- Email: [email protected]
P75. A gene therapy for a gene mutation in human iPS cell using helper-dependent adenoviral vector
S42
Abstract / Differentiation 80 (2010) S17–S63
isolated satellite cells from limb muscles of aged mice (aged satellite cells) and young mice (young satellite cells) by FACS using a monoclonal antibody SM/C-2.6. Satellite cells remarkably decreased in both number/g muscle weight and number/muscle cross section with age. To clarify the molecular mechanisms leading to loss of satellite cells with age, we performed DNA microarray analysis using freshly isolated aged and young satellite cells. Compared with young satellite cells, 95 genes were down-regulated (o1/4-fold) in aged satellite cells. In contrast, 85 genes were up-regulated (44-fold) in aged satellite cells. Detailed analyses of some identified genes will be reported. On the other hand, when evaluated myogenic potential in vitro, there was no difference in proliferation and differentiation ability between residual aged satellite cells and young satellite cells. In vivo intramuscular transplantation experiment also showed that muscle reconstitution ability of aged satellite cells was similar to that of young satellite cells. To elucidate whether the decline of regenerative potential in aged muscle results at least from deterioration of aged muscle environment, we transplanted satellite cells freshly isolated from young GFP-Tg mice into CTX-injected young and aged muscle. The muscle reconstitution ability in aged host significantly decreased compared with that in young host. Therefore, it is thought that the decline of muscle regenerative potential in sarcopenia is attributed to decrease in satellite cell number and deterioration of aged muscle environment. doi: 10.1016/j.diff.2010.09.080
P75 A gene therapy for a gene mutation in human iPS cell using helper-dependent adenoviral vector
T. Yoshida a, H. Koizumi b, K. Yuki a, S. Kubota a, Y. Hirabayashi b, K. Suzuki b, K. Mitani b, T. Kobayashi a, M. Ohyama a, M. Amagai a, Y. Okada a, W. Akamatsu a, K. Tsubota a, S. Shimmura a, Y. Ozawa a, H. Okano a a
Keio University School of Medicine, Tokyo, Japan Saitama Medical University Research Center of Gene Medicine, Saitama, Japan E-mail address: [email protected] (T. Yoshida) b
Retinitis pigmentosa (RP) is one of the hereditary neurodegenerative diseases, which finally causes photoreceptor cell death. Several kinds of mutations in genes, e.g. rhodopsin, peripherin and cGMP phophodiesterase, are reported to cause the disease, however, the fundamental therapeutic approach has not established yet. Recent progress in the stem cell science has led us to a possibility of applying cell transplantation for this disease. Embryonic stem (ES) cells were the first candidate cell source of the transplantation; however, there were some concerns in using ES cells; immunological responses and ethical issues. On the other hand, induced pluripotent stem (iPS) cells may overcome these problems. Patients may use their own somatic cells to obtain cells for transplantation. However, there is another concern; if the disease were caused by genetic reason, the mutation would be inherited into iPS cells, and would cause the degeneration again. Thus, we try to cure the gene mutation in iPS cells before differentiation and transplantation. It has been reported that gene targeting of human ES or iPS cells is quite difficult compared with that of mouse ES or iPS cells. However, we have recently reported that introducing targeting construct using helper-dependent (HD) adenoviral vector resulted in effective recombination rate. We established iPS cells from skin cells of a RP patient who has a mutation in rhodopsin gene allele. We try to replace the wild type rhodopsin allele into the mutated rhodopsin allele of the iPS cells using a HD adenoviral vector. We also attempt to differentiate the iPS cells into rod photoreceptor cells by the methods reported by other groups, and injected into subretinal space of the eyes of the model mice of retinal
degenerative disease. This study could be applicable to the regenerative medicine not only for RP patient, but also for the patients of any kind of neurodegenerative diseases caused by genetic reasons. doi: 10.1016/j.diff.2010.09.081
P76 Imaging hematopoietic regeneration in real-time
Jeffrey R. Harris, Tannishtha Reya Duke University Medical Center, Durham, NC, USA Although we have learned a great deal about the phenotype and function of hematopoietic stem and progenitor cells, we have remained largely in the dark about the dynamic behavior of these cells in context of their native microenvironment. Here we describe a strategy that combines the use of transgenic mice with live microscopy to allow in vivo imaging of hematopoietic cell behavior in real time. The high spatial and temporal resolution of the system has allowed us to visualize the living hematopoietic tissue with exceptional clarity and track the architecture and dynamics of cell division, cell migration and cell death. Using this approach we have compared the kinetics of regeneration following two distinct forms of injury: chemotherapy and radiation. While chemotherapy led to a clear loss of hematopoietic cells, much of the microenvironment remained intact allowing for rapid recovery; in contrast, radiation exposure resulted in extensive destruction of both hematopoietic cells and the microenvironment, and was associated with delayed recovery. Imaging of the early events that occurred in each type of injury revealed an unexpected activation of myeloid cells in the bone marrow following chemotherapy, but not radiation. Importantly, ectopic delivery of these cells following radiation markedly enhanced both short-term hematopoietic recovery as well as long-term survival of mice receiving a limiting transplant dose, identifying myeloid cells as an important component of injury repair and regeneration. Thus, the development of this high-resolution in vivo imaging approach provides a unique view within the living organism and can be a powerful tool for gaining new insight into the regulation of fundamental biological processes such as homeostasis and regeneration. doi: 10.1016/j.diff.2010.09.082
P77 Epiplakin1 (Eppk1) marks the progenitor population in adult liver
Akira Matsuo a,b, Tetsu Yoshida a, Rika Miki a,b, Sakuhei Fujiwara c, Kazuhiko Kume a, Shoen Kume a,b a
Division of Stem Cell Research, Department of Stem Cell Biology, IMEG, Kumamoto University, Kumamoto, Japan b Global COE ‘‘Cell Fate Regulation Research and Education Unit’’, Kumamoto University, Kumamoto, Japan c Department of Anatomy, Biology and Medicine (Dermatology), Faculty of Medicine, Oita University, Oita, Japan E-mail address: [email protected] (A. Matsuo) In liver, oval cells appear in the portal area in response to injury and are considered as transit amplifying cells. The current
Abstract / Differentiation 80 (2010) S17–S63
isolated satellite cells from limb muscles of aged mice (aged satellite cells) and young mice (young satellite cells) by FACS using a monoclonal antibody SM/C-2.6. Satellite cells remarkably decreased in both number/g muscle weight and number/muscle cross section with age. To clarify the molecular mechanisms leading to loss of satellite cells with age, we performed DNA microarray analysis using freshly isolated aged and young satellite cells. Compared with young satellite cells, 95 genes were down-regulated (o1/4-fold) in aged satellite cells. In contrast, 85 genes were up-regulated (44-fold) in aged satellite cells. Detailed analyses of some identified genes will be reported. On the other hand, when evaluated myogenic potential in vitro, there was no difference in proliferation and differentiation ability between residual aged satellite cells and young satellite cells. In vivo intramuscular transplantation experiment also showed that muscle reconstitution ability of aged satellite cells was similar to that of young satellite cells. To elucidate whether the decline of regenerative potential in aged muscle results at least from deterioration of aged muscle environment, we transplanted satellite cells freshly isolated from young GFP-Tg mice into CTX-injected young and aged muscle. The muscle reconstitution ability in aged host significantly decreased compared with that in young host. Therefore, it is thought that the decline of muscle regenerative potential in sarcopenia is attributed to decrease in satellite cell number and deterioration of aged muscle environment. doi: 10.1016/j.diff.2010.09.080
P75 A gene therapy for a gene mutation in human iPS cell using helper-dependent adenoviral vector
T. Yoshida a, H. Koizumi b, K. Yuki a, S. Kubota a, Y. Hirabayashi b, K. Suzuki b, K. Mitani b, T. Kobayashi a, M. Ohyama a, M. Amagai a, Y. Okada a, W. Akamatsu a, K. Tsubota a, S. Shimmura a, Y. Ozawa a, H. Okano a a
Keio University School of Medicine, Tokyo, Japan Saitama Medical University Research Center of Gene Medicine, Saitama, Japan E-mail address: [email protected] (T. Yoshida) b
Retinitis pigmentosa (RP) is one of the hereditary neurodegenerative diseases, which finally causes photoreceptor cell death. Several kinds of mutations in genes, e.g. rhodopsin, peripherin and cGMP phophodiesterase, are reported to cause the disease, however, the fundamental therapeutic approach has not established yet. Recent progress in the stem cell science has led us to a possibility of applying cell transplantation for this disease. Embryonic stem (ES) cells were the first candidate cell source of the transplantation; however, there were some concerns in using ES cells; immunological responses and ethical issues. On the other hand, induced pluripotent stem (iPS) cells may overcome these problems. Patients may use their own somatic cells to obtain cells for transplantation. However, there is another concern; if the disease were caused by genetic reason, the mutation would be inherited into iPS cells, and would cause the degeneration again. Thus, we try to cure the gene mutation in iPS cells before differentiation and transplantation. It has been reported that gene targeting of human ES or iPS cells is quite difficult compared with that of mouse ES or iPS cells. However, we have recently reported that introducing targeting construct using helper-dependent (HD) adenoviral vector resulted in effective recombination rate. We established iPS cells from skin cells of a RP patient who has a mutation in rhodopsin gene allele. We try to replace the wild type rhodopsin allele into the mutated rhodopsin allele of the iPS cells using a HD adenoviral vector. We also attempt to differentiate the iPS cells into rod photoreceptor cells by the methods reported by other groups, and injected into subretinal space of the eyes of the model mice of retinal
degenerative disease. This study could be applicable to the regenerative medicine not only for RP patient, but also for the patients of any kind of neurodegenerative diseases caused by genetic reasons. doi: 10.1016/j.diff.2010.09.081
P76 Imaging hematopoietic regeneration in real-time
Jeffrey R. Harris, Tannishtha Reya Duke University Medical Center, Durham, NC, USA Although we have learned a great deal about the phenotype and function of hematopoietic stem and progenitor cells, we have remained largely in the dark about the dynamic behavior of these cells in context of their native microenvironment. Here we describe a strategy that combines the use of transgenic mice with live microscopy to allow in vivo imaging of hematopoietic cell behavior in real time. The high spatial and temporal resolution of the system has allowed us to visualize the living hematopoietic tissue with exceptional clarity and track the architecture and dynamics of cell division, cell migration and cell death. Using this approach we have compared the kinetics of regeneration following two distinct forms of injury: chemotherapy and radiation. While chemotherapy led to a clear loss of hematopoietic cells, much of the microenvironment remained intact allowing for rapid recovery; in contrast, radiation exposure resulted in extensive destruction of both hematopoietic cells and the microenvironment, and was associated with delayed recovery. Imaging of the early events that occurred in each type of injury revealed an unexpected activation of myeloid cells in the bone marrow following chemotherapy, but not radiation. Importantly, ectopic delivery of these cells following radiation markedly enhanced both short-term hematopoietic recovery as well as long-term survival of mice receiving a limiting transplant dose, identifying myeloid cells as an important component of injury repair and regeneration. Thus, the development of this high-resolution in vivo imaging approach provides a unique view within the living organism and can be a powerful tool for gaining new insight into the regulation of fundamental biological processes such as homeostasis and regeneration. doi: 10.1016/j.diff.2010.09.082
P77 Epiplakin1 (Eppk1) marks the progenitor population in adult liver
Akira Matsuo a,b, Tetsu Yoshida a, Rika Miki a,b, Sakuhei Fujiwara c, Kazuhiko Kume a, Shoen Kume a,b a
Division of Stem Cell Research, Department of Stem Cell Biology, IMEG, Kumamoto University, Kumamoto, Japan b Global COE ‘‘Cell Fate Regulation Research and Education Unit’’, Kumamoto University, Kumamoto, Japan c Department of Anatomy, Biology and Medicine (Dermatology), Faculty of Medicine, Oita University, Oita, Japan E-mail address: [email protected] (A. Matsuo) In liver, oval cells appear in the portal area in response to injury and are considered as transit amplifying cells. The current