasomotor assessment revealed a significant level of endothelium-dependent impairment in MSC-treated vessels. Our results suggest that naïve MSC delivery to injured vasculature as a model for cell delivery during endarterectomy procedures may have risks of potential adverse events including thrombosis. Such risks are important and need to be assessed prior to the use of MSCs in any therapeutic approach involving luminal dwell.
272. Is Intravascular Transplantation of Mesenchymal Stem Cells Safe?
Murat Ugurlucan,1 Dario Furlani,1 Erik Pittermann,1 Lee Lee Ong,1 Can Yerebakan,1 Ralf Gaebel,1 Christian Klopsch,1 Wei Wei Wang,1 Wenzhong Li,1 Nan Ma,1 Gustav Steinhoff.1 1 Cardiac Surgery, University of Rostock, Rostock, Germany. Introduction: Cell based therapies have been widely used for the regeneration of the damaged organs. Stem cells can be applied to these tissues by various methods. Intravascular administration of cells is one of the commonly executed routes for stem cell transplantation. We aimed to investigate the consequences of mesenchymal stem cells (MSC) when they are administered through an intravascular route. Materials and Methods: Human MSC were obtained from adipose tissue, fluorescent labeled and prepared in PBS with two different concentrations as 1x106 cells in 200µl or 1ml for intravital microscopy. Twenty SCID mouse were divided into 3 groups as Group 1 (Sham, n: 4, PBS injection), Group 2 (n: 8, 1x106 cells in 200µl PBS injection group) and Group 3 (n: 8, 1x106 cells in 1ml PBS injection). Intravascular cell injection was performed through an arterial catheter inserted retrograde into the abdominal aorta via the left femoral artery. The right cremaster muscle was dissected and prepared for intravital fluorescence microscopy. A total of five consecutive injections were planned. Following the procedure animals were sacrificed and cremaster muscle, aorta and iliac arteries were Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Mesenchymal, Muscle and Bone Stem Cells harvested for histopathologic analysis. Results: Injection of PBS into the aorta did not lead to any effect nor changed the flow parameters in the cremaster muscle. In Group 2, following injection of 100µl of the cell/PBS solution, the flow in both arteries and veins stopped in all animals. In Group 3, following injection of 100µl of the cell/PBS solution, blood flow stopped both in arteries and veins in four animals after the first injection and in one animal after the second injection; and did not re-start. In two animals, blood flow stopped after the first injection but re-started spontaneously after 2-3 minutes but stopped following the second injection and did not re-start. In one animal the blood flow stopped after first injection but restarted in a slower manner in 2 minutes and stopped again spontaneously. Passage of cells from arterioles, occlusion of distal capillaries and no return back from the venules were observed in Group 2 and 3 under intravital microscopy. Histopathologic examination of tissues showed occlusion of distal capillaries in Group 2 and 3 as well as cellular blockage in common iliac, external iliac and inferior epigastric arteries. Conclusion: Our in vivo experiments with intra-arterial injection of human MSC indicate targeted injection of these cells into circulation may lead to occlusion in the distal vasculature due to their size; hence, may be hazardous.
273. Efficient Differentiation into Osteoblastic Lineage from Both Mouse Embryoid Bodies and Bone Marrow Stromal Cells by Adenovirus Vectors
Katsuhisa Tashiro,1,2 Kenji Kawabata,1 Asami Ino,1 Haruna Sakurai,1,2 Fuminori Sakurai,1 Hiroyuki Mizuguchi.1,2 1 Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan; 2Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan. Embryonic stem (ES) cells and bone marrow stromal cells (BMSCs) are expected to be a source of the cell and the tissue for regenerative medicine because they can differentiate into multiple cell types. To differentiate into pure target cells from ES cells and BMSCs, it is necessary to develop the efficient transduction methods into them. In the present study, we optimized adenovirus (Ad) vector-mediated transduction into embryoid bodies (EBs), which are often formed to differentiate into functional cells from ES cells, and BMSCs by using variety types of Ad vectors. We prepared β-galactosidase (LacZ)-expressing Ad vectors under the control of four different promoters, and also constructed fiber-modified Ad vectors, AdRGD and AdK7, which contain Arg-Gly-Asp (RGD) peptide in the HI loop of the fiber knob and polylysin (KKKKKKK: K7) in the C-terminal of the fiber knob, respectively. When five days-cultured EBs and primary murine BMSCs were transduced with various types of Ad vectors, which had different promoters, the CA (the cytomegalovirus enhancer/β-actin promoter with β-actin intron) promoter exhibited the highest transduction efficiency in both EBs and BMSCs. When BMSCs were transduced with fiber-modified Ad vectors to increase transgene expression, transduction efficiency was markedly improved by using AdK7 vector. On the other hand, high transduction efficiency was obtained in EBs even using unmodified Ad vector. Next, we examined whether functional gene transduction into EBs and BMSCs by means of optimized Ad vector could promote the differentiation efficiency. As a model for cellular differentiation, EBs and BMSCs were differentiated into osteoblasts by optimized Ad vector-mediated transduction of Runx2 gene, which is an essential transcription factor for controlling osteoblast differentiation. The EBs and BMSCs transduced with Runx2-expressing Ad vector showed higher alkaline phophatase activity and calcium accumulation than the cells transduced with LacZ-expressing Ad vector (control vector). Furthermore, the expression of marker genes of osteoblast differentiation, such as osterix and osteocalcin, was significantly up-regulated in Runx2-transduced cells. These results indicate that efficient Runx2 gene transfer into EBs and BMSCs by using optimized Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Ad vector can be effective to promote the osteogenic potential, and that this transduction method can be a powerful tool for therapeutic applications based on stem cells.
274. Stable Transduction of rAAV-GFP in Cultured Dividing Myo-Endo Cells
Michael Y. Mi,1 Ying Tang,1 Bo Zheng,1 Johnny Huard,1 Bing Wang.1 1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA. Myogenic endothelial (myo-endo) cells, isolated from adult human skeletal muscle, posses the capacity for self-renewal and the multilineage potential to regenerate tissues of the musculoskeletal system such as myofibers in the injured skeletal muscle of SCID mice (Bo Zheng et al. 2007, Nature Biotechnology). Viral vectors are commonly used in ex vivo approaches for stem cell therapy modalities. The potential for insertional mutagenesis by random DNA integration limits the application of retro-and lenti-viral vectors for stem cell therapy in vivo. On the other hand, rAAV vectors that have all of their viral replication (Rep) genes deleted exist as extrachromosomal episomes rather than as DNA integrated genomes, which ensures that the rAAV transduction remains innocuous. In order to develop rAAVbased, myo-endo progenitor cell gene therapy to treat musculoskeletal degenerative diseases, we investigated the efficiency of transduction of an AAV2 vector in myo-endo cells cultured over 10 passages. In this study, we infected myo-endo cells (Passage 3) with AAV2GFP and continuously split the cells (1:3) to keep them at a cellular confluency of approximately 50%. The results demonstrated that over 50% of cells remained GFP positive through each passage examined by fluorescence microscopy and flow cytometry, which is similar to the transduction efficiency observed by the Lenti-GFP viral vector. These results indicate that rAAV mediated transduction is not affected by the splitting of the cell cultures. It is evidenced by the current study that the rAAV genome, in its episomal form, can be evenly distributed into the daughter cell nuclei after mitotic division, with no appreciable loss of the episomic transgene. This observation will help us to further our understanding of AAV-based ex vivo stem cell therapy and allow us to better engineer these systems to treat musculoskeletal degenerative diseases.
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270. Angiogenesis in Response to Local Delivery of Human Embryonic Stem Cell Derived Mesenchymal Stem Cells into Ischemic Rat Hind Limb
Juha P. Laurila,1,2 Lilja Laatikainen,1 Peiman Hematti,3 Mikko O. Laukkanen.1 1 Medicity Research Laboratory, University of Turku, Turku, Finland; 2Turku Graduate School of Biomedical Sciences, University of Turku, Turku, Finland; 3Department of Medicine, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI. Mesenchymal stem cells (MSCs) have caused a lot of excitement among stem cell researchers owing to their multipotential differentiation capacity and apparent lack of immunogenecity. In addition, MSCs have been found to enhance tissue regeneration and angiogenesis upon transplantation, although the mechanism remains poorly defined. MSCs derived from embryonic stem cells (hESCderived MSCs) would provide an appealing source for therapeutic material if they were to function like adult MSCs. The aim of our study was to characterize the in vivo potential of hESC-derived MSCs, and provide insight into how MSCs enhance regeneration and angiogenesis. The study was executed utilizing rat hind limb ischemia model, wherein the distribution of hESC-derived MSCs was studied by fluorescence microcopy and bioluminescent imaging. We found 70% of the transplanted cells to be lost within 6 hours of local delivery, while after three days only a few cells remained, scattered throughout the tissue. However, no cells were detected in the lungs or other tissues. Interestingly, analysis of capillary density by immunohistochemistry showed significantly enhanced angiogenesis three days after transplantation. Quantitative RT-PCR suggests this effect to be due to increased endogeneous VEGF-D expression whereas VEGF-A appears to play a lesser role. While the transplanted cells did express low level of VEGF-A 6 hours after transplantation, it was overwhelmed by recipients own VEGF-A expression. Furthermore, no graft-derived VEGF expression could be detected three days post-transplantation. The study shows hESCderived MSCs to be able to enhance angiogenesis by stimulating endogeneous VEGF-D expression although only a fraction of transplanted cells remain in the area of injury after 24 hours. The current study shows hESC-derived MSCs to harbor similar functional properties as compared to adult MSCs, thus highlighting the great promise hESC-derived MSCs hold as a potential source of allogeneic material for therapeutic applications. S102
271. Adverse Effects of Mesenchymal Stem Cell Transplantation in a Denuded Rabbit Carotid Artery
Ciara O’Shea,1 Sean O. Hynes,1 Georgina Shaw,1 Barbara A. Coen,2 Mary Murphy,1 Ailish C. Hynes,2 Frank Barry,1 Timothy O’Brien.1 1 Regenerative Medicine Institute, National University of Ireland, Galway, Ireland; 2Department of Physiology, National University of Ireland, Galway, Ireland. Endothelial dysfunction is an important factor in cardiovascular pathology. Endothelial progenitor cells have been shown to improve endothelial repair following vessel injury. It has been suggested that pluripotent mesenchymal stem cells(MSCs) may also differentiate to endothelial cells and thereby potentially contribute to endothelial repair. More rapid re-endothelialisation could be associated with a better outcome following percutaneous angioplasty procedures. The present investigation examined the delivery of MSCs to a denuded vessel in vivo. The right carotid arteries of male New Zealand white rabbits were denuded by passing an uninflated 3 French Fogarty balloon catheter along the artery three times. 1x105 MSCs in a bolus of 200 µl was then delivered intraluminally and allowed to dwell for twenty minutes. MSC engraftment was assessed using PKH26 labelling and transduction with adenoviral reporter genes. Vessels were examined at two weeks for levels of endothelialisation, as well as for neointimal hyperplasia and vasomotor function. Labelled MSCs and those transduced with adenoviral lacZ as a reporter gene were noted to be engrafted in the vessel wall following local arterial delivery. Endothelialisation was not improved following MSC delivery at two weeks. Furthermore, an increase in adverse events occurred in MSC-treated vessels with an increased number of occlusions due to thrombosis(75%) and neointimal hyperplasia (25%). Histological sections revealed an increased neointima in MSC-treated vessels. Vasomotor assessment revealed a significant level of endothelium-dependent impairment in MSC-treated vessels. Our results suggest that naïve MSC delivery to injured vasculature as a model for cell delivery during endarterectomy procedures may have risks of potential adverse events including thrombosis. Such risks are important and need to be assessed prior to the use of MSCs in any therapeutic approach involving luminal dwell.
272. Is Intravascular Transplantation of Mesenchymal Stem Cells Safe?
Murat Ugurlucan,1 Dario Furlani,1 Erik Pittermann,1 Lee Lee Ong,1 Can Yerebakan,1 Ralf Gaebel,1 Christian Klopsch,1 Wei Wei Wang,1 Wenzhong Li,1 Nan Ma,1 Gustav Steinhoff.1 1 Cardiac Surgery, University of Rostock, Rostock, Germany. Introduction: Cell based therapies have been widely used for the regeneration of the damaged organs. Stem cells can be applied to these tissues by various methods. Intravascular administration of cells is one of the commonly executed routes for stem cell transplantation. We aimed to investigate the consequences of mesenchymal stem cells (MSC) when they are administered through an intravascular route. Materials and Methods: Human MSC were obtained from adipose tissue, fluorescent labeled and prepared in PBS with two different concentrations as 1x106 cells in 200µl or 1ml for intravital microscopy. Twenty SCID mouse were divided into 3 groups as Group 1 (Sham, n: 4, PBS injection), Group 2 (n: 8, 1x106 cells in 200µl PBS injection group) and Group 3 (n: 8, 1x106 cells in 1ml PBS injection). Intravascular cell injection was performed through an arterial catheter inserted retrograde into the abdominal aorta via the left femoral artery. The right cremaster muscle was dissected and prepared for intravital fluorescence microscopy. A total of five consecutive injections were planned. Following the procedure animals were sacrificed and cremaster muscle, aorta and iliac arteries were Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Mesenchymal, Muscle and Bone Stem Cells harvested for histopathologic analysis. Results: Injection of PBS into the aorta did not lead to any effect nor changed the flow parameters in the cremaster muscle. In Group 2, following injection of 100µl of the cell/PBS solution, the flow in both arteries and veins stopped in all animals. In Group 3, following injection of 100µl of the cell/PBS solution, blood flow stopped both in arteries and veins in four animals after the first injection and in one animal after the second injection; and did not re-start. In two animals, blood flow stopped after the first injection but re-started spontaneously after 2-3 minutes but stopped following the second injection and did not re-start. In one animal the blood flow stopped after first injection but restarted in a slower manner in 2 minutes and stopped again spontaneously. Passage of cells from arterioles, occlusion of distal capillaries and no return back from the venules were observed in Group 2 and 3 under intravital microscopy. Histopathologic examination of tissues showed occlusion of distal capillaries in Group 2 and 3 as well as cellular blockage in common iliac, external iliac and inferior epigastric arteries. Conclusion: Our in vivo experiments with intra-arterial injection of human MSC indicate targeted injection of these cells into circulation may lead to occlusion in the distal vasculature due to their size; hence, may be hazardous.
273. Efficient Differentiation into Osteoblastic Lineage from Both Mouse Embryoid Bodies and Bone Marrow Stromal Cells by Adenovirus Vectors
Katsuhisa Tashiro,1,2 Kenji Kawabata,1 Asami Ino,1 Haruna Sakurai,1,2 Fuminori Sakurai,1 Hiroyuki Mizuguchi.1,2 1 Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan; 2Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan. Embryonic stem (ES) cells and bone marrow stromal cells (BMSCs) are expected to be a source of the cell and the tissue for regenerative medicine because they can differentiate into multiple cell types. To differentiate into pure target cells from ES cells and BMSCs, it is necessary to develop the efficient transduction methods into them. In the present study, we optimized adenovirus (Ad) vector-mediated transduction into embryoid bodies (EBs), which are often formed to differentiate into functional cells from ES cells, and BMSCs by using variety types of Ad vectors. We prepared β-galactosidase (LacZ)-expressing Ad vectors under the control of four different promoters, and also constructed fiber-modified Ad vectors, AdRGD and AdK7, which contain Arg-Gly-Asp (RGD) peptide in the HI loop of the fiber knob and polylysin (KKKKKKK: K7) in the C-terminal of the fiber knob, respectively. When five days-cultured EBs and primary murine BMSCs were transduced with various types of Ad vectors, which had different promoters, the CA (the cytomegalovirus enhancer/β-actin promoter with β-actin intron) promoter exhibited the highest transduction efficiency in both EBs and BMSCs. When BMSCs were transduced with fiber-modified Ad vectors to increase transgene expression, transduction efficiency was markedly improved by using AdK7 vector. On the other hand, high transduction efficiency was obtained in EBs even using unmodified Ad vector. Next, we examined whether functional gene transduction into EBs and BMSCs by means of optimized Ad vector could promote the differentiation efficiency. As a model for cellular differentiation, EBs and BMSCs were differentiated into osteoblasts by optimized Ad vector-mediated transduction of Runx2 gene, which is an essential transcription factor for controlling osteoblast differentiation. The EBs and BMSCs transduced with Runx2-expressing Ad vector showed higher alkaline phophatase activity and calcium accumulation than the cells transduced with LacZ-expressing Ad vector (control vector). Furthermore, the expression of marker genes of osteoblast differentiation, such as osterix and osteocalcin, was significantly up-regulated in Runx2-transduced cells. These results indicate that efficient Runx2 gene transfer into EBs and BMSCs by using optimized Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Ad vector can be effective to promote the osteogenic potential, and that this transduction method can be a powerful tool for therapeutic applications based on stem cells.
274. Stable Transduction of rAAV-GFP in Cultured Dividing Myo-Endo Cells
Michael Y. Mi,1 Ying Tang,1 Bo Zheng,1 Johnny Huard,1 Bing Wang.1 1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA. Myogenic endothelial (myo-endo) cells, isolated from adult human skeletal muscle, posses the capacity for self-renewal and the multilineage potential to regenerate tissues of the musculoskeletal system such as myofibers in the injured skeletal muscle of SCID mice (Bo Zheng et al. 2007, Nature Biotechnology). Viral vectors are commonly used in ex vivo approaches for stem cell therapy modalities. The potential for insertional mutagenesis by random DNA integration limits the application of retro-and lenti-viral vectors for stem cell therapy in vivo. On the other hand, rAAV vectors that have all of their viral replication (Rep) genes deleted exist as extrachromosomal episomes rather than as DNA integrated genomes, which ensures that the rAAV transduction remains innocuous. In order to develop rAAVbased, myo-endo progenitor cell gene therapy to treat musculoskeletal degenerative diseases, we investigated the efficiency of transduction of an AAV2 vector in myo-endo cells cultured over 10 passages. In this study, we infected myo-endo cells (Passage 3) with AAV2GFP and continuously split the cells (1:3) to keep them at a cellular confluency of approximately 50%. The results demonstrated that over 50% of cells remained GFP positive through each passage examined by fluorescence microscopy and flow cytometry, which is similar to the transduction efficiency observed by the Lenti-GFP viral vector. These results indicate that rAAV mediated transduction is not affected by the splitting of the cell cultures. It is evidenced by the current study that the rAAV genome, in its episomal form, can be evenly distributed into the daughter cell nuclei after mitotic division, with no appreciable loss of the episomic transgene. This observation will help us to further our understanding of AAV-based ex vivo stem cell therapy and allow us to better engineer these systems to treat musculoskeletal degenerative diseases.
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