- Email: [email protected]
738. Cyclooxygenase-2 Plays a Critical Role in BMP4GFP Transduced Muscle Derived Stem Cell Mediated Bone Healing in the Critical Sized Calvarial Defect Model
MESENCHYMAL STEM CELLS which targets a 21 nt sequence in nNOS mRNA coded by exon 28, in association with neomycin, an apoptotic agent. The effects on cell viability were determined by MTT (3-(4, 5-dimetiltiazol2yl)-2, 5-difenil bromate of tetrazoline) assay. For that, glioma cells U-251MG in culture were previously injured with neomycin (Sigma, 300 μg/ml) for 24 h and then treated with e28_hnNOS for 4 hour. Non-injured and scrambled control groups were also included. The control siRNA was the commercial scramble All-Stars™ (Qiagen™) and the transfection occurred with cationic liposomes. The use of siRNA e28_hnNOS in association with neomycin reduced the cell viability to 96.36% while the scramble sequence increased it to 103,69% (figure 1). In conclusion, our study revealed the potential use of RNAi in combination with antibiotic therapy to reduce the glioma cells viability. The protocol based on nNOS siRNAs in combination with the apoptotic agent neomycin may point a promising future for cancer therapeutics. Further studies using animal models of cancer may confirm whether the effects found in our study will occur “in vivo” and may reveal potential side effects of this protocol.
Figure 1. Effects of siRNA e28_hnNOS in combination with neomycin on glioma cell viability “in vitro” at 4 h post-transfection, determined by MTT assay. Dotted lines represent 100% viability from non-injured group (n=7).
Mesenchymal Stem Cells 737. Defective Proliferation and Myogenic Differentiation Potential of Muscle-Derived Stem Cells Isolated from Dystrophin/Utrophin Double Knock-Out Mice
Aiping Lu,1 Ying Tang,1 Minakshi Poddar,1 Bing Wang,1 Johnny Huard.1 1 Orthopaedic Surgery, University of Pittsburgh, PA. It is critical to find a successful therapy that will improve the histopathology of the muscles of Duchenne muscular dystrophy (DMD) patients. Most of the current treatments have been centered on the restoration of dystrophin within the dystrophic muscle; however, these technologies have faced serious limitations, including immunological reactions, which have hindered their success. Despite the lack of dystrophin at birth, the initiation of any signs of muscle weakness does not occur until 6-10 years of age. Hence, it has been posited that muscle weakness corresponds to the exhaustion of muscle progenitor cells which leads to the accumulation of fibrosis and fatty deposits that further exacerbates the muscle wasting process in DMD patients. The mouse model of DMD, known as the dystrophin/ utrophin double Knock-Out (dKO, dys-/-utro-/-) is a severe and more S284
reliable DMD mouse model than the mdx, making it a more relevant model to human DMD patients in terms of disabling and lethal phenotypes. In this study we isolated muscle derived stem cells (MDSCs) from 6 week old dKO mice and dystrophin deficient mice but heterozygote for utrophin (dys-/-utro+/-, dKO-hetero), as previously described via a modified preplate technique. The phenotype of dKOhetero mice is similar to mdx, so we compared MDSCs from these two animal models. The proliferation behavior of the MDSCs isolated from dKO and dKO-hetero muscle was examined by using live Cell Imaging system (LCI). The myogenic differentiation capacity of the MDSCs was assessed by staining of fast myosin heavy chain (MyHCf). We found that MDSCs isolated from the skeletal muscle of 6 week old dKO mice have a reduced ability to proliferate and differentiate compared to MDSCs isolated from dKO-hetero mice. Cellular resistance to hydrogen peroxide-induced oxidative stress was also examined and we found that dKO MDSCs displayed a reduced resistance to oxidative stress, compared to the dKO-hetero MDSCs. In addition, the single muscle fibers were isolated from 6 weeks old dKO mice and 9 week old WT control mice. The results showed that there are more cell nuclei in WT muscle fibers compared to dKO muscle fibers, five days post culturing, the WT muscle fibers were able to release myogenic progenitor cells forming new myotubes, in contrasts to that observed with dKO muscle fibers. These results support both a reduction in the number and myogenic potential of the MDSCs derived from dKO mice when compared to WT MDSCs. Together, these results suggested that the MDSCs from dKO mice are defective in their proliferation and myogenic differentiation capacities which may contributes to the histopathology associated with DMD. These observations suggest that blocking the exhaustion of muscle progenitor cells and stem cell-mediated therapy may represent a potential strategy to prevent or delay the onset of debilitating DMD disease-related changes.
738. Cyclooxygenase-2 Plays a Critical Role in BMP4GFP Transduced Muscle Derived Stem Cell Mediated Bone Healing in the Critical Sized Calvarial Defect Model
Xueqin Gao,1 Arvydas Usas,1 Aiping Lu,1 Ying Tang,1 Minakshi Podar,1 Mathieu Huard,1 James H. Cummins,1 Johnny Huard.1 1 Orthopaedic Surgery, University of Pittsburgh, PA. Introduction: Muscle derived stem cells (MDSC) have been shown to contribute and interact with the host cells to regenerate bone. The mechanism behind the interaction is unknown. This study aims to investigate if cyclooxygenase-2 (COX-2) plays a role in the donor and host interaction during MDSC mediated bone healing in a mouse critical sized calvarial defect model. Methods: (1) COX-2 expression during MDSC mediated bone healing. MDSC isolated from C57BL/10J mice were transduced with retro-BMP4GFP and transplanted (5×105) into a 5mm critical sized calvarial defect created in C57BL/6J mice using fibrin sealant(FS) as scaffold. Control mice received PBS and FS. Animals were sacrificed at 3, 7, 14, 21 post implantation(PI) and COX-2 expression was detected by immunofluorescence and colocalized with GFP(donor). (2) The role of host COX-2 in the MDSC mediated bone formation. BMP4GFP transduced MDSC were transplanted into COX-2(-/-) and COX2(+/+) animals (n=4) using the skull defect model as stated above. The bone formation was evaluated by micro CT biweekly for 8 weeks. Immunohistochemistry of GFP was performed to evaluate donor contribution. (3) The role of donor COX-2 in MDSC mediated bone healing[/bold]. MDSC isolated from 5 week old COX-2(+/+) and COX-2(-/-) mice were transduced with retro-BMP4GFP and implanted into a calvarial defect created in CD-1 nude mice (n=4) and the bone formation was evaluated by microCT biweekly for 6 weeks. Results: (1) COX-2 is expressed dynamically during MDSC mediated bone regeneration. At 3 days PI, COX-2 was expressed in Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
MESENCHYMAL STEM CELLS host cells in both groups and in the donor cells in the MDSC transplant group. At 7 days, COX-2 was strongly expressed in the newly formed chondrocytic nodule in MDSC group and colocalized with GFP, but only in few cells in control group. At 2 weeks, COX-2 expression was found in chondrocytes but not in the osteoblasts in MDSC group and no expression was found in the control group. At 3 weeks, when more mature bone formed, COX-2 expression was significantly reduced. (2) COX-2 deficient mice regenerated less bone. BMP4GFP transduced MDSC regenerated significantly less bone in COX-2(-/-) host mice than in COX-2(+/+) mice as revealed by microCT(P<0.01). However, the defect was not completely healed even in the COX2(+/+) recipient mice. Few donor cells (GFP) were present in COX2(+/+) group, and almost no donor cells were found in one COX-2(-/-) group. We found many lymphocytes in the regenerated tissues in the COX-2(+/+) mice indicating immune rejection while the COX-2(-/-) mice formed granulation tissue over the defect area. (3) COX-2(-/-) MDSC regenerate markedly less bone in CD-1 nude mice. BMP4GFP transduced COX-2(-/-) MDSC formed significantly less bone in CD-1 mice than COX-2(+/+) MDSC at 2,4 and 6 weeks PI. Conclusions: Our findings indicate COX-2 is a key factor for the MDSC mediated bone regeneration. Both donor and host the COX-2 are important for MDSC mediated bone healing. COX-2 inhibitors should be used with caution when performing stem cell transplantation. We will further investigate the mechanism.
739. Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate Wound Healing in Skin
David L. Mack,1 Aleksander Skardal,1 James Yoo,1 Shay Soker.1 Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC.
1
Mesenchymal stem cells (MSCs) have shown therapeutic potential for repair and regeneration of tissues damaged by injury or disease. Amniotic fluid-derived stem (AFS) cells possess properties found also in MSCs such as multipotent differentiation, immunomodulatory activity and the lack of significant immunogenicity. MSC treatment of acute and chronic wounds result in accelerated wound closure, increased epithelialization, granulation tissue and angiogenesis. Given that AFS cells can be obtained less invasively than MSCs and show greater proliferative capacity in culture, we investigated whether they could augment wound healing in a similar fashion as MSCs. A full thickness skin wound was surgically created on the dorsum of nu/nu mice. A bioprinter was used to deposit 2 layers of a fibrin-collagen gel, containing either 5 x 106 AFS cells, MSCs, or no cells over the surgical site. At day 7 and 14, AFS and MSC-driven wound closure was significantly greater compared to that of the gel-only treatment.
Re-epithelialization of the wound area was also found to be significantly greater in AFS and MSC groups at day 7 and 14 compared to gel-only group. H&E staining of harvested skin at day 7 revealed a 3-fold increase in microvessel density (MVD) in the Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
AFS and MSC-regenerated skin compared to gel only. At day 14, AFS-treated wounds had increased MVD compared to both MSC and gel-only groups. Furthermore, more mature vessels were observed in the AFS-treated group. Histology revealed that near the center of the wound area, both AFS and MSC-treated animals had robust layers of keratinocytes present that had formed organized epidermis. Finally, GFP-labeled AFS cells were employed to determine whether the printed cells were permanently integrating or were transient in the regenerating tissue. Immunofluorescence showed that AFS cell numbers decreased over time with no visible cells present at day 14.
Staining for von Willebrand factor revealed that AFS cells did not co-localize with developing blood vessels, despite the increased neovascular activity. These observations suggest that the increased wound closure rates and angiogenesis may be due to the localized, sustained secretion of trophic factors, rather than direct cell-cell interactions. AFS cells perform as well as MSCs in terms of wound closure. However, AFS cells seem to induce greater neovascularization of the regenerating skin. Therefore, bioprinting AFS cells in a hydrogel carrier might address the clinical need for more effective treatments for large-scale wounds and burns.
740. Prevention of Radiation-Induced Lung Injury by Administration of Gene-Modified Mesenchymal Stem Cells
Jiaxin Xue,1 Xin Li,2 You Lu,1 Lin Zhou,1 Yongsheng Wang,1 Li Jia,1 Xianming Mo.3 1 Department of Thoracic Oncology, West China Hospital, Sichuan University, Chengdu, China; 2Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University, Xi’an, China; 3 Laboratory of Stem Cell Biology, West China Hospital, Sichuan University, Chengdu, China. Radiation-induced lung injury (RILI), a common and serious side effect of radiotherapy for thoracic cancers, can limit therapy itself and may even cause mortality. The aim of this study was to investigate the prevention and repairing efficacy of RILI by adenovirus-mediated soluble transforming growth factor-beta type II receptor gene (Ad-sTβR) modified mesenchymal stem cells (Ad-sTβR-MSCs). Mesenchymal stem cells (MSCs) from male mice were isolated, identified, and transduced with Ad-sTβR. It was confirmed that MSCs could specifically home into radiation injured lung and the engrafted MSCs levels had a 20-fold increase as compared with non-irradiated lung. Transduction of Ad-sTβR did not alter the migration capacity of MSCs to injured lung in vivo and ex vivo. Migration assay and blocking experiments revealed that SDF-1α/CXCR4 axis played an important role in this process, suggesting that the increased levels of MSCs in lung stemmed from the radiation-induced homing of MSCs rather than the physical entrapment of circulating MSCs. Administration of Ad-sTβR-MSCs exerted a significant reduction for lung injury, as demonstrated not only by survival and histopathology, but also by assay of malondialdehyde (MDA), hydroxyproline, plasma cytokines (IL-1β, TNF-α, IL-6, IL-10 and active TGF-β1), and the expression of CTGF and α-SMA. S285
Figure 1. Effects of siRNA e28_hnNOS in combination with neomycin on glioma cell viability “in vitro” at 4 h post-transfection, determined by MTT assay. Dotted lines represent 100% viability from non-injured group (n=7).
Mesenchymal Stem Cells 737. Defective Proliferation and Myogenic Differentiation Potential of Muscle-Derived Stem Cells Isolated from Dystrophin/Utrophin Double Knock-Out Mice
Aiping Lu,1 Ying Tang,1 Minakshi Poddar,1 Bing Wang,1 Johnny Huard.1 1 Orthopaedic Surgery, University of Pittsburgh, PA. It is critical to find a successful therapy that will improve the histopathology of the muscles of Duchenne muscular dystrophy (DMD) patients. Most of the current treatments have been centered on the restoration of dystrophin within the dystrophic muscle; however, these technologies have faced serious limitations, including immunological reactions, which have hindered their success. Despite the lack of dystrophin at birth, the initiation of any signs of muscle weakness does not occur until 6-10 years of age. Hence, it has been posited that muscle weakness corresponds to the exhaustion of muscle progenitor cells which leads to the accumulation of fibrosis and fatty deposits that further exacerbates the muscle wasting process in DMD patients. The mouse model of DMD, known as the dystrophin/ utrophin double Knock-Out (dKO, dys-/-utro-/-) is a severe and more S284
reliable DMD mouse model than the mdx, making it a more relevant model to human DMD patients in terms of disabling and lethal phenotypes. In this study we isolated muscle derived stem cells (MDSCs) from 6 week old dKO mice and dystrophin deficient mice but heterozygote for utrophin (dys-/-utro+/-, dKO-hetero), as previously described via a modified preplate technique. The phenotype of dKOhetero mice is similar to mdx, so we compared MDSCs from these two animal models. The proliferation behavior of the MDSCs isolated from dKO and dKO-hetero muscle was examined by using live Cell Imaging system (LCI). The myogenic differentiation capacity of the MDSCs was assessed by staining of fast myosin heavy chain (MyHCf). We found that MDSCs isolated from the skeletal muscle of 6 week old dKO mice have a reduced ability to proliferate and differentiate compared to MDSCs isolated from dKO-hetero mice. Cellular resistance to hydrogen peroxide-induced oxidative stress was also examined and we found that dKO MDSCs displayed a reduced resistance to oxidative stress, compared to the dKO-hetero MDSCs. In addition, the single muscle fibers were isolated from 6 weeks old dKO mice and 9 week old WT control mice. The results showed that there are more cell nuclei in WT muscle fibers compared to dKO muscle fibers, five days post culturing, the WT muscle fibers were able to release myogenic progenitor cells forming new myotubes, in contrasts to that observed with dKO muscle fibers. These results support both a reduction in the number and myogenic potential of the MDSCs derived from dKO mice when compared to WT MDSCs. Together, these results suggested that the MDSCs from dKO mice are defective in their proliferation and myogenic differentiation capacities which may contributes to the histopathology associated with DMD. These observations suggest that blocking the exhaustion of muscle progenitor cells and stem cell-mediated therapy may represent a potential strategy to prevent or delay the onset of debilitating DMD disease-related changes.
738. Cyclooxygenase-2 Plays a Critical Role in BMP4GFP Transduced Muscle Derived Stem Cell Mediated Bone Healing in the Critical Sized Calvarial Defect Model
Xueqin Gao,1 Arvydas Usas,1 Aiping Lu,1 Ying Tang,1 Minakshi Podar,1 Mathieu Huard,1 James H. Cummins,1 Johnny Huard.1 1 Orthopaedic Surgery, University of Pittsburgh, PA. Introduction: Muscle derived stem cells (MDSC) have been shown to contribute and interact with the host cells to regenerate bone. The mechanism behind the interaction is unknown. This study aims to investigate if cyclooxygenase-2 (COX-2) plays a role in the donor and host interaction during MDSC mediated bone healing in a mouse critical sized calvarial defect model. Methods: (1) COX-2 expression during MDSC mediated bone healing. MDSC isolated from C57BL/10J mice were transduced with retro-BMP4GFP and transplanted (5×105) into a 5mm critical sized calvarial defect created in C57BL/6J mice using fibrin sealant(FS) as scaffold. Control mice received PBS and FS. Animals were sacrificed at 3, 7, 14, 21 post implantation(PI) and COX-2 expression was detected by immunofluorescence and colocalized with GFP(donor). (2) The role of host COX-2 in the MDSC mediated bone formation. BMP4GFP transduced MDSC were transplanted into COX-2(-/-) and COX2(+/+) animals (n=4) using the skull defect model as stated above. The bone formation was evaluated by micro CT biweekly for 8 weeks. Immunohistochemistry of GFP was performed to evaluate donor contribution. (3) The role of donor COX-2 in MDSC mediated bone healing[/bold]. MDSC isolated from 5 week old COX-2(+/+) and COX-2(-/-) mice were transduced with retro-BMP4GFP and implanted into a calvarial defect created in CD-1 nude mice (n=4) and the bone formation was evaluated by microCT biweekly for 6 weeks. Results: (1) COX-2 is expressed dynamically during MDSC mediated bone regeneration. At 3 days PI, COX-2 was expressed in Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
MESENCHYMAL STEM CELLS host cells in both groups and in the donor cells in the MDSC transplant group. At 7 days, COX-2 was strongly expressed in the newly formed chondrocytic nodule in MDSC group and colocalized with GFP, but only in few cells in control group. At 2 weeks, COX-2 expression was found in chondrocytes but not in the osteoblasts in MDSC group and no expression was found in the control group. At 3 weeks, when more mature bone formed, COX-2 expression was significantly reduced. (2) COX-2 deficient mice regenerated less bone. BMP4GFP transduced MDSC regenerated significantly less bone in COX-2(-/-) host mice than in COX-2(+/+) mice as revealed by microCT(P<0.01). However, the defect was not completely healed even in the COX2(+/+) recipient mice. Few donor cells (GFP) were present in COX2(+/+) group, and almost no donor cells were found in one COX-2(-/-) group. We found many lymphocytes in the regenerated tissues in the COX-2(+/+) mice indicating immune rejection while the COX-2(-/-) mice formed granulation tissue over the defect area. (3) COX-2(-/-) MDSC regenerate markedly less bone in CD-1 nude mice. BMP4GFP transduced COX-2(-/-) MDSC formed significantly less bone in CD-1 mice than COX-2(+/+) MDSC at 2,4 and 6 weeks PI. Conclusions: Our findings indicate COX-2 is a key factor for the MDSC mediated bone regeneration. Both donor and host the COX-2 are important for MDSC mediated bone healing. COX-2 inhibitors should be used with caution when performing stem cell transplantation. We will further investigate the mechanism.
739. Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate Wound Healing in Skin
David L. Mack,1 Aleksander Skardal,1 James Yoo,1 Shay Soker.1 Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC.
1
Mesenchymal stem cells (MSCs) have shown therapeutic potential for repair and regeneration of tissues damaged by injury or disease. Amniotic fluid-derived stem (AFS) cells possess properties found also in MSCs such as multipotent differentiation, immunomodulatory activity and the lack of significant immunogenicity. MSC treatment of acute and chronic wounds result in accelerated wound closure, increased epithelialization, granulation tissue and angiogenesis. Given that AFS cells can be obtained less invasively than MSCs and show greater proliferative capacity in culture, we investigated whether they could augment wound healing in a similar fashion as MSCs. A full thickness skin wound was surgically created on the dorsum of nu/nu mice. A bioprinter was used to deposit 2 layers of a fibrin-collagen gel, containing either 5 x 106 AFS cells, MSCs, or no cells over the surgical site. At day 7 and 14, AFS and MSC-driven wound closure was significantly greater compared to that of the gel-only treatment.
Re-epithelialization of the wound area was also found to be significantly greater in AFS and MSC groups at day 7 and 14 compared to gel-only group. H&E staining of harvested skin at day 7 revealed a 3-fold increase in microvessel density (MVD) in the Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
AFS and MSC-regenerated skin compared to gel only. At day 14, AFS-treated wounds had increased MVD compared to both MSC and gel-only groups. Furthermore, more mature vessels were observed in the AFS-treated group. Histology revealed that near the center of the wound area, both AFS and MSC-treated animals had robust layers of keratinocytes present that had formed organized epidermis. Finally, GFP-labeled AFS cells were employed to determine whether the printed cells were permanently integrating or were transient in the regenerating tissue. Immunofluorescence showed that AFS cell numbers decreased over time with no visible cells present at day 14.
Staining for von Willebrand factor revealed that AFS cells did not co-localize with developing blood vessels, despite the increased neovascular activity. These observations suggest that the increased wound closure rates and angiogenesis may be due to the localized, sustained secretion of trophic factors, rather than direct cell-cell interactions. AFS cells perform as well as MSCs in terms of wound closure. However, AFS cells seem to induce greater neovascularization of the regenerating skin. Therefore, bioprinting AFS cells in a hydrogel carrier might address the clinical need for more effective treatments for large-scale wounds and burns.
740. Prevention of Radiation-Induced Lung Injury by Administration of Gene-Modified Mesenchymal Stem Cells
Jiaxin Xue,1 Xin Li,2 You Lu,1 Lin Zhou,1 Yongsheng Wang,1 Li Jia,1 Xianming Mo.3 1 Department of Thoracic Oncology, West China Hospital, Sichuan University, Chengdu, China; 2Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University, Xi’an, China; 3 Laboratory of Stem Cell Biology, West China Hospital, Sichuan University, Chengdu, China. Radiation-induced lung injury (RILI), a common and serious side effect of radiotherapy for thoracic cancers, can limit therapy itself and may even cause mortality. The aim of this study was to investigate the prevention and repairing efficacy of RILI by adenovirus-mediated soluble transforming growth factor-beta type II receptor gene (Ad-sTβR) modified mesenchymal stem cells (Ad-sTβR-MSCs). Mesenchymal stem cells (MSCs) from male mice were isolated, identified, and transduced with Ad-sTβR. It was confirmed that MSCs could specifically home into radiation injured lung and the engrafted MSCs levels had a 20-fold increase as compared with non-irradiated lung. Transduction of Ad-sTβR did not alter the migration capacity of MSCs to injured lung in vivo and ex vivo. Migration assay and blocking experiments revealed that SDF-1α/CXCR4 axis played an important role in this process, suggesting that the increased levels of MSCs in lung stemmed from the radiation-induced homing of MSCs rather than the physical entrapment of circulating MSCs. Administration of Ad-sTβR-MSCs exerted a significant reduction for lung injury, as demonstrated not only by survival and histopathology, but also by assay of malondialdehyde (MDA), hydroxyproline, plasma cytokines (IL-1β, TNF-α, IL-6, IL-10 and active TGF-β1), and the expression of CTGF and α-SMA. S285