217

STRATEGIES TOWARD CNS-REGENERATION USING INDUCEDPLURIPOTENT STEM CELLSHIDEYUKI OKANO
hidokano@sc.itc.keio.ac.jp
Department of Physiology, Keio University School of Medicine, 35 Shinanomachi,
Shinjuku-ku, Tokyo 112-0012, Japan
Induced pluripotent stem (iPS) cells are pluripotent stem cells directly reprogrammed from cultured
mouse fibroblast by introducing Oct3/4, Sox2, c-Myc, and Klf4. Cells obtained using this technology,
which allows the ethical issues and immunological rejection associated with embryonic stem (ES)cells to be avoided, might be a clinically useful source for cell replacement therapies. Here wedemonstrate that murine iPS cells formed neurospheres that produced electrophysiologicallyfunctional neurons, astrocytes, and oligodendrocytes. Secondary neurospheres (SNSs) generated
from various mouse iPS cell showed their neural differentiation capacity and teratoma formation
after transplantation into the brain of immunodeficient NOD/SCID mice. We found that origin(source of somatic cells) of the iPS cells are the crucial determinant for the potential tumorigenicityof iPS-derived neural stem/progenitor cells and that their tumorigenicity results from the persistentpresence of undifferentiated cells within the SNSs. Furthermore, transplantation of non-tumorigenic
Nanog-iPS-derived SNSs into mouse spinal cord injury (SCI) model promoted locomotor function
recovery. Surprisingly, SNSs derived from c-Myc minus iPS cells generated without drug selection
showed robust tumorigenesis, in spite of their potential to contribute adult chimeric mice without
tumor formation..
Keywords: neural stem/precursor cells, induced pluripotent stem (iPS) cells, regeneration, spinal
cord injury1.

Introduction

It had been long believed that adult mammalian central nervous system (CNS) do notregenerate upon their injury. However, we wanted to challenge this dogma taking
advantage of stem cell technology. The major strategies for the regeneration of thedamage CNS would include, i) Transplantation of stem/ progenitor cells, and ii)
Activation of endogenous stem cells and self-repair mechanisms[1-4]. We haveinvestigated transplantation therapy for spinal cord injury (SCI) model for more than tenyears using cell sources including fetal CNS-derived neural stem/progenitor cells(NS/PCs) [5-8], ES-derived NS/PCs, induced pluripotent stem (iPS) cells-derived
NS/PCs and neural crest stem cells [9, 10]. Here, I will mainly talk about cell therapy for
SCI model using iPS cells-derived NS/PCs.

2.1. Cell Therapy for SCI Model Using iPS Cells-Derived NS/PCs
The iPS cells are pluripotent stem cells directly reprogrammed from cultured mouse
fibroblasts by the introduction of Oct3/4, Sox2, c-Myc, and Klf4 [11]. Various types of218 H. Okano
induced pluripotent stem (iPS) cells, each exhibiting different biological properties, havebeen established by different methods [12]. Their properties, including theirdifferentiation potentials and tumorigenic activities (teratoma-forming potentials) indifferent contexts, should be investigated in detail from the aspects of safety and efficacy
of cell transplantation therapies.

2.2. Evaluation of the Safety of NS/PCs Derived from iPS Cells
First, we evaluated the safety of mouse-derived induced pluripotent stem (iPS) cells
through secondary neurosphere (SNS) formation, according to the method for neuraldifferentiation of mouse ES cells we reported [13, 14]. We generated SNSs from various
mouse iPS cell clones and examined their neural differentiation capacity and teratomaforming potential after transplantation into the brains of immunodeficient NOD/SCID
mice. In collaboration with Prof. Shinya Yamanaka’s laboratory, we used 36 iPS cellclones, which differ in (i) their origin (source somatic cells), (ii) presence or absence ofc-Myc retroviral transduction, and (iii) presence or absence of drug selection for Nanog
expression. While the NS/PCs generated from the various mouse iPS cells showedsimilar neural differentiation capacity, they showed enormously different teratomaforming potential after transplantation into the brain (striatum) of NOD/SCID mice.
Accordingly, we came to the conclusion that the origin of the iPS cells are a crucial
determinant of the tumorigenic potential of iPS-derived NS/PCs and that theirtumorigenic potential is co-related to the persistent presence of undifferentiated cells
within the iPS-cell-derived NS/PCs[15].
2.3. Transplantation of NS/PCs Derived from Pre-Evaluated Safe iPS Cells
Next, we also demonstrated that transplantation of pre-evaluated "safe" iPS-cell-derived
NS/PCs into a mouse model of spinal cord injury (SCI) resulted in graft-derived
neurogenesis and myelination, various non-cell autonomous trophic effects against thehost spinal cords, and long-lasting recovery of locomotor function, without tumor
formation. On the other hand, transplantation of NS/PCs (SNSs), derived from preevaluated ‘unsafe’ iPS cells, judged from the persistent presence of undifferentiated cells
within the iPS-derived neurpsheres and/or robust teratoma formation potential upontransplantation into NOD/SCID mice brains, resulted in a certain degree of functional
recovery for a short period. However, it was eventually followed by teratoma formation
within the spinal cord and deterioration of locomotor function, possibly due to the tumor
mass effects.
Thus, properly pre-evaluated iPS clone-derived cells must be a promising cell source for
future transplantation therapy. Our present study is the first demonstration of thetherapeutic potential of iPS-derived NS/PCs for the repair of SCI. Taken together, prior
to the start of clinical trials of human CNS disorders using iPS cells, it is essential to preevaluate each iPS cell clone carefully, and to conduct preclinical transplantation tests
using appropriate primate models under stringent clinical settings. I would also like toStrategies Toward CNS-Regeneration Using Induced Pluripotent Stem Cells 219
describe about the results of our recent experiments involving transplantation of human
iPS-derived NS/PCs into a mouse model of SCI.

Acknowledgments
This work was done by Okano laboratory in Department of Physiology, Keio University
School of Medicine in collaboration with Spinal Cord Injury Research Team atDepartment of Orthopedic Surgery, Keio University School of Medicine and Yamanaka
Laboratory at Kyoto University. This work was supported by Grants-in-Aid for Scientific
Research from JSPS and the Ministry of Education, Culture, Sports, Science andTechnology of Japan (MEXT) and by a Grant-in-aid for the Global COE program fromMEXT to Keio University.

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