Grafted human-induced pluripotent stem-cell–derived
neurospheres promote motor functional recovery after
spinal cord injury in mice
Satoshi Noria,b,1, Yohei Okadaa,c,1, Akimasa Yasudaa,b, Osahiko Tsujib, Yuichiro Takahashia,b, Yoshiomi Kobayashia,b,Kanehiro Fujiyoshib, Masato Koiked, Yasuo Uchiyamad, Eiji Ikedae,f, Yoshiaki Toyamab, Shinya Yamanakag,
Masaya Nakamurab,2, and Hideyuki Okanoa,2a

Department of Physiology, bDepartment of Orthopaedic Surgery, cKanrinmaru-Project, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan;
Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Bunkyoku, Tokyo 113-8421, Japan; eDepartment of
Pathology, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan; fDepartment of Pathology, Yamaguchi University Graduate School of
Medicine, Ube, Yamaguchi 755-8505, Japan; and gCenter for iPS Cell Research and Application, Kyoto University, Shogoin, Sakyoku, Kyoto 606-8507, Japand

Edited by Fred H. Gage, The Salk Institute, San Diego, CA, and approved September 1, 2011 (received for review May 20, 2011)

stem-cell–based medicine
synaptic connection| cell transplantation | neurotrauma |S

tem-cell–based approaches, such as the transplantation ofneural stem/progenitor cells (NS/PCs), are promising sourcesof therapies for various central nervous system disorders (1–3).
Previous studies reported functional recovery after transplantation of NS/PCs into the injured spinal cord of rodents andnonhuman primates (4–9). Furthermore, recent studies revealed
that embryonic stem cells (ESCs) can generate neural cells including NS/PCs (10–12) and oligodendrocyte precursor cells(OPCs) (13, 14). Therefore, human ESC-based therapies are
moving out of the laboratory and into clinical treatments forspinal cord injury (SCI) (12, 13, 15). However, the use of human
ESC-based therapies is complicated by ethical concerns in certain countries. To avoid the problems associated with ESCs, wepreviously established induced pluripotent stem cells (iPSCs)
from mouse ﬁbroblasts (16, 17) and conﬁrmed the therapeuticpotential of iPSC-derived neurospheres (iPSC-NSs) for treating
SCI in animal models (18).
Here, aiming at human iPSC-based therapies for SCI patients,
we examined the therapeutic potential of human iPSC-NSs bytransplanting them into nonobese diabetic severe combined immunodeﬁcient (NOD-SCID) SCI model mice. We used a clone
from human iPSCs (hiPSCs) that we established from adulthuman dermal ﬁbroblasts by the retroviral transduction of four
reprogramming factors; for the clone used in this study, 201B7,
www.pnas.org/cgi/doi/10.1073/pnas.1108077108

the factors were Oct3/4, Sox2, Klf4, and c-Myc (19). These
grafted hiPSC-NSs survived, migrated, and differentiated into
the three neural lineages in the injured spinal cord. They promoted angiogenesis and axonal regrowth and preserved myelination, and some formed synapses with host mouse neurons.
These positive effects promoted functional recovery that persisted for up to 112 d after SCI, without tumor formation.
These ﬁndings indicated that neurospheres derived from
hiPSCs are a potential cell source for transplantation therapy
for SCI.
Results
Grafted hiPSC-NSs Survived, Migrated, and Differentiated into Three
Neural Lineages. Contusive SCI was induced at the Th10 level in

NOD-SCID mice, and 5 × 105 Venus+ hiPSC-NSs or PBS was
injected into the lesion epicenter, 9 d after injury. To examine
the effects of grafted hiPSC-NSs in the injured spinal cord, histological analyses were performed 56 d after SCI [after functional recovery, based on the Basso mouse scale (BMS) score,
was observed to plateau]. Ten mice in each group were killed on
day 56, and 18 mice grafted with hiPSC-NSs and 16 PBS-injected
mice remained. These mice were assessed by BMS and for longterm safety of the grafted hiPSC-NSs, 112 d after SCI (Table S1).
On day 56, the grafted hiPSC-NSs had survived and migrated
into the host spinal cord (Fig. 1 A and B). To examine theirdifferentiation potentials, we performed immunohistochemical
analyses and quantiﬁed the proportion of Venus+ cells immunopositive for cell-type–speciﬁc markers. The engrafted hiPSCNSs differentiated into neuronal nuclei (NeuN)+ and β-tubulin
isotype III (βIII tubulin)+ neurons, glial ﬁbrillary acidic protein(GFAP)+ astrocytes, and adenomatous polyposis coli CC-1
(APC)+ oligodendrocytes (Fig. 1 C–F). The βIII tubulin+/Venus+ neurons comprised 49.1 ± 2.0% of the Venus+ cells, andthe mature NeuN+/Venus+ neurons comprised 22.9 ± 1.0%.
Thus, 56 d after SCI, about 50% of the grafted hiPSC-NSs had
differentiated into neurons, about half of which were matureneurons. GFAP+/Venus+ astrocytes comprised 17.0 ± 1.2%, but
APC+/Venus+ oligodendrocytes were rare (3.0 ± 0.4%). Nestin+/Venus+ NS/PCs made up 10.7 ± 2.2% of the total (Fig. 1G).

Author contributions: Y.U., Y. Toyama, S.Y., M.N., and H.O. designed research; S.N., Y.O.,
A.Y., O.T., Y. Takahashi, Y.K., K.F., M.K., and E.I. performed research; S.N., M.K., and E.I.
analyzed data; and S.N., Y.O., M.K., M.N., and H.O. wrote the paper.
The authors declare no conﬂict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1

S.N. and Y.O. contributed equally to this work.

2

To whom correspondence may be addressed. E-mail: hidokano@a2.keio.jp or masa@
sc.itc.keio.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1108077108/-/DCSupplemental.

PNAS | October 4, 2011 | vol. 108 | no. 40 | 16825–16830

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Once their safety is conﬁrmed, human-induced pluripotent stem
cells (hiPSCs), which do not entail ethical concerns, may becomea preferred cell source for regenerative medicine. Here, we investigated the therapeutic potential of transplanting hiPSC-derived neurospheres (hiPSC-NSs) into nonobese diabetic (NOD)severe combined immunodeﬁcient (SCID) mice to treat spinal cord
injury (SCI). For this, we used a hiPSC clone (201B7), established bytransducing four reprogramming factors (Oct3/4, Sox2, Klf4, and cMyc) into adult human ﬁbroblasts. Grafted hiPSC-NSs survived,
migrated, and differentiated into the three major neural lineages(neurons, astrocytes, and oligodendrocytes) within the injured
spinal cord. They showed both cell-autonomous and noncellautonomous (trophic) effects, including synapse formation between hiPSC-NS–derived neurons and host mouse neurons, expression of neurotrophic factors, angiogenesis, axonal regrowth,and increased amounts of myelin in the injured area. These positive effects resulted in signiﬁcantly better functional recovery
compared with vehicle-treated control animals, and the recovery
persisted through the end of the observation period, 112 d postSCI. No tumor formation was observed in the hiPSC-NS–grafted
mice. These ﬁndings suggest that hiPSCs give rise to neural
stem/progenitor cells that support improved function post-SCI
and are a promising cell source for its treatment.

caudal

A

B

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NeuN

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hSyn(h)/ßIIIhSyn(h)tub/ Bsn(m)Bsn(m)/ßIII tub/
HNu(h)/Hoechst HNu(h)/Hoechst

rostralhSyn(h)/ChAT/
Hoechstt

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(m): mouse specific antibody (h): human specific antibody

VenusßIII tubulin

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F-2

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APC

% of Venus positive cells

G

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Venus

Venus

Fig. 2. Evidence for synapse formation between hiPSC-derived neurons andhost mouse spinal cord neurons. (A) Sections were triple-stained with HNu(green), βIII tubulin (red), and the presynaptic marker Bassoon (Bsn, white).
The Bsn antibody used here recognized the rat and mouse, but not human,protein. (B) Sections triple-stained for HNu (green), βIII tubulin (red), andthe human-speciﬁc presynaptic marker hSyn (white). (C) Confocal images
showing a large number of somatic and dendritic terminals from graft-derived nerve cells on host motor neurons at the ventral horns. (D) Electron
microscopy showing synapse formation between host mouse neurons andgraft-derived Venus+ (black) human neurons: the pre- and postsynaptic
structures indicated transmission from a host neuron to a graft-derived
neuron (D-1) and from a graft-derived neuron to a graft-derived neuron (D2). H, host neuron; G, graft-derived neuron; arrowheads, postsynaptic density. (Scale bars, 20 μm in A-1, A-2, B-1, B-2, C-1, and C-2; 10 μm in A-3, B-3,
and C-3; and 0.5 μm in D.)

60

H-1

50

H-2
H-3

40
30
20
10
0
NeuN ßIII tub GFAP APC Nestin

Fig. 1. In vivo differentiation of hiPSC-NSs. (A and B) Venus+ hiPSC-NSs were
integrated at or near the lesion epicenter (arrowheads). (Scale bars, 1000 μm
in A; 100 μm in B.) (C–F) Representative images of Venus+-grafted cells labeled with the neural markers NeuN+ (mature neurons) (C); βIII tubulin+ (allneurons) (D); GFAP+ astrocytes (E); and APC+ oligodendrocytes (F). (Scale bar,
20 μm.) (G) Percentages of cell-type–speciﬁc marker-positive cells among the
Venus+-grafted cells 56 d after SCI. Values are means ± SEM (n = 4). (H) Most
hiPSC-derived neurons differentiated into GAD67+ (GABAergic) neurons.
(Scale bars, 50 μm in H-1; 20 μm in H-2; and 10 μm in H-3.)

Because 22.9% of the hiPSC-NSs differentiated into matureneurons, we next examined their neurotransmitter phenotype,using neurotransmitter-speciﬁc markers. Of the Venus+ cells,
15.8 ± 2% were glutamic acid decarboxylase 67 (GAD67)+, indicating that 69% (15.8/22.9% = 69.0%) of the hiPSC-NS–
derived mature neurons were GABAergic (Fig. 1H). We also
found small numbers of Venus+ tyrosine hydroxylase (TH)+neurons and choline acetyltransferase (ChAT)+ cholinergic neurons (Fig. S1 A and B).
Synapse Formation Between hiPSC-Derived Neurons and Host Mouse
Neurons. To evaluate the ability of the hiPSC-NS–derived

neurons to integrate with the host neural circuitry, triple immunostaining was performed with antibodies to human nuclear
protein (HNu), βIII tubulin, and the presynaptic protein Bassoon(Bsn). The anti-Bsn antibody is a monoclonal that selectively
recognizes mouse and rat, but not human epitopes. Grafted βIII
tubulin+/HNu+ cells in the neural parenchyma were observedin contact with the synaptic boutons of host neurons (Fig. 2A).
In addition, triple immunostaining for HNu, βIII tubulin, andhuman-speciﬁc synaptophysin (hSyn) revealed dense ﬁelds of
16826 | www.pnas.org/cgi/doi/10.1073/pnas.1108077108

boutons apposed to βIII tubulin+/HNu− host mouse neurons(Fig. 2B). These host neurons in the ventral gray matter were
ChAT+, and some of the boutons represented graft-speciﬁc
terminals (Fig. 2C). Immunoelectron microscopy revealed Venus+ (i.e., human) presynaptic and postsynaptic structures andsynapses between host mouse neurons and Venus+ hiPSC-derived neurons at the injured site (Fig. 2D).
Transplantation of hiPSC-NSs Enhanced Angiogenesis and Axonal
Regrowth but Did Not Induce Abnormal Innervation of Pain-Related
CGRP+ Afferents After SCI. To evaluate the effects of hiPSC-NS

transplantation on angiogenesis after SCI, immunohistochemical analyses for platelet endothelial cell adhesion molecule-1(PECAM-1) were performed. There were signiﬁcantly more
PECAM-1+ blood vessels at the lesion epicenter in the hiPSCNS group than in the control group (Fig. 3 A–C). To determine
the source of the angiogenic signals, we examined the vascularendothelial growth factor (VEGF) expression in the grafted
spinal cord by immunohistochemistry (Fig. 3D). Quantitative
analyses revealed that the VEGF+ area at the lesion epicenter
was signiﬁcantly larger in the hiPSC-NS group than in the control
group (Fig. 3E). Furthermore, both GFAP+/Venus+ hiPSC-derived astrocytes (Fig. 3F) and GFAP+/Venus− host mouse
astrocytes expressed VEGF (Fig. 3G), consistent with the resultsof RT-PCR (Fig. 3 H and I). Note that the mouse Vegf mRNA
expression level was higher in the hiPSC-NS–grafted mice thanin PBS-injected mice.
Because angiogenesis generally improves tissue sparing, we
examined the atrophic changes of the injured spinal cord byhematoxylin–eosin (H&E) staining. Unlike the hiPSC-NS group,
atrophic changes of the injured spinal cord were prominent in
the control group (Fig. 3 J and K). Quantitative analysis revealed
signiﬁcant differences in the transverse area of the spinal cord
between the control and hiPSC-NS group, suggesting that thehiPSC-NS transplantation prevented atrophy of the injured spinal cord (Fig. 3L). Luxol fast blue (LFB) staining also revealed
Nori et al.

hiPSC-NS
A-2

B-2

Number of PECAM-1
positive blood vessels

B-1

300

*

200

100

0
hiPSC-NS

control

VEGF positive area(mm2)

PECAM-1 / HNu

C

E

D-2

VEGF

D-1

0.02

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0.015
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hiPSC-NS

control

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F-1

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hiPSC-NS-grafted mice
cultured hiSPC-NSs

Mouse Vegf mRNA levels

Human VEGF mRNA levels

H

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**

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**

0.9 0.6 0.3
Rostral (mm)00.3 0.6 0.9
Caudal (mm)

0.5
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N-2

LFB positivemyelinated area(mm2)

LFB staining

Vegfc

control
**

O

N-1

Vegfb

Vegfa, b and c: mouse Vegf family members

Spinal cord area(mm2)control

hiPSC-NS-grafted micecontrol (PBS) mice

2

VEGFC

VEGFA, B and C: human VEGF family members

J-1

3

control
0.025

hiPSC-NS

0.02
**
*

0.015

*

**
**

0.01
0.005
0

0.9 0.6 0.3 0 0.3 0.6 0.9
Rostral (mm) Caudal (mm)Fig. 3. Transplanted hiPSC-NSs enhanced angiogenesis and prevented
atrophic changes and demyelination after SCI. (A and B) Representative
images of PECAM-1+ blood vessels. (C) Quantitative analysis of PECAM-1+
blood vessels at the lesion epicenter. Values are means ± SEM (n = 4). (D)Representative images of axial sections stained for VEGF. (E) Quantitative
analysis of the VEGF+ area at the lesion epicenter. Values are means ± SEM(n = 4). (F and G) Expression of VEGF in GFAP+ astrocytes among Venus+graft–derived human cells (yellow indicates VEGF+, GFAP+, and Venus+ cells)(F) and host mouse-derived cells (G) in the spinal cord. (H) Expression ofhuman VEGF mRNA (VEGFA, -B, and -C are the human VEGF family members)
5 d after the hiPSC-NSs were transplanted (black bars) compared with cultured hiPSC-NSs before transplantation (gray bars). Values are means ± SEM(n = 3, each). Human VEGF expression was undetectable in the spinal cord ofmice treated with PBS. (I) Expression of mouse Vegf mRNA (Vegfa, -b, and -c
are the mouse Vegf family members) 5 d after hiPSC-NS transplantation(black bars) or PBS injection (gray bars) into the spinal cord. The mouse Vegf
expression level was higher in the hiPSC-NS–grafted mice than in PBSinjected mice. Values are means ± SEM (n = 3, each). (J and K) Representative
H&E-stained images of sagittal and axial sections at the lesion epicenter 56 dafter SCI. (L) Quantitative analysis of the spinal cord area measured in H&E-

Nori et al.

a greater preservation of myelinated areas in the hiPSC-NS
group compared with the control group (Fig. 3 M–O).
To evaluate the effects of hiPSC-NS transplantation on axonal
regrowth after SCI, we examined the expression of neuroﬁlament 200 kDa (NF-H), 5-hydroxytrytamine (5HT), and growthassociated protein 43 (GAP43) in the injured spinal cord byimmunohistochemistry. There were signiﬁcantly more NF-H+
neuronal ﬁbers in the hiPSC-NS group than in the control group(Fig. 4 A and B). 5HT+ ﬁbers, descending serotonergic raphespinal tract axons that are important for the motor functional
recovery of hind limbs (20–22), were observed at the lumbar
intumescence in all mice (Fig. 4C). Contusive SCI resulted ina signiﬁcant decrease in the number of 5HT+ ﬁbers. Relative to
mice at 7 d after SCI (2 d before the transplantation), by 56
d after SCI a slight but signiﬁcant increase in the 5HT+ ﬁber area
was seen in the control group, and the hiPSC-NS group showed
an even greater enhancement (Fig. 4 C and D).
GAP43+ axons, which are regrowing (23), were detected in the
distal cords of all mice, but in the hiPSC-NS group, there weresigniﬁcantly more GAP43+ ﬁbers in the ventral region 1 mm
caudal to the lesion epicenter (Fig. 4 E and F), suggesting thatthe hiPSC-NS transplantation promoted axonal regrowth in the
injured spinal cord. We also observed hiPSC-NS–derived astrocytes closely associated with NF-H+ ﬁbers and 5HT+ ﬁbers (Fig.
4 G and H). Moreover, RT-PCR revealed the expression ofneurotrophic factors (NGF, BDNF, and hepatocyte growth factor, HGF), which are associated with the axonal growth andsurvival of existing neurons, by both the grafted human cells andthe host mouse tissues (Fig. 4 I and J).
To examine the effect of hiPSC-NSs on structural changes inpain afferents entering the dorsal horn of the spinal cord aboveand below the injured spinal segments, we investigated the distribution of calcitonin gene-related peptide (CGRP+) ﬁbers,
which are involved in peripheral and spinal pain mechanisms (6,24, 25). We quantiﬁed the areas of CGRP+ ﬁbers in lamina III, 4
mm rostral and 4 mm caudal to the lesion epicenter. There wereno signiﬁcant differences in the areas of CGRP+ ﬁbers in lamina
III between the hiPSC-NS and control groups (Fig. S2 A–C).
Transplanted hiPSC-NSs Promoted Motor Functional and Electrophysiological Recovery After SCI. We evaluated the motor func-

tional recovery by BMS score, Rotarod test, and the DigiGait
system. The BMS score showed signiﬁcantly better functional
recovery in the hiPSC-NS than the control group 21 d after SCI
and thereafter (Fig. 5A). In the Rotarod test, the hiPSC-NS
group showed a signiﬁcantly longer running time than the control group 56 d post-SCI (Fig. 5B). The DigiGait system measures the treadmill gait, as an objective evaluation of motor function (26, 27). Whereas all of the hiPSC-NS–grafted mice (n =18) could walk on the treadmill at 8 cm/s, a subset of the control
mice (4 out of 16) could not maintain this speed. The proﬁle ofstride length at 8 cm/s clearly demonstrated a signiﬁcantly betterrecovery of motor function in the hiPSC-NS–grafted mice compared with the 12 control mice that could walk at this speed(Fig. 5C).
Motor-evoked potential (MEP) was used to measure thefunctional recovery in all of the mice electrophysiologically. The
latency of the motor-evoked potential was also measured, fromthe onset of stimulus to the ﬁrst response of each wave. At 112 dafter SCI, waves were detected in most of the hiPSC-NS group(14 of 17 mice), but none were detected in the control group (0of 15 mice) (Fig. 5D). The average signal-to-response latency inthe hiPSC-NS group was 4.6 ± 0.1 ms. Consistent with thestained axial sections at different regions. Values are means ± SEM (n = 6).
(M and N) Representative LFB-stained images of the axial sections at thelesion epicenter 56 d after SCI. (O) Quantitative analysis of the myelinatedarea by LFB-stained axial sections at different regions. Values are means ±SEM (n = 6). *P < 0.05, **P < 0.01. (Scale bars, 500 μm in A, K, and M; 200 μm
in B; 100 μm in D and N; 20 μm in F-1 and G; 10 μm in F-2; and 1,000 μm in J.)

PNAS | October 4, 2011 | vol. 108 | no. 40 | 16827

NEUROSCIENCE

control
A-1

D

F
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G-2

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1200

Human trophic factors
mRNA levels

I

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cultured hiPSC-NSs

1000
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BDNF

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6

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7 14 21 28 35 42 49 56

Days after injury
2 ms

0
0.003

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SCI(56 d after SCI)*

0.00250.002
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8control

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6
4
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Bdnf

Hgf

Fig. 4. Transplanted hiPSC-NSs enhanced axonal growth after SCI. (A)Representative images of sagittal sections stained for NF-H at the lesion
epicenter. (B) Quantitative analysis of the NF-H+ area. Values are means ±SEM (n = 4). (C) Representative images of axial sections stained for 5HT at
the lumbar intumescence. (D) Quantitative analysis of the 5HT+ area at the
lumbar intumescence in axial sections. Values are means ± SEM (n = 6 each inthe 7 d and control 56 d after SCI groups, and n = 5 in the hiPSC-NS (56d after SCI) group). (E) Representative images of midsagittal sections stainedfor GAP43 in the ventral region 1 mm caudal to the lesion epicenter. (F)Quantitative analysis of the GAP43+ area in midsagittal sections. Values aremeans ± SEM (n = 4). (G and H) NF-H+ neural ﬁbers and 5HT+ (serotonergic)ﬁbers extended in association with GFAP+/Venus+-graft–derived humanastrocytes. (I) Expression of human neurotrophic factor mRNAs (NGF, BDNF,
and HGF) 5 d after hiPSC-NS transplantation (black bars) compared with
cultured hiPSC-NSs before transplantation (gray bars). Values are means ±SEM (n = 3, each). (J) Expression of mouse neurotrophic factor mRNAs (Ngf,
Bdnf, and Hgf) 5 d after hiPSC-NS transplantation (black bars) or PBS injection (gray bars) into the spinal cord. Values are means ± SEM (n = 3, each).
*P < 0.05, **P < 0.01. (Scale bars, 100 μm in A; 50 μm in C, E, and H-1; 20 μm
in G-1, H-2, and H-3; and 10 μm in G-2.)

electrophysiology results, α-CaM kinase 2+ descending motor
axons were observed to persist in the lesion epicenter in thehiPSC-NS group (Fig. S3).
Long-Term Observation Revealed No Tumor Formation After hiPSC-NS
Transplantation. To investigate the long-term safety of the grafted

hiPSC-NSs, we extended the follow-up period to 112 d after SCI.
Motor functional recovery was maintained in the hiPSC-NS
group for the entire period (Fig. 6A), the hiPSC-NS–grafted
mice showed no tumor formation (Fig. 6B), and the grafted
cells exhibited normal neural differentiation (Fig. 6C). We
determined the proportion of Venus+ grafted cells that were
immunopositive for each cell-type–speciﬁc marker 112 d after
SCI. The grafted hiPSC-NSs had differentiated into NeuN+(40.2 ± 2.8%), βIII tubulin+ (50.7 ± 2.0%), GFAP+ (18.1 ±
2.2%), APC+ (8.9 ± 1.6%), and Nestin+ (7.5 ± 1.0%) cells. For
comparison, at 56 d after SCI, the grafted cells had differentiated16828 | www.pnas.org/cgi/doi/10.1073/pnas.1108077108**

2Transplantation

0.0005**C
100

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0

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**

4

0.001

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HGF

**

**

Rotarod test

0.1

B

A

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Stride length (cm)hiPSC-NS

Treadmill gait analyses

control*

total run time (sec)

0.15

GFAP/Venus/5HT
5HT/Hoechst

Mouse trophic factors
mRNA levels

GFAP/Venus/NF-H

5HT positive area(mm2)

NF-H

C-2

5HT

C-1

GAP43 positive area(mm2)B

NF-H positive area(mm2)hiPSC-NS
A-2

BMS score

control
A-1

control

hiPSC-NS

Fig. 5. Transplanted hiPSC-NSs promoted motor functional and electrophysiological recovery after SCI. (A) Motor function in the hindlimbs wasassessed weekly by the BMS score for 56 d. Values are means ± SEM. (B)Rotarod test 56 d after SCI. Graph shows the total run time. Values aremeans ± SEM. (C) Treadmill gait analysis using the DigiGait system 56 d after
SCI. Graph shows stride length. Values are means ± SEM. (D) Electrophysiological analysis performed 112 d after SCI. MEP waves were detected inmost of the hiPSC-NS group (14 out of 17), whereas they were not detectedin the control group (0 out of 15). **P < 0.01. Behavioral analyses wereassessed by two observers who were blind to the treatment.

into NeuN+ (22.9 ± 1.0%), βIII tubulin+ (49.1 ± 2.0%), GFAP+(17.0 ± 1.2%), APC+ (3.0 ± 0.4%), and Nestin+ (10.7 ± 2.2%)
cells (Fig. 6D). Notably, signiﬁcantly higher percentages of
NeuN+ mature neurons and APC+ oligodendrocytes were
present at 112 d than at 56 d after SCI, whereas the percentage
of graft-derived human Nestin+ NS/PCs slightly decreased. We
also examined the amount of proliferation among the grafted
cells by Ki-67 labeling. The percentage of Ki-67+/HNu+ cells was
signiﬁcantly decreased between 56 d post-SCI (1.1 ± 0.2%) and
112 d post-SCI (0.7 ± 0.1%). The Ki-67+/HNu+ cells were dispersed throughout the graft area without evidence of clustering
at particular sites.
Discussion
In the present study, we used clone 201B7 of hiPSC-derived
neurospheres as a cell source to treat SCI in adult NOD-SCID
mice. We demonstrated that the hiPSC-NSs differentiated into
neurons, astrocytes, and oligodendrocytes in the injured spinal
cord and promoted motor functional recovery. Hypothetically,
the transplantation of hiPSC-NSs could result in a wide range of
positive effects, including angiogenesis, axonal regeneration, andlocal-circuitry reconstruction, which have been reported in previous studies using rodent ESCs or iPSCs for SCI treatment (12,18). All these mechanisms could contribute to motor functional
recovery after hiPSC-NS transplantation for SCI.
Angiogenesis after SCI promotes endogenous repair andsupports axonal outgrowth (28, 29). Here we observed that thetransplantation of hiPSC-NSs enhanced angiogenesis and tissue
sparing after SCI. Astrocytes express angiogenic growth factors
such as VEGF under hypoxic conditions (12, 30, 31), and we
observed that the hiPSC-derived astrocytes and host astrocytes
expressed VEGF, suggesting that the transplantation of hiPSCNSs promoted VEGF expression in both host- and graftderived astrocytes.
Motor functional recovery was also supported by the axonal
regrowth that was promoted by the transplanted hiPSC-NSs. Our
immunohistochemical analyses revealed grafted hiPSC-derived
GFAP+ astrocytes closely associated with NF-H+ and 5HT+ﬁbers. Graft-derived astrocytes are reported to promote theregrowth of NF-H+ and 5HT+ ﬁbers by offering a growth-permissive substrate (12, 18, 32). Furthermore, neurotrophic factors
such as NGF, BDNF, and HGF play critical roles in axonal
growth and in the survival of existing neurons (33–38). Consistent with these reports, we observed that hiPSC-NS transplantation promoted axonal regrowth at the distal spinal cord.
Because our treatment induced axonal regrowth, we also examNori et al.

BMS score**** ** **** ** ** ** ** ** ** ** **

**

4

2
Transplantation
00

hiPSC-NS

control

14 28 42 56 70 84 98 112

60
50

C

**

40
30
20

**

10
0
NeuN ßIII tub GFAP APC Nestin

Days after injury

B

56 d
112 d

C-1

C-2

C-3

Venus

NeuN

Merge

C

Fig. 6. Long-term observation revealed no tumor formation after hiPSC-NS
transplantation. (A) For 112 d, motor function in the hindlimbs was assessed
weekly by the BMS score. Values are means ± SEM. (B) Representative H&E
image of hiPSC-NS–grafted mice. (C) Boxed area in B. (C-1–C-3) Immunohistochemistry showing normal neural differentiation of the grafted cells.
(D) Percentages of cell-type–speciﬁc marker-positive cells among the Venus+
human cells 56 and 112 d after SCI. Values are means ± SEM (n = 4 and 5,
respectively). **P < 0.01. (Scale bars, 500 μm in B; and 50 μm in C.)

ined the pain afferents entering lamina III of the spinal cord,
which is closely associated with NS/PC-transplantation–induced
allodynia (6). Our analysis revealed no signiﬁcant differences
between the hiPSC-NS and control groups. Thus, althoughallodynia was not assessed, our morphological data suggested
that hiPSC-NS transplantation did not induce abnormal innervation by pain afferents, in contrast to a previous study inwhich NS/PCs derived from adult rat spinal cord were transplanted into a rat SCI model (6).
The motor functional recovery observed in this study may also
be due to the formation of synapses between hiPSC-derived
neurons and host mouse spinal cord neurons. Our present data
support the potential for grafted hiPSC-NSs to form synapses
with host neurons in the injured spinal cord. Previous studies
have shown post-SCI functional improvements associated withintraspinal grafts containing neuronal progenitors either alone orin combination with other cells or interventions (4, 7, 39–43).
Such studies also report that graft-derived human neurons canreceive projections from host mouse neurons and that their extended processes make synapses with host neurons (39). It is
likely that local neurons in the lesioned area (a mixture of preserved host neurons and graft-derived neurons) transmit signals
by relay.
We observed that most of the hiPSC-derived mature neurons
were GABAergic, a neurotransmitter type that plays importantroles in the spinal cord by controlling the levels of motoneuronal
output and sensory input, by modulating primary afferent
transmitter release, and by direct postsynaptic inhibition of
motoneurons (44, 45). Furthermore, hypofunctioning spinal
GABAergic inhibition is involved in pathological pain states thatdevelop due to SCI (46). Thus, synapse formation by donorderived GABAergic neurons might be important for motor coordination within the spinal neuronal network and for the suppression of SCI-induced spasticity (8, 47) and pain (46).
The greatest potential drawback of hiPSC-based therapies istheir potential for tumorigenicity. Therefore, we observed thetreated animals for an extended period. Not only was functional
recovery maintained in the hiPSC-NS group for 112 d after SCI,
but no tumor formation occurred at all. Quantitative analysis of
the phenotype of the grafted hiPSC-NSs revealed an increase in
the percentages of NeuN+ mature neurons and APC+ oligodendrocytes at 112 d compared with 56 d after SCI, showing thatmost of the grafted hiPSC-NSs successfully differentiated into
mature neural cells over time. Some Nestin+/HNu+ cells and Ki67+/HNu+ cells were still present at 112 d post-SCI, although
their proportions were lower than at 56 d after SCI, indicating
Nori et al.

that some grafted cells remained as NS/PCs. However, no evidence of excessive proliferation, clusters of proliferating cells, orother signs of tumor formation were observed in any of thetransplant-receiving mice. Collectively, the lack of tumors, increase in NeuN numbers over time, and low Nestin and Ki-67
numbers support the possibility that this approach will be safein humans.
Consistent with our ﬁndings, previous studies using human
fetal NS/PCs showed some Nestin+/HNu+ cells that were negative for the proliferative markers Ki-67 and proliferating cell
nuclear antigen in the striatum of NOD-SCID mice, 6 mo after
transplantation (48), 28–34% Nestin+/HNu+, and 2–4% Ki-67+/
HNu+ cells were in the spinal cord of NOD-SCID mice 4 moafter transplantation (5), and 11–14% Nestin+/HNu+ and 3–5%
Ki-67+/HNu+ cells were in the spinal cord of nude rats 6 moafter transplantation (49). Notably, no tumor formation wasobserved in any of these studies, despite the presence of Nestin+and Ki-67+–grafted cells, even after long-term observation.
Recently, clinical trials of human stem-cell–based therapy for
SCI have been launched, using human NS/PCs (4, 5) or human
ESC-derived OPCs (15). In this paradigm, OPC-mediated restoration of myelination and trophic effects are the most likely
mechanisms for the resulting beneﬁts. Besides overcoming concerns about immune responses and the ethics of using human
ESCs (hESCs), our present study suggests that grafted hiPSCNSs might be more beneﬁcial than hESCs, because the hiPSCNSs gave rise to GABAergic neurons, which can help to suppress
SCI-induced spasticity and pain (8, 46, 47). In addition, we foundthat hiPSC-NSs differentiated into astrocytes and oligodendrocytes, which can exert positive effects through both cell-autonomous and noncell-autonomous (trophic) mechanisms.
Nevertheless, our present results are only a ﬁrst step towardclinical applications. In our future studies, the safety and effectiveness of hiPSC-derived NS/PCs will be more intensively investigated, for example, using nonhuman primate SCI models (8).
In particular, hiPSCs established by delivering the reprogramming
factors using a different method, such as an integration-free virus
system (50), or a virus-free (51) or transgene-free system (52–54),
or by using (HLA)-homozygous donor-derived cells (55), shouldbe evaluated.
Materials and Methods
Cell Culture, Neural Induction, and Lentivirus Transduction. The cell culture andneural induction of hiPSCs (201B7) were performed as described previously(11, 18, 56) with slight modiﬁcations.
Lentivirus was prepared and transduced into neurospheres as described
previously (11). Brieﬂy, hiPSC-derived primary neurospheres were dissociatedand infected with lentivirus expressing Venus ﬂuorescent protein undercontrol of the EF promoter (pCSII-EF–Venus). These primary neurospheres
were passaged into secondary and tertiary neurospheres and used fortransplantation.
Spinal Cord Injury Model and Transplantation. Adult female NOD-SCID mice
(20–22 g) were anesthetized via an i.p. injection of ketamine (100 mg/kg)
and xylazine (10 mg/kg). Contusive SCI was induced at the Th10 level using
an IH impactor (60 kdyn; Precision Systems and Instrumentation), as described previously (57).
Nine days after the injury, 5 × 105 hiPSC-NSs were transplanted into thelesion epicenter of each mouse (n = 31) using a glass micropipette and stereotaxic injector (KDS310; Muromachi-Kikai). An equal volume of PBS was
injected instead into control mice (n = 29).
Behavioral and Histological Analyses. Behavioral analyses were evaluatedusing the BMS, Rotarod apparatus (Muromachi Kikai), and the DigiGait
system (Mouse Speciﬁcs) (detailed protocols are described in SI Materials andMethods). For histological analyses, mice were deeply anesthetized and intracardially perfused with 4% paraformaldehyde (PFA; pH 7.4). The dissected spinal cords were then sectioned into axial/sagittal sections using
a cryostat (detailed conditions are in SI Materials and Methods). All behavioral and histological analyses were conducted by observers blind to thetreatment. All animal experiments were approved by the ethics committee

PNAS | October 4, 2011 | vol. 108 | no. 40 | 16829

NEUROSCIENCE

D
6% of Venus positive cells

A

ACKNOWLEDGMENTS. We thank Drs. K. Kitamura, N. Nagoshi, M. Mukaino,
A. Iwanami, F. Renault-Mihara, S. Shibata, H. Shimada, T. Harada, S. Miyao,
and H.J. Okano for technical assistance and scientiﬁc discussions; S. Kaneko
for critical proofreading of the manuscript; and all the members of the H.O.
and S.Y. laboratories for encouragement and generous support. We also
thank Dr. K. Takahashi for the undifferentiated iPS cells. This work was
supported by grants from Grants-in-Aid for Scientiﬁc Research from Japan

Society for the Promotion of Science (SPS) and the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (MEXT); the Project forRealization of Regenerative Medicine; and Support for Core Institutes for iPS
Cell Research from the MEXT; Japan Science and Technology–California Institute for Regenerative Medicine collaborative program; the Kanrinmaru
project from Keio University; Research Fellowships for Young Scientists from
JSPS; Keio Gijuku Academic Development Funds; by a Grant-in-Aid for the
Global COE program from MEXT to Keio University; and by a Grant-in-Aid for
Scientiﬁc Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education, Science, Sports and Culture of Japan.

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of Keio University and were in accordance with the Guide for the Care andUse of Laboratory Animals (National Institutes of Health, Bethesda, MD).

16830 | www.pnas.org/cgi/doi/10.1073/pnas.1108077108

Nori et al.