- Email: [email protected]
262. Genetic Modification of Neural Stem Cells
STEM CELL BIOLOGY We could show that all of four carriers tested were able to transfect MSCs with mRNA encoding CXCR4 very efciently. Receptor expression was again compared to that obtained by pDNA delivery, and revealing that the number of transfected cells was considerably higher with mRNA than with pDNA, except when DOTAP/DOPE was used as a carrier. The expression of CXCR4 was still detectable 48 hours post transfection, i.e. long enough to be sufcient for the MSCs to migrate to the target tissue.
262.
Genetic Modication of Neural Stem Cells
Ravi K. Chandra. Pharmacy, Acharya & B.M. Reddy College of Pharmacy, Bangalore, Karnataka, India.
Neural stem cells (NSCs) are the main vehicle for genetic and molecular therapies in the central nervous system (CNS). The sustainability of NSCs has been ensured through genetic manipulation both in vitro and in vivo. NSC lines have also been immortalized and controlled for cell growth in similar fashion. Their potential to differentiate and their genetic plasticity make them the modality of choice for cellular transplantation. After transplantation, NSCs also exhibit inherent long-distance migratory capabilities and a remarkable capacity to integrate into brain structures. This makes NSCs the ideal candidate for delivery and expression of therapeutic genes. Finally, the imaging possibilities with NSC transplants are endless, and they will be a pivotal component to safe and effective human transplantation. This paper provides an overview on NSCs and the various methods in which they have been genetically manipulated for biological investigation.
263. APOA-1 Is a Novel Marker of Erythroid Cell Maturation from Hematopoietic Stem Cells in Mice and Humans
Tomoko Inoue,1 Daisuke Sugiyama,2 Ryo Kurita,1 Tatsuo Oikawa,1 Kasem Kulkeaw,2 Hirotaka Kawano,1 Yoshie Miura,1 Michiyo Okada,1 Youko Suehiro,1 Atsushi Takahashi,1 Tomotoshi Marumoto,1 Hiroyuki Inoue,1 Norio Komatsu,3 Kenzaburo Tani.1 1 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Department of Hematopoietic Stem Cells, SSP Stem Cell Unit, Kyushu University, Fukuoka, Japan; 3Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University School of Medicine, Tokyo, Japan. The mechanism that regulates terminal erythroid cell maturation from hematopoietic stem cells is poorly defined. Therefore, identifying genes and surface markers that are restricted to specic stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identied 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identied and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression level of Apoa-1 gene, an apolipoprotein family member, was signicantly increased as hematopoietic stem cells differentiate into terminal differentiated erythroid cells of mouse FL. Apoa-1 protein was more abundant in differentiated erythroid cells than hematopoietic stem and progenitor cells in mouse FL by ELISA. Moreover, the expression of APOA-1 gene was detected in terminal differentiated erythroid cells of human peripheral blood. We conclude S100
that APOA-1 is a novel marker of terminal erythroid maturation from hematopoietic stem cells in mice and humans. APOA-1 can potentially be used to identify mature erythrocytes in combination with Ter119 antigen or glycophorinA antigen from in vitro cultured erythroid cell sources such as ES or iPS cells. Also, gene transfer of APOA-1to hematopoietic stem cells derived from ES/iPS cells may be helpful for ex vitro expansion of red cells from the standpoint of regenerative medicine. Now, we are trying to investigate whether APOA-1 plays pivotal roles in erythroid cell maturation.
264. A Stem Cell Gene Therapy Approach for Enhancing the Efcacy of Breast Cancer Therapy by Trastuzumab (Herceptin)
Ines Beyer,1 Jonas Persson,1 ZongYi Li,1 Ying Liu,1 Roma Yumul,1 Andre Lieber.1 1 Medical Genetics, University of Washington, Seattle, WA. Trastuzumab is an essential targeted anticancer drug, which is the rst-line treatment of Her2/neu-overexpressing breast cancer. Unfortunately, a signicant number of Her2/neu-positive patients do not respond to treatment with trastuzumab-containing regimens. Mechanisms for resistance include obstacles preventing trastuzumab binding to its target – Her2/neu. In breast cancer, tumor cells are often surrounded by tumor stroma consisting of stroma cells, tumorassociated macrophages and matrix proteins such as collagen or laminin. We observed extensive extracellular matrix and intercellular junctions in breast cancer patients and xenograft models, which block access to Her2/neu and, potentially, the intratumoral dissemination of trastuzumab after systemic application. Recently, we established a new stem cell gene therapy approach for breast cancer therapy. This approach is based on the ex vivo modication of hemapoietic stem cells, which, after transplantation, home in on the tumor and deliver therapeutic transgenes to the tumor stroma in models with transplanted mouse and human tumors (Blood; 2009; 113:5423-33). Here we tested whether this approach could be used to transiently degrade tumor stroma and facilitate trastuzumab therapy in a xenograft model for breast cancer. To degrade tumor stroma proteins we utilized the peptide hormone relaxin. In in vitro studies, we demonstrated that the expression of relaxin in the Her2/neu-positive breast cancer cell line BT474-M1 improved the therapeutic effect of trastuzumab. For in vivo studies, we established tumors by the injection of BT474-M1 cells into the mammary fat pad of CB17/SCID/beige mice. We demonstrated that these tumors were massively inltrated by mouse CD45+ and F4/80 positive macrophages. Towards the testing of a combination of our stem cell gene therapy with trastuzumab therapy, we showed that intraperitoneal trastuzumab injection delayed the growth of BT474-M1 tumors. To test a stem cell gene therapy-based approach, we transduced syngeneic mouse bone marrow cells with insulated SIN lentivirus vectors expressing relaxin under tight doxycyclin (Dox) control and transplanted them into sub-lethally irradiated mice. After engraftment of transplanted cells in the bone marrow and BT474-M1 tumor establishment, mice will be treated with Dox and trastuzumab. Results will be presented. We expect a signicant prolongation of survival in mice that received trastuzumab therapy and Dox, compared to mice that were treated with trastuzumab without Dox or control mice.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
262.
Genetic Modication of Neural Stem Cells
Ravi K. Chandra. Pharmacy, Acharya & B.M. Reddy College of Pharmacy, Bangalore, Karnataka, India.
Neural stem cells (NSCs) are the main vehicle for genetic and molecular therapies in the central nervous system (CNS). The sustainability of NSCs has been ensured through genetic manipulation both in vitro and in vivo. NSC lines have also been immortalized and controlled for cell growth in similar fashion. Their potential to differentiate and their genetic plasticity make them the modality of choice for cellular transplantation. After transplantation, NSCs also exhibit inherent long-distance migratory capabilities and a remarkable capacity to integrate into brain structures. This makes NSCs the ideal candidate for delivery and expression of therapeutic genes. Finally, the imaging possibilities with NSC transplants are endless, and they will be a pivotal component to safe and effective human transplantation. This paper provides an overview on NSCs and the various methods in which they have been genetically manipulated for biological investigation.
263. APOA-1 Is a Novel Marker of Erythroid Cell Maturation from Hematopoietic Stem Cells in Mice and Humans
Tomoko Inoue,1 Daisuke Sugiyama,2 Ryo Kurita,1 Tatsuo Oikawa,1 Kasem Kulkeaw,2 Hirotaka Kawano,1 Yoshie Miura,1 Michiyo Okada,1 Youko Suehiro,1 Atsushi Takahashi,1 Tomotoshi Marumoto,1 Hiroyuki Inoue,1 Norio Komatsu,3 Kenzaburo Tani.1 1 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Department of Hematopoietic Stem Cells, SSP Stem Cell Unit, Kyushu University, Fukuoka, Japan; 3Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University School of Medicine, Tokyo, Japan. The mechanism that regulates terminal erythroid cell maturation from hematopoietic stem cells is poorly defined. Therefore, identifying genes and surface markers that are restricted to specic stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identied 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identied and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression level of Apoa-1 gene, an apolipoprotein family member, was signicantly increased as hematopoietic stem cells differentiate into terminal differentiated erythroid cells of mouse FL. Apoa-1 protein was more abundant in differentiated erythroid cells than hematopoietic stem and progenitor cells in mouse FL by ELISA. Moreover, the expression of APOA-1 gene was detected in terminal differentiated erythroid cells of human peripheral blood. We conclude S100
that APOA-1 is a novel marker of terminal erythroid maturation from hematopoietic stem cells in mice and humans. APOA-1 can potentially be used to identify mature erythrocytes in combination with Ter119 antigen or glycophorinA antigen from in vitro cultured erythroid cell sources such as ES or iPS cells. Also, gene transfer of APOA-1to hematopoietic stem cells derived from ES/iPS cells may be helpful for ex vitro expansion of red cells from the standpoint of regenerative medicine. Now, we are trying to investigate whether APOA-1 plays pivotal roles in erythroid cell maturation.
264. A Stem Cell Gene Therapy Approach for Enhancing the Efcacy of Breast Cancer Therapy by Trastuzumab (Herceptin)
Ines Beyer,1 Jonas Persson,1 ZongYi Li,1 Ying Liu,1 Roma Yumul,1 Andre Lieber.1 1 Medical Genetics, University of Washington, Seattle, WA. Trastuzumab is an essential targeted anticancer drug, which is the rst-line treatment of Her2/neu-overexpressing breast cancer. Unfortunately, a signicant number of Her2/neu-positive patients do not respond to treatment with trastuzumab-containing regimens. Mechanisms for resistance include obstacles preventing trastuzumab binding to its target – Her2/neu. In breast cancer, tumor cells are often surrounded by tumor stroma consisting of stroma cells, tumorassociated macrophages and matrix proteins such as collagen or laminin. We observed extensive extracellular matrix and intercellular junctions in breast cancer patients and xenograft models, which block access to Her2/neu and, potentially, the intratumoral dissemination of trastuzumab after systemic application. Recently, we established a new stem cell gene therapy approach for breast cancer therapy. This approach is based on the ex vivo modication of hemapoietic stem cells, which, after transplantation, home in on the tumor and deliver therapeutic transgenes to the tumor stroma in models with transplanted mouse and human tumors (Blood; 2009; 113:5423-33). Here we tested whether this approach could be used to transiently degrade tumor stroma and facilitate trastuzumab therapy in a xenograft model for breast cancer. To degrade tumor stroma proteins we utilized the peptide hormone relaxin. In in vitro studies, we demonstrated that the expression of relaxin in the Her2/neu-positive breast cancer cell line BT474-M1 improved the therapeutic effect of trastuzumab. For in vivo studies, we established tumors by the injection of BT474-M1 cells into the mammary fat pad of CB17/SCID/beige mice. We demonstrated that these tumors were massively inltrated by mouse CD45+ and F4/80 positive macrophages. Towards the testing of a combination of our stem cell gene therapy with trastuzumab therapy, we showed that intraperitoneal trastuzumab injection delayed the growth of BT474-M1 tumors. To test a stem cell gene therapy-based approach, we transduced syngeneic mouse bone marrow cells with insulated SIN lentivirus vectors expressing relaxin under tight doxycyclin (Dox) control and transplanted them into sub-lethally irradiated mice. After engraftment of transplanted cells in the bone marrow and BT474-M1 tumor establishment, mice will be treated with Dox and trastuzumab. Results will be presented. We expect a signicant prolongation of survival in mice that received trastuzumab therapy and Dox, compared to mice that were treated with trastuzumab without Dox or control mice.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy