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955. Increased Numbers of Bronchioalveolar Stem Cells Following Adenovirus-Mediated Expression of Sonic Hedgehog in the Lung
Hematopoietic, Lung, Liver, Skin and Cancer Stem Cells 955. Increased Numbers of Bronchioalveolar Stem Cells Following Adenovirus-Mediated Expression of Sonic Hedgehog in the Lung
Anja Krause,1 Ju Joh,1 Austen Gess,1 Yaqin Xu,1 Howard Lou,1 Ronald G. Crystal,1 Stefan Worgall.1 1 Weill Cornell Medical College, NY, NY. Bronchioalveolar stem cells (BASC) residing in the bronchioalveolar duct junction (BADJ) are characterized as hematopoietic (CD45neg) and endothelial (pecamneg) lineage-negative cells that express the surface markers CD34 and Sca-1 (CD34pos, Sca-1pos) and the Clara cell marker CC10. Based on our prior data that adenovirus (Ad) overexpression of sonic hedgehog N (ShhN), a genetically engineered form of Shh, in the lung is associated with an increase in the number of CD34pos, Sca-1pos, CD45neg, pecamneg cells in the lung, the present study analyzes the number of CD34pos cells in the BADJ that co-express the Clara cell marker CC10 following administration of AdShhN and the potential of CD34pos, Sca-1pos, CD45neg, pecamneg cells from the AdShhN-stimulated lungs to be transplanted to syngeneic mice. AdShhN or the control vector AdNull (5x1010 particle units/mouse) were administered intratracheally to C57Bl/6 mice. Ten days later, CD34 and CC10 double-positive cells in the lung were analyzed by immunohistochemistry. Following administration of AdShhN the number of CC10pos, CD34pos cells in the bronchioalveolar duct regions were increased by 1.7-fold compared to the AdNull group (p<0.05), suggesting that overexpression of ShhN increases the number of BASC expressing CC10. To analyze if the CD34pos, Sca-1pos, CD45neg, pecamneg cells isolated from AdShhN-stimulated lungs could be transplanted to syngeneic mice, AdShhN was administered to the lungs of ROSA/C57Bl/6 mice, that express β-galatosidase (β-gal), by intratracheal administration. Ten days later, β-galpos, Sca-1pos, CD45neg, pecamneg cells or β-galpos, Sca-1neg, CD45neg, pecamneg cells were purified using magnetic beads and transplanted via the intravenous and intratracheal route to syngeneic C57Bl/6 mice that had undergone unilateral pneumonectomy 2 days prior to the transplantation. Four wk or 3 mo later, the lungs were analyzed histologically for β-galpos CC10pos cells. The mice that had received the β-galpos, Sca-1pos, CD45neg pecamneg or β-galpos, CD34pos, Sca-1pos, CD45neg, pecamneg cells via the intravenous route showed only few β-galpos cells present in the lungs at both time points. In contrast, following intratracheal administration of β-galpos, CD34pos, Sca-1pos, CD45neg, pecamneg cells numerous β-galpos cells were present in the alveoli and airways of the recipient mice at both time points. Only a few β-galpos cells were found in the lungs of the mice that received β-galpos, Sca-1neg, CD45neg, pecamneg cells intratracheally (p<0.01 compared to the mice that had received β-galpos, CD34pos, Sca-1pos, CD45neg, pecamneg cells). A subset of the β-galpos cells present in the lungs following intratracheal transplantation of the β-galpos, CD34pos, Sca-1pos, CD45neg, pecamneg cells expressed the Clara cell marker CC10. This data demonstrates that administration of AdShhN to the respiratory tract increases the number of BASC in the lung and that Sca-1pos, CD45neg, pecamneg cells from the lung cells can be transplanted to syngeneic mice to persist for up to 3 months. This has implications for the expansion of pulmonary stem cells and their use in the development of novel strategies enhancing lung repair.
956. Adult Stem Cell Mediated Liver Gene Delivery of Alpha 1-Antitrypsin
Hong Li,1 Yuanqing Lu,1 Bin Zhang,1 Bryon Petersen,2 Sihong Song.1 1 Pharmaceutics, University of Florida, Gainesville, FL; 2 Pathology, University of Florida, Gainesville, FL. Alpha 1-antitrypsin (AAT) deficiency is a genetic disorder caused mostly by a single base substitution in the AAT gene, and leads to hepatocyte dysfunction and lung destruction. Replacing dysfunctional S356
hepatocytes with genetically modified stem cells is considered to be a potentially powerful approach for the treatment of this disease. In this study, we examed long-term and stable AAT transgene expression in liver by ex vivo transduction and liver transplantation of liver progenitor cells (oval cells), bone marrow (BM) cells, or adipose tissue-derived mesenchymal stem cells (AT-MSCs). Freshly isolated stem cells (oval cells, BM cells and AT-MSCs) from C57BL/6 male mice were infected with recombinant adeno-associated virus serotype 1 vector – expressing human AAT (rAAV1-CB-hAAT) and rAAV8CB-hAAT at 104 particles/cell and lentiviral vector – expressing human AAT (Lenti-CB-hAAT) at 102 particles/cell, respectively. 2 hours after infection, 1-5 x106 stem cells were transplanted into the liver of monocrotaline treated and partial-hepatectomized C57BL/6 female recipients. Serum samples were collected every week for hAAT ELISA assay to evaluate transgene expression. To evaluate the engraftment efficiency of transplanted adult stem cells, recipient liver tissues were collected 10-14 weeks post transplantation for immunostaining. Oval cell transplantation studies showed that transgene (hAAT) expression can be detected in serum by ELISA (up to2.5 ug/ml) and in liver cells by immunostaining (up to 5% hAAT positive cells) with rAAV1-CB-AAT vector. Consisting with our previous observation, these results indicate it is feasible to use adult stem cell for liver gene delivery. Considering clinical practices, we next investigated the possibility of transplanting genetically modified BM cells. Immunostaining revealed that about 5% of hAAT positive cells were derived from rAAV8-CB-hAAT infected donor BM cells, while 1-2% hAAT positive cells were from rAAV1-CBhAAT or Lenti-CB-hAAT infected donor BM cells. Data showed that BM cells can be transduced by rAAV and lentiviral vectors and engrafted into liver resulting in transgene expression. Recent studies have demonstrated that adipose tissue may represent an ideal source of autologous stem cells, in terms of easy isolation, substantial cell number and minimal patient discomfort. In order to test the feasibility of using AT-MSCs liver transplantation, we have performed the third set of studies. In vitro study showed that AT-MSCs can be efficiently transduced by rAAV1 vectors. After transplantation of rAAV1-CBhAAT infected AT-MSCs to the recipient liver, serum levels of hAAT leveling the recipients were detected by hAAT specific ELISA. In conclusion, results from these studies indicated that oval cells, BM cells and AT-MSCs can be transduced by rAAV and lentiviral vector and served as platform for transgene expression. AT-MSC-based gene therapy presents a novel approach for the treatment of human genetic diseases, such as AAT deficiency. Future studies will focus on achieving therapeutic levels of transgene expression, and gene correction in AT-MSC for liver regeneration.
957. Epidermal Stem Cell Gene Targeting for the Treatment of Epidermolysis Bullosa Lisa M. Petek, Daniel G. Miller. 1 Pediatrics, University of Washington, Seattle, WA.
Epidermal stem cells having a holoclone phenotype are present in adult skin and have been shown to have remarkable proliferative capacity with the ability to double 100 times and generate over 1020 cells. Genetic manipulation of this cell population would allow expansion, characterization, and autologous transplantation of modified cells using techniques already established for the treatment of severe full thickness burns. The simplex form of Epidermolysis Bullosa (EB) is a dominantly inherited skin disease caused by mutations of either keratin 5 or keratin 14. These mutations result in the production of aberrantly folded keratins with dominant negative activity and cause collapse of the intermediate filament network in basal keratinocytes. This weakens the basal cell layer of the epidermis and results in frequent blister formation. There is no effective treatment for EB, and individuals experience a life of frequent painful blisters that often become secondarily infected by Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Anja Krause,1 Ju Joh,1 Austen Gess,1 Yaqin Xu,1 Howard Lou,1 Ronald G. Crystal,1 Stefan Worgall.1 1 Weill Cornell Medical College, NY, NY. Bronchioalveolar stem cells (BASC) residing in the bronchioalveolar duct junction (BADJ) are characterized as hematopoietic (CD45neg) and endothelial (pecamneg) lineage-negative cells that express the surface markers CD34 and Sca-1 (CD34pos, Sca-1pos) and the Clara cell marker CC10. Based on our prior data that adenovirus (Ad) overexpression of sonic hedgehog N (ShhN), a genetically engineered form of Shh, in the lung is associated with an increase in the number of CD34pos, Sca-1pos, CD45neg, pecamneg cells in the lung, the present study analyzes the number of CD34pos cells in the BADJ that co-express the Clara cell marker CC10 following administration of AdShhN and the potential of CD34pos, Sca-1pos, CD45neg, pecamneg cells from the AdShhN-stimulated lungs to be transplanted to syngeneic mice. AdShhN or the control vector AdNull (5x1010 particle units/mouse) were administered intratracheally to C57Bl/6 mice. Ten days later, CD34 and CC10 double-positive cells in the lung were analyzed by immunohistochemistry. Following administration of AdShhN the number of CC10pos, CD34pos cells in the bronchioalveolar duct regions were increased by 1.7-fold compared to the AdNull group (p<0.05), suggesting that overexpression of ShhN increases the number of BASC expressing CC10. To analyze if the CD34pos, Sca-1pos, CD45neg, pecamneg cells isolated from AdShhN-stimulated lungs could be transplanted to syngeneic mice, AdShhN was administered to the lungs of ROSA/C57Bl/6 mice, that express β-galatosidase (β-gal), by intratracheal administration. Ten days later, β-galpos, Sca-1pos, CD45neg, pecamneg cells or β-galpos, Sca-1neg, CD45neg, pecamneg cells were purified using magnetic beads and transplanted via the intravenous and intratracheal route to syngeneic C57Bl/6 mice that had undergone unilateral pneumonectomy 2 days prior to the transplantation. Four wk or 3 mo later, the lungs were analyzed histologically for β-galpos CC10pos cells. The mice that had received the β-galpos, Sca-1pos, CD45neg pecamneg or β-galpos, CD34pos, Sca-1pos, CD45neg, pecamneg cells via the intravenous route showed only few β-galpos cells present in the lungs at both time points. In contrast, following intratracheal administration of β-galpos, CD34pos, Sca-1pos, CD45neg, pecamneg cells numerous β-galpos cells were present in the alveoli and airways of the recipient mice at both time points. Only a few β-galpos cells were found in the lungs of the mice that received β-galpos, Sca-1neg, CD45neg, pecamneg cells intratracheally (p<0.01 compared to the mice that had received β-galpos, CD34pos, Sca-1pos, CD45neg, pecamneg cells). A subset of the β-galpos cells present in the lungs following intratracheal transplantation of the β-galpos, CD34pos, Sca-1pos, CD45neg, pecamneg cells expressed the Clara cell marker CC10. This data demonstrates that administration of AdShhN to the respiratory tract increases the number of BASC in the lung and that Sca-1pos, CD45neg, pecamneg cells from the lung cells can be transplanted to syngeneic mice to persist for up to 3 months. This has implications for the expansion of pulmonary stem cells and their use in the development of novel strategies enhancing lung repair.
956. Adult Stem Cell Mediated Liver Gene Delivery of Alpha 1-Antitrypsin
Hong Li,1 Yuanqing Lu,1 Bin Zhang,1 Bryon Petersen,2 Sihong Song.1 1 Pharmaceutics, University of Florida, Gainesville, FL; 2 Pathology, University of Florida, Gainesville, FL. Alpha 1-antitrypsin (AAT) deficiency is a genetic disorder caused mostly by a single base substitution in the AAT gene, and leads to hepatocyte dysfunction and lung destruction. Replacing dysfunctional S356
hepatocytes with genetically modified stem cells is considered to be a potentially powerful approach for the treatment of this disease. In this study, we examed long-term and stable AAT transgene expression in liver by ex vivo transduction and liver transplantation of liver progenitor cells (oval cells), bone marrow (BM) cells, or adipose tissue-derived mesenchymal stem cells (AT-MSCs). Freshly isolated stem cells (oval cells, BM cells and AT-MSCs) from C57BL/6 male mice were infected with recombinant adeno-associated virus serotype 1 vector – expressing human AAT (rAAV1-CB-hAAT) and rAAV8CB-hAAT at 104 particles/cell and lentiviral vector – expressing human AAT (Lenti-CB-hAAT) at 102 particles/cell, respectively. 2 hours after infection, 1-5 x106 stem cells were transplanted into the liver of monocrotaline treated and partial-hepatectomized C57BL/6 female recipients. Serum samples were collected every week for hAAT ELISA assay to evaluate transgene expression. To evaluate the engraftment efficiency of transplanted adult stem cells, recipient liver tissues were collected 10-14 weeks post transplantation for immunostaining. Oval cell transplantation studies showed that transgene (hAAT) expression can be detected in serum by ELISA (up to2.5 ug/ml) and in liver cells by immunostaining (up to 5% hAAT positive cells) with rAAV1-CB-AAT vector. Consisting with our previous observation, these results indicate it is feasible to use adult stem cell for liver gene delivery. Considering clinical practices, we next investigated the possibility of transplanting genetically modified BM cells. Immunostaining revealed that about 5% of hAAT positive cells were derived from rAAV8-CB-hAAT infected donor BM cells, while 1-2% hAAT positive cells were from rAAV1-CBhAAT or Lenti-CB-hAAT infected donor BM cells. Data showed that BM cells can be transduced by rAAV and lentiviral vectors and engrafted into liver resulting in transgene expression. Recent studies have demonstrated that adipose tissue may represent an ideal source of autologous stem cells, in terms of easy isolation, substantial cell number and minimal patient discomfort. In order to test the feasibility of using AT-MSCs liver transplantation, we have performed the third set of studies. In vitro study showed that AT-MSCs can be efficiently transduced by rAAV1 vectors. After transplantation of rAAV1-CBhAAT infected AT-MSCs to the recipient liver, serum levels of hAAT leveling the recipients were detected by hAAT specific ELISA. In conclusion, results from these studies indicated that oval cells, BM cells and AT-MSCs can be transduced by rAAV and lentiviral vector and served as platform for transgene expression. AT-MSC-based gene therapy presents a novel approach for the treatment of human genetic diseases, such as AAT deficiency. Future studies will focus on achieving therapeutic levels of transgene expression, and gene correction in AT-MSC for liver regeneration.
957. Epidermal Stem Cell Gene Targeting for the Treatment of Epidermolysis Bullosa Lisa M. Petek, Daniel G. Miller. 1 Pediatrics, University of Washington, Seattle, WA.
Epidermal stem cells having a holoclone phenotype are present in adult skin and have been shown to have remarkable proliferative capacity with the ability to double 100 times and generate over 1020 cells. Genetic manipulation of this cell population would allow expansion, characterization, and autologous transplantation of modified cells using techniques already established for the treatment of severe full thickness burns. The simplex form of Epidermolysis Bullosa (EB) is a dominantly inherited skin disease caused by mutations of either keratin 5 or keratin 14. These mutations result in the production of aberrantly folded keratins with dominant negative activity and cause collapse of the intermediate filament network in basal keratinocytes. This weakens the basal cell layer of the epidermis and results in frequent blister formation. There is no effective treatment for EB, and individuals experience a life of frequent painful blisters that often become secondarily infected by Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy