KEIO UNIVERSITY GLOBAL COE PROGRAM Education and Research Center for Stem Cell Medicine
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Hideyuki Okano

Hideyuki Okano

Hideyuki Okano

Professor, Department of Physiology, Graduate School of Medicine, Keio University / Chairman, Graduate School of Medicine, Keio University
Hideyuki Okano, MD, PhD
http://www.okano-lab.com/

Brief background description / Profile

March 1977

Graduated from Keio Shiki Senior High School

April 1977

Entered Keio University School of Medicine

March 1983

Graduated from the Keio University School of Medicine

April 1983

Research Associate, Department of Physiology, Keio University School of Medicine (Prof. Yasuzo Tsukada)

August 1985

Research Associate, Institute for Protein Research, Osaka University (Prof. Katsuhiko Mikoshiba)

October 1989

Studied at the Department of Biological Chemistry, Johns Hopkins University School of Medicine, USA (Dr. Craig Montell)

August 1991

Research Associate, Institute for Protein Research, Osaka University (Prof. Katsuhiko Mikoshiba)

April 1992

Research Associate, Department of Chemistry, Institute of Medical Science, University of Tokyo (Prof. Katsuhiko Mikoshiba)

September 1994

Professor, Department of Molecular Neurobiology, Institute of Basic Medical Sciences, University of Tsukuba

April 1997

Professor, Department of Neuroanatomy, Osaka University Medical School

April 1999

Professor, Graduate School of Medicine, Osaka University

April 2001

Professor, Department of Physiology, Keio University School of Medicine

From 2003 to 2008

Program Leader, 21st-Century COE Program "Basic Study and Clinical Application of the Human Stem Cell Biology and Immunology"

October 2007

Chair, Graduate School of Medicine, Keio University

Since 2008

Program Leader, Global COE Program "Education and Research Center for Stem Cell Medicine"

License, Certification・Ph.D

1983

Medical license

1988

Doctor of Medicine (Keio University)

Theme

Neural stem cells have self-renewal capacity and are multipotential cells that can produce neurons, astrocytes and oligodendrocytes. Embryo-derived neural stem cells can be cultured semipermanently, and are expected as the source of cell transplantation therapy for neurodegenerative diseases. In addition, it has recently been revealed that neural stem cells remain in the adult brain, continuously producing neurons, and great attention has been paid to the nerve regeneration strategy in non-invasive treatment by activating endogenous stem cells.
For realizing treatment by cell transplantation, it is indispensable to efficiently obtain the necessary neural cells, to obtain appropriate differentiation of these cells and to reconstruct neural networks. However, with the progress in the research on the mechanisms of neurogenesis, it has been revealed that there is still a large obstacle to implementation of this treatment; namely, neural stem cells produce only neurons in early development and their differentiation into glial cells begins in later development. In addition, projection-type neurons with specific neurotransmitters, such as motor neurons, are generated only within a relatively narrow time window on the long developmental time axis. These phenomena have been observed in repeated subcultures in vitro, and represent a barrier to the use of neural stem cells for transplantation. On the other hand, as for the use of endogenous neural stem cells, although the phenomenon of adult neurogenesis, which is the basis of such use, has been confirmed, there have not been sufficient physiological and anatomical discussions, and clinical application is still a distance away.
Therefore, to address the above problems, first, we aim to construct an in vitro experimental culture system that would reproduce the in vivo developmental processes. Next, by analyzing the developmental mechanisms of the central nervous system based on the observations, we would like to elucidate the mechanisms regulating the developmental potential of neural stem cells, which changes in a time-sequence-specific manner. In addition, we propose to investigate in detail the phenomenon of adult neurogenesis at the individual level by using knockout and knockin mice, and furthermore to elucidate the mechanisms underlying maintenance of the undifferentiated state, self-renewal mechanisms and molecular mechanisms of differentiation and fate determination in adult neural stem cells, by combining comprehensive analytical data obtained by techniques such as DNA microarrays and ChIP sequencing. In the future, we would like to expand the possibilities of clinical application of neural stem cells and propose new treatment methods for neurodegenerative diseases.

Research activities

Development of safe transplantation therapy using mouse iPS cell-derived neural stem cells for spinal cord injury

In collaboration with Prof. Shinya Yamanaka (Kyoto University) and others, we induced neural stem cells from various mouse iPS cells using ’safe’ clones. By transplanting them into the injured spinal cord, we succeeded in obtaining good functional recovery. We found that this functional recovery was caused by remyelination and axon guidance by the transplanted cells. On the other hand, when neural stem cells derived from "dangerous" clones, which have been demonstrated to show tumorigenicity in prior investigations, were transplanted into the injured spinal cord, functional recovery was temporarily obtained, but the beneficial effect was lost with the formation and expansion of tumors derived from the transplanted cells. These results underscore the importance of performing sufficient safety evaluation before using iPS cells for transplantation therapy (Tsuji et al., PNAS, 2010).

Success in the production of genetically modified primates

In collaboration with Dr. Erika Sasaki (Central Institute for Experimental Animals), we succeeded in producing genetically modified animals using a primate, the common marmoset. Expression of the transgene was observed not only in the first generation, in which gene transfer was carried out, but also in the second generation. Thus, primates in which a transgene was transmitted to the next generation were produced for the first time in the world.
Although genetically modified animals, such as mice and rats, have contributed to research in the life sciences, animal experiments using primates, which are functionally and anatomically much more similar to humans than rodents, have been sought for experimental studies of human diseases. The Central Institute for Experimental Animals has addressed this issue since the 1970s, and succeeded in establishing common marmosets, which are the smallest primates and have high fecundity, as standardized experimental animals in 1980; the institute has continued to carry out planned breeding of these animals as experimental animals until date. Our research group has performed animal experiments using these marmosets and has obtained great results for developing treatment methods to allow regeneration of the injured spinal cord, etc. In addition, Dr. Sasaki and others have conducted collaborative research with us and have progressed very far in the development and study of animal models of human disease, with much success.
In the genetically modified primates used in this study, an exogenous gene encoding green fluorescent protein (GFP) was introduced into marmoset embryos in vitro using a viral vector. The embryos were returned to the uteri of foster marmosets to establish pregnancy, and as a result, 4 foster mothers gave birth to 5 children, all of which were genetically modified marmosets. Moreover, transgene integration was confirmed in the germ cells in 2 out of these 5 marmosets, and from one of them, second-generation GFP-integrated marmosets were obtained.
In the future, it is expected that model marmosets for human intractable neurological diseases, such as Parkinson's disease and amyotrophic lateral sclerosis (ALS), can be produced using this genetic modification technology, and that preclinical studies would advance greatly with the use of these animals (Sasaki et al., Nature, 2009).

Evaluation and verification of the relationship between the source of somatic cells used for establishing iPS cells and the safety of their transplantation in mice

In collaboration with Prof. Shinya Yamanaka (Kyoto University) and others, we induced differentiation of various mouse iPS cells established by different methods from different sources of somatic cells into neural progenitor cells and transplanted them into mouse brains. As a result, we found that the source of the somatic cells used for establishing the iPS cells greatly influenced the safety of transplantation.
In this study, 4 genes (Oct3/4, Klf4, Sox2 and c-Myc) or 3 genes excluding c-Myc (according to the research group of Prof. Shinya Yamanaka and others, reactivation of c-Myc has been confirmed to be one of the causes of tumorigenesis) were introduced into mouse embryo-derived fibroblasts and tail-derived fibroblasts, hepatocytes and gastric epithelial cells from adult mice, to establish iPS cells with or without a cell selection process. Neural progenitor cells induced as neurospheres2) from these iPS cells were transplanted into the brains of immunodeficient mice. It was noted that tumors were rarely observed in mice transplanted with neurospheres derived from iPS cells established from mouse embryonic fibroblasts and adult gastric epithelial cells. This was considered to be equivalent to the results obtained in similar experiments using ES cells. On the other hand, tumors were formed in many of the mice transplanted with neurospheres from iPS cells established from adult mouse tail-derived fibroblasts. Tumors were also formed in some of the mice transplanted with iPS cells derived from hepatocytes. The tumor formation was considered to be related to the presence of residual undifferentiated cells even after the induction of differentiation into neural cells. In addition, histological analyses revealed that these tumors were teratomas or teratocarcinomas. The above results were not related to the presence or absence of c-Myc in factors used for establishing the iPS cells, or the presence or absence of a cell selection process. In addition, activation of the c-Myc gene, which is a tumor-causing factor in chimeric mice, was not observed in the iPS cells used in the transplantation experiments, confirming that this gene was not involved in the teratoma formation. Thirty-six kinds of mouse iPS cells were evaluated in this experiment, and such a large-scale simultaneous safety evaluation was performed for the first time in the world. These results clearly indicate the importance and direction of the methods used to evaluate and select iPS cell lines with excellent safety for transplantation among many cell lines, as well as the importance of somatic cells used for the establishment of iPS cells to ensure the safety of human iPS cells for future application to regenerative medicine (Miura et al., Nature Biotech, 2009).

Development of a culture system that reproduces the developmental processes of the central nervous system

First, we developed a culture system in which differentiation of mouse embryonic stem cells into neural stem cells can be induced and changes in their differentiation potential can be reproduced and analyzed in vitro (Okada Y et al., Stem Cells, 2008). In this culture system, as well as in neurogenesis in vivo, neural stem cells first produce exclusively neurons that appear only in early development (motor neurons, cholinergic neurons, dopaminergic neurons, etc.). After successive subcultures, they begin to produce neurons (GABAergic neurons, etc.) and glial cells that appear during or after mid-development. Using this system, it became possible to distinguish and deal with immature and mature neural stem cells and to perform comparative analyses of the differences in the gene expression levels and therapeutic effects of transplantation among these neural stem cells.

Time-sequence-specific regulatory mechanisms of the differentiation potential of neural stem cells by COUP-TFI/II

Next, to elucidate the mechanisms regulating the changes in the developmental potential of neural stem cells in a time-sequence-specific manner, we analyzed genes expressed at different levels in immature and mature neural stem cells by DNA microarrays using the above-mentioned differentiation system for differentiation of mouse ES cells to neural stem cells. Functional screening was performed by overexpression of genes using a lentiviral vector or by gene knockdown, particularly focusing on the regulatory factors for gene expression, such as chromatin remodeling factors and transcription factors, of the genes expressed at different levels. As a result, it was revealed that the knockdown of COUP-TFI and II genes, which are members of the orphan nuclear receptor family, inhibited the maturation of neural stem cells, i.e., their acquisition of the differentiation potential into glial cells. In addition, it was also revealed that knockdown of these genes resulted in maintenance of the differentiation potential of the cells into specific neurons that are generated only during early neurogenesis. Knockdown of COUP-TFI/II was also performed in the embryonic mouse brain, and inhibition of the acquisition of differentiation potential of the cells into glial cells and maintenance of the productivity of early-type neurons were confirmed in the neural stem cells, consistent with the results obtained in vitro. The above results suggest that COUP-TFI/II is required for the transition from immature to mature neural stem cells. On the basis of this finding, neurons that appear only in early development, such as motor neurons, can be prepared continuously from ES cells by knocking down COUP-TFI/II; this is expected to lead to the development of regenerative medicine technologies (Naka H et al. Nat Neurosci 2008).

Selected Paper

  1. Sasaki E, Suemizu H, Shimada A, Hanazawa K, Oiwa R, Kamioka M, Sotomaru Y, Hirakawa R, Eto T, Shiozawa S, Maeda T, Ito R, Kito C, Yagihashi C, Kawai K, Miyoshi H, Tanioka Y, Tamaoki N, Habu S, Okano H, Nomura T. : Generation of transgenic non-human primates with germ line transmission. Nature, 459(7246):523-527, 2009. (*H. Okano is the corresponding author in this paper)
  2. Naka H, Nakamura S, Shimazaki T, Okano H: Requirement for COUP-TFI and II in the temporal specification of neural stem cells in CNS development. Nat. Neurosci. 11(9): 1014-1023, 2008.
  3. Nagoshi N, Shibata S, Kubota Y, Nakamura M, Nagai Y, Satoh E, Okada Y, Mabchi Y, Katoh H, Okada S, Fukuda K, Suda T, Matsuzaki Y, Toyama Y, Okano H.: Ontogeny and Multipotency of Neural Crest-Derived Stem Cells in Bone Marrow, Dorsal Root Ganglia and Whisker Pad of Adult Rodents. Cell Stem Cell. 2: 392-403, 2008.
  4. Kaneko S, Iwanami A, Nakamura M, Kishino A, Kikuchi K, Shibata S, Okano HJ, Ikegami T, Moriya A, Konishi O, Nakayama C, Kumagai K, Kimura T, Sato Y, Goshima Y, Taniguchi M, Ito M, He Z, Toyama Y, and Okano H: A selective Sema3A-inhibitor enhances regenerative responses and functional recovery of the injured spinal cord. Nat. Med. 12 (12): 1380-1389, 2006.
  5. Okada S, Ishii K, Miyao T, Shimzakai T, Katoh H, Yamane J, Yoshimura A, Iwamoto Y, Nakamura M, Toyama Y, Okano H.: Conditional ablation of STAT3 or SOCS3 discloses a dual role for reactive astrocytes after spinal cord injury. Nat. Med. 12 (7): 829-834, 2006.

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