Available online at www.sciencedirect.

com

Experimental Neurology 209 (2008) 368 – 377

www.elsevier.com/locate/yexnr

Review
Stem cells for the treatment of spinal cord injury
Margaret Coutts, Hans S. Keirstead ⁎
Reeve-Irvine Research Center, Stem Cell Research Center, Department of Anatomy and Neurobiology, 2111 Gillespie Neuroscience Research Facility,
College of Medicine, University of California Irvine, Irvine, CA, USA

Received 21 August 2007; revised 29 August 2007; accepted 1 September 2007

Available online 12 September 2007

Abstract

This article reviews stem cell-based strategies for spinal cord injury repair, and practical issues concerning their translation to the clinic. Recent
progress in the stem cell field includes clinically compliant culture conditions and directed differentiation of both embryonic stem cells and
somatic stem cells. We provide a brief overview of the types of stem cells under evaluation, comparing their advantages and disadvantages for use
in human clinical trials. We review the practical considerations and risks that must be addressed before human treatments can begin. With a
growing understanding of these practical issues, stem cell biology, and spinal cord injury pathophysiology, stem cell-based therapies are moving
closer to clinical application.
© 2007 Elsevier Inc. All rights reserved.

Keywords: Human embryonic stem cell; Neural stem cell; Bone marrow stem cell; Differentiation; Clinical trial; Regulatory

Introduction
This review summarizes stem cell-based therapeutic strate-
gies to treat spinal cord injury (SCI). Here we present the
emerging perspective that stem cell administration is a viable
therapeutic strategy and discuss the challenges to its develop-
ment. Pre-clinical research has demonstrated the effectiveness of
some stem cell-based treatments; however, several significant
obstacles must be overcome before this research translates to the
clinic. These hurdles include: identifying the best source of stem
cells, optimizing their characteristics prior to transplant, re-
ducing the risks of stem cell therapy, developing large-scale
manufacturing technologies, and fulfilling regulatory considera-
tions for government approval.
Current treatments for SCI (Baptiste and Fehlings, 2007)
include surgery to stabilize the injury site, high doses of corti-
costeroids to help limit secondary injury processes and reha-
bilitative care. Preclinical studies with neuroprotective agents
(such as minocycline and the Rho antagonist, Cethrinr) are
encouraging, and clinical trials with these agents will soon
begin. While these treatment options may provide benefits,
⁎ Corresponding author. Fax: +1 949 824 5352.
E-mail address: hansk@uci.edu (H.S. Keirstead).
0014-4886/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2007.09.002
clinical improvements are modest and many patients still face
significant neurologic dysfunction and disability.
Stem cells offer several approaches for SCI repair. Stem cell-
based therapies could, 1) replace damaged or diseased cells, 2)
provide a cell-based electrical ‘relay’ between neurons above and
below the injury, 3) ameliorate clinical deterioration and/or
facilitate regeneration by providing neuroprotective or growth
factors, or 4) play other indirect roles such as promoting neovas-
cularization or providing a permissive substrate for regeneration
of endogenous cells.
Pathophysiology of SCI
Transplanting stem cell derivates will not be sufficient to
achieve complete functional restoration following SCI. The
complex, reactive, and oftentimes multifocal nature of SCI
presents several clinical challenges that must be overcome before
stem cell-based therapies can become clinical realities.
SCI results in inflammation, progressive hemorrhagic ne-
crosis, edema, demyelination and cellular destruction. In the
earlier stages of SCI, there is vascular destruction, a loss of
neurons within the grey matter, and a loss of myelinating
oligodendrocytes in the white matter. Axonopathy leads to a loss
of functional connections (denervation) and retraction of the
 M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377
proximal axon. Cell death as a result of traumatic insult leads to
further cell death via excitotoxicity. Excitotoxicity occurs as a
result of the accumulation of excitatory molecules such as
glutamate in the extracellular fluid, leading to overactivation of
neurotransmitter receptors. High levels of calcium enter the cell
and activate enzymes (phospholipases, proteases, etc.) that go on
to damage cell structures. This cell damage and other changes
often lead to induced cell death (apoptosis) and free radical-
mediated lipid oxidation (Liu et al., 1997). Thus, during the first
few days after injury there are many features detrimental to the
survival and integration of transplanted cells (Hausmann, 2003).
Inflammation plays a role in both the early and chronic stages
of spinal cord injury (Hausmann, 2003; Fleming et al., 2006;
Donnelly and Popovich, in press). Macrophages, neutrophils
and T cells migrate in from the peripheral circulation and become
activated. Microglia (normally resident in the spinal cord) also
become activated. Both activated microglia and macrophages
remove dead cells and debris via phagocytosis. The cytokines
and chemokines that these cells produce propagate the in-
flammatory processes, as does complement activation (Ander-
son et al., 2004). Thus, inflammation in both the early and
chronic stages of spinal cord injury could also damage trans-
planted cells.
After the initial injury, the damage site expands from the
injury epicenter (many centimeters in a human). Analysis of
chronic SCI shows that, typically, portions of the more external
white matter are spared while there is extensive damage of the
more internally located grey matter. Within white matter, there is
degeneration of both ascending and descending axons, and
demyelination due to loss of oligodendrocytes. Chronic, prog-
ressive demyelination is a persistent feature of SCI (Totoiu and
Keirstead, 2005). The astrocyte response begins immediately
after injury (proliferation, hypertrophy, etc.) and evolves over
time. Reactive astrocytes produce extracellular matrix compo-
nents such as chondroitin and keratan sulfate proteoglycans.
Ultimately, a scar-encapsulated cavity many times the size of the
initial injury forms. The glial scar presents both a physical barrier
and an inhibitory environment for axonal regeneration and
remyelination (Fitch and Silver, 2008; Silver and Miller, 2004).
Work from our laboratory strongly suggests that the gliotic
environment of scarred, chronic lesions contributes to the failure
of remyelination by prohibiting transplant-derived oligoden-
drocytes from remyelinating (Keirstead et al., 2005). Therefore,
effective cell therapies for SCI likely necessitate transplantation
during a brief therapeutic window: following acute inflamma-
tion, and prior to glial scar formation (Keirstead et al., 2005;
Okano et al., 2003).
Types of stem cells
Stem cells can be divided into two broad categories, em-
bryonic stem cells and somatic stem cells. Somatic stem cells
include endogenous progenitor cells that repair and replace
tissues in our bodies. The category of somatic stem cells also
includes cells and cell lines derived from fetal tissues, neonatal
tissues and adult tissues (e.g. neural stem cells, mesenchymal
stem cells). These different types of stem cells will be briefly
369
reviewed in this section, including a discussion of their po-
tential for the treatment of SCI. Considerations include their
potential for self-renewal, multipotency for providing a range
of differentiated cell types, and the potential to meet clinical
needs.
Human embryonic stem cells
Embryonic stem cells (ESCs) owe their discovery and
potential to the early work of Kleinsmith and Pierce (1964).
They demonstrated that a single embryonal carcinoma cell had
the capacity to self-renew, and to generate multiple mature cell
types. Later work with cell lines derived from the inner cell mass
of cultured blastocyst stage embryos underscored the plasticity
and potential of ESCs (Evans and Kaufman, 1981; Martin,
1981). A landmark experiment used ESCs to generate an entire
mouse (Nagy et al., 1993). This astonishing plasticity was of
interest not only to the scientific community, but also to medical
community interested in clinical approaches to repair and
replace damaged tissues. Successful isolation of human ESCs
(hESCs) (Thomson et al., 1998) further heightened the interest in
the field. ESCs can be prepared from pre-implantation or
blastocyst-stage embryos (from unused embryos created during
in vitro fertilization procedures), by somatic cell nuclear transfer,
or by parthenogenetic activation of eggs (Cibelli et al., 2002;
Vrana et al., 2003). Unlike normal somatic cells, ESCs can be
grown in virtually unlimited quantities because they do not
undergo senescence, retain high telomerase activity and normal
cell cycle signaling. With proper culture techniques, they do not
undergo the genomic, mitochondrial and epigenetic changes that
lead to transformation (Zeng and Rao, 2007).
Of all stem cell types, ESCs currently show the greatest
potential for the widest range of cell replacement therapies.
They can be propagated in vitro almost indefinitely, can be
stably banked, and maintain a normal karyotype and
differentiation potential even after years of culture. These
attributes make hESCs a commercially viable possibility for
production-scale processes. Originally, hESCs where cultivat-
ed on mouse fibroblast feeder layers with medium containing
bovine serum. Great progress has been made in cultivating
these cells, as current approaches use defined media with few
or no reagents derived from animal sources (Richards et al.,
2002; Lee et al., 2005). This is important to preclude the
possibility of cellular incorporation of zoonotic pathogens
from the media. Other types of stem cells are not as thoroughly
characterized, and the methods for directing hESC differen-
tiation are most advanced. Several studies have indicated that
rodent ESCs can be directed in their differentiation to neuronal
(Finley et al., 1996; Lang et al., 2004) or glial fates (Liu et al.,
2000; Billon et al., 2006). ESC-derived neurons can survive,
integrate and help restore function following transplantation
into spinal cord injured rats (Deshpande et al., 2006). Human
ESCs have been directed to differentiate into multipotent neural
precursors (Carpenter et al., 2001; Reubinoff et al., 2001), into
low-purity motor neurons (Li et al., 2005) and recently into high-
purity oligodendrocyte progenitors (Keirstead et al., 2005; Nistor
et al., 2005). Cumulatively, these studies demonstrate that the
370 M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377
directed differentiation of hESCs into high-purity neural popula-
tions is possible.
Neural stem cells
Endogenous neural stem cells (NSCs) exist within the central
nervous system (CNS) of higher mammals, and recently, several
groups have successfully isolated and expanded human NSCs
from specific regions of the developing and adult brain (Snyder
et al., 1992; Lois and Alvarez-Buylla, 1993; Uchida et al., 2000)
(Reynolds and Weiss, 1992), spinal cord (Mayer-Proschel et al.,
1997) and optic nerve (Shi et al., 1998). NSCs are of particular
interest for SCI repair. It is believed that they are already com-
mitted to a neural fate and hence will be easier to differentiate
into mature neural phenotypes, and less likely than ESCs to
become neoplastic.
NSCs are neurogenic and gliogenic within the CNS
throughout both development and adulthood. They can be
expanded in vitro by exposure to different growth factors,
maintain some capacity for self-renewal even after several
freeze–thaw cycles, and are capable of generating differenti-
ated progeny that can functionally integrate (Caldwell et al.,
2001) and repair the damaged CNS (Nunes et al., 2003;
Cummings et al., 2006; Ogawa et al., 2002; Iwanami et al.,
2005). NSCs can also protect against excitotoxicity and secrete
neurotrophic growth factors (Llado et al., 2004; Lu et al.,
2003). Multipotent NSCs have recently been isolated from
adult human subcortical white matter, and can be maintained in
vitro before being transplanted into fetal rat brain, where they
generate functionally competent neurons and glia (Nunes et al.,
2003). However, adult NSCs divide less frequently than their
embryonic counterparts and therefore may be more difficult to
expand into large cultures required for clinical applications
(Doetsch et al., 1999; Morshead et al., 1998). Besides their
limited replication potential, there is evidence that the
differentiation potential of NSCs decreases with time in culture
(Wright et al., 2006). In addition, differentiation of NSCs into
high purity populations has not been demonstrated, although
progress has been made to increase the percentage of either
neurons or astrocytes during in vitro differentiation using
different combinations of growth factors (Caldwell et al., 2001;
Han et al., 2002) or alternate growth conditions (Yan et al.,
2007). Nonetheless, transplanted multipotent NSCs have been
shown to reach regions of tissue damage, differentiate into
myelinating oligodendrocytes, and cause clinical improvement
following intraventricular, intravenous, intraspinal, or intra-
peritoneal delivery to various demyelinating or dysmyelinating
animal models (Einstein et al., 2003; Pluchino et al., 2003)
(Ben-Hur et al., 2003; Bulte et al., 2003).
Approximately 35–40 reports have described neural stem
cell treatments for SCI (reviewed in Enzmann et al., 2006); most
have used brain-derived NSC. Many of these studies showed
that transplanted NSCs are able to generate astrocytes and
oligodendrocytes very effectively. Generally, the production of
neurons was low or not detectable. It has been suggested that the
adult SCI environment is conducive for the differentiation of
NSCs to oligodendrocytes or astrocytes, but for undefined
reasons, it is not as conducive for neuronal differentiation. Some
studies show that more mature populations of neural precursors
will generate neurons, either because these cells do not respond
to inhibitory signals, or alternatively, no longer need positive
environmental influences to attain a neuronal phenotype (Han
et al., 2002; Roy et al., 2004; Yan et al., 2007).
Human neural tissue is generally obtained from cadavers,
which restricts supply. Furthermore, human neural tissue has a
limited expansion potential in vitro. These factors limit the
usefulness of NSCs in the clinical setting.
Olfactory ensheathing cells
Olfactory ensheathing cells (OECs) are support cells that
wrap olfactory axons and facilitate their regeneration throughout
the life of mammalian species. They are reported to have
exceptional plasticity, and importantly, allow neurons to cross a
glial scar as well as the PNS–CNS boundary (reviewed in
(Richter and Roskams, 2008) and (Raisman and Li, 2007)).
These cells are relatively easy to obtain from nasal biopsies and
could provide a source of autologous cells for transplant. Over
the last decade, a number of labs have used OECs in several
different acute and chronic models of rodent SCI. In some cases,
remyelination of axons and regeneration of damaged axons was
reported along with a surprising degree of functional recovery.
Other groups have not been able to reproduce these results, due
perhaps in part to differences in biological properties of primary
OECs with increasing age and/or passage number (Pastrana
et al., 2006). While there is great contention in the field, the
majority of reports hold that these cells are supportive in repair
processes, but the evidence that they facilitate regeneration of
long axonal tracts is limited. In addition, it is not yet clear
whether they can be expanded in sufficient numbers for use in
human cell replacement strategies.
Mesenchymal stem cells
Mesenchymal stem cells (MSC), bone marrow mononuclear
cells and umbilical cord blood are potentially rich sources of
stem cells and a number of studies have used them to treat CNS
damage. Some of these studies have shown promising results,
however, basic knowledge concerning their mechanism of
action and therapeutic potential is lacking. Despite the lack of
understanding of the underling mechanisms, clinical trials for
SCI treatments are beginning (Yoon et al., 2007; Callera and do
Nascimento, 2006).
MSC and bone marrow-derived cells have epitomized a
central question in stem cell biology: whether stem cells from
one tissue can generate cells of another. The discovery of Y-
chromosome-labeled neurons (Mezey et al., 2003), Purkinje
cells (Weimann et al., 2003) or hippocampal cells (Cogle et al.,
2004) within the brains of women who had received bone
marrow transplants from men ignited controversy of whether
this was a rare fusion event, or evidence of stem cell plasticity
that could lead to useful therapies.
The application of these types of stem cells for CNS repair
has been reviewed recently (Parr et al., 2007; Enzmann et al.,
 M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377
2006). These reviews take on the difficult task of comparing
studies with greatly heterogeneous starting materials and dif-
ferences in injury models, immunosuppression regimes, meth-
ods of transplantation, etc. The effort to evaluate these studies is
well warranted, given the practical advantages of MSC and bone
marrow-derived cells: they are easily obtainable, autologous
transplantation is possible, they may be immuno-privileged, and
they have the ability to migrate to areas of damage and in-
flammation. Most of these studies report improved function as a
result of implantation (usually determined by a subjectively-
scored locomotor test, though some use field potential recording
(Akiyama et al., 2002a,b), and differentiation to oligodendrocytes,
or less frequently neurons, weeks to months after transplantation.
Some of the most convincing studies demonstrated that LacZ or
GFP pre-labeled cells localized to Schwann- and oligodendro-
cyte-like cells following transplantation (Akiyama et al., 2002a,b)
(Akiyama et al., 2002a,b; Sasaki et al., 2001). Opposite results
were obtained in other studies: there was no detection of trans-
differentiation in the transplanted cells, even though functional
improvement was noted (Koda et al., 2005).
How can these disparate results be reconciled? A broad
survey of the literature indicates that differentiation of
transplanted MSC and bone marrow stem cells is dictated by
the environment, a concern that is made more relevant by the fact
that means of in vivo differentiation to high-purity neural
populations are lacking. Some degree of differentiation may well
be possible, especially given that a subset of ex vivo bone
marrow-derived cells express neuronal and oligodendroglial
markers (Goolsby et al., 2003; Steidl et al., 2002). Besides
directly replacing damaged oligodendrocytes and neurons, bone
marrow cells and MSC could play an important supportive role
in SCI therapies. They could create a more favorable envi-
ronment for limiting damage and promoting regeneration, via
immunoregulation (Aggarwal and Pittenger, 2005; Noel et al.,
2007), expression of growth factors and cytokines (Song et al.,
2004), improved vascularization, providing a permissive growth
substrate, and/or suppressing cavity formation (Hofstetter et al.,
2002). These different mechanisms are not mutually exclusive
and a number of them could contribute to improved outcomes.
Indeed, naive and genetically-modified MSC have been used in
combinatorial therapies in animal models of SCI (Lu et al., 2005;
Lu et al., 2004).
Endogenous stem cells and progenitors
Neural stem cells (NSC) are present in the adult spinal cord,
however, it is clear that the capacity of endogenous NSCs to
replace lost cells after SCI is poor. Axonal regeneration from
pre-existing neurons is also poor, and it is likely that many of
the same factors that prevent axonal regeneration also inhibit the
function of endogenous NSC, neural progenitors and mature
neurons. These factors include the formation of the glial scar,
the lack of neurotrophic factors, inhibitory sulfated proteogly-
cans, and inhibitory myelin-associated molecules (reviewed in
(Ramer et al., 2005) and (Fitch and Silver, 2008)). Falling
cAMP levels may also be inhibitory to regenerating cells
(Pearse et al., 2004) and differentiating progenitors.
371
In contrast to the lack of neuronal regeneration within the adult
CNS, glial regeneration is successful following some insults to the
CNS. Endogenous oligodendrocyte precursors can proliferate and
differentiate in response to various types of injuries (Keirstead
et al., 1998; McTigue et al., 2001; Wolswijk, 2000). Cell division
is a prerequisite for remyelination (Keirstead et al., 1998),
which is characterized by thin and short myelin sheaths (Totoiu
and Keirstead, 2005) (Prineas et al., 1993). Interestingly, remy-
elination is dependent on the re-expression of developmentally-
regulated genes (Capello et al., 1997), though it differs in some
respects from transcription in development (Ibanez et al., 2003).
While oligodendrocytes proliferate and differentiate in response
to SCI, there is a net loss of myelin. Demyelination as a chron-
ic, progressive problem in SCI (Guest et al., 2005; Totoiu and
Keirstead, 2005).
Remyelination is less efficient in old animals than in young
animals (Shields et al., 2000) and less efficient after repeated
episodes of demyelination (Mason et al., 2004). It is possible
that depletion of myelinogenic progenitors contributes to
remyelination failure. Mature oligodendrocytes are incapable
of remyelinating axons (Keirstead and Blakemore, 1997; Crang
et al., 1998) and there is no convincing evidence that diffe-
rentiated oligodendrocytes are able to revert to a progenitor
state. In addition, astrogliosis could contribute to the failure of
remyelination, forming a physical barrier and blocking access of
oligodendrocyte progenitors to demyelinated axons (Keirstead
et al., 2005; Ibanez et al., 2003). Astrocytes can also express
inhibitory molecules such as Jagged1 (which inhibits oligo-
dendrocyte differentiation and process outgrowth) (John et al.,
2002). Failure of remyelination is probably due to a com-
bination of environmental factors and innate characteristics of
endogenous oligodendrocyte progenitors.
In addition to directly replacing damaged neurons and
oligodendrocytes, stem cell therapies could also play an indirect
role by supporting endogenous stem cells. Transplanted cells
could provide trophic factors (Zhang et al., 2006) or serve as a
substrate permissive for growth, differentiation, elongation or
connection to other cells. Because the glial scar takes weeks to
form a thick, rubbery obstruction, there is likely a ‘window of
opportunity’ following injury during which the impediments to
endogenous or transplant-mediated regeneration are fewer. This
period of time presents an opportunity for stem cell-derived
therapies to repair SCI.
Clinical and scientific challenges
Potential stem cell therapies have a number of clinical and
technical issues that need to be resolved before human clinical
trials can begin. It will not be necessary to answer all of the
questions regarding underlying molecular mechanisms of
action, but there must be clear evidence that stem cell therapies
are safe, provide improved function and that the benefits
outweigh the risks. Most important is an understanding of the
potential dangers of stem cell-based therapy. Preclinical studies
will be essential in answering these questions; however, there
will always be some uncertainties when translating preclinical
animal studies to the human condition. Hence, collective ‘buy-
372 M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377
in’ from the scientific and medical communities is a critical
prerequisite to a clinical trial.
Do no harm
A number potential adverse effects must be evaluated before
a stem cell-based therapeutic can be applied to humans. The
risks include tumorigenesis, immunological complications,
allodynia (pain), or complications associated with an unexpect-
ed change in phenotype of the transplanted cells (for example,
dedifferentiation or excess proliferation).
Tumor formation is a significant concern for transplant
strategies involving embryonic stem cells; however, this risk
decreases as the cells become more highly differentiated (i.e. less
multipotent). Transplanted ESCs by definition form teratomas,
which consist of endodermal, mesodermal and ectodermal
lineages (Reubinoff et al., 2001). However, no rational medical
researcher would consider using undifferentiated ESCs in a
human therapeutic strategy. Somatic stem cells are less pro-
liferative and less multipotent; hence they are considered less
tumorigenic.
Another important concern is immune rejection of the
transplant. The host's immune system could destroy the
implanted cells and thereby eliminate any benefit that they
conferred. Furthermore, the inflammatory response associated
with cell rejection could cause additional harm to the patient.
HLA matching of the stem cell transplant to the host is one
method to avoid immune rejection. Unfortunately, the number of
available stem cell lines is limited, and it is likely that life-long
immunosuppression will be necessary to prevent rejection of the
transplanted cells. The side-effects and risks of immunosup-
pression are significant and include nausea, vomiting, diarrhea,
liver and kidney toxicity, lowered counts of leukocytes and
platelets, and increased susceptibility to infections and malig-
nancies (Habwe, 2006). Also, it is not clear what effect im-
munosuppression will have on the transplanted cells. This is not
a trivial issue, as evidenced by the fact that pancreatic islet
transplantation was not successful until immunosuppression
regimes were developed that were less toxic to the transplanted
cells (Nanji and Shapiro, 2004). It is intriguing that ESC and
ESC-derived cells may not generate the same degree of immune
response associated with other transplanted tissues (Drukker
et al., 2006; Utermohlen and Kronke, 2007). Such studies should
be repeated with the differentiated cells of interest.
It is conceivable that transplant rejection may be overcome by
tolerizing recipients prior to transplantation (Salama et al., 2001;
Rosengard and Turka, 2001). Other potential means of avoiding
transplant rejection include generating stem cells that are
immunologically compatible with recipients using somatic cell
nuclear transfer (SCNT—deriving a stem cell line by trans-
ferring nuclei of recipient into donor stem cell or egg) or by
developing a “universal donor” ESC line (Lengerke et al., 2007).
It remains to be seen if these later approaches will be successful
or commercially viable.
Allodynia is a significant concern for stem cell-based
strategies to treat SCI. In a thorough and thoughtful investiga-
tion, Hoffstetter and colleagues recently underscored the ben-
efits and dangers of cell-based therapies for SCI (Hofstetter
et al., 2005). In these studies, NSCs were transplanted into the
low-thoracic spinal cord of rats 1 week after injury. Functional
recovery was noted in the affected hind limbs, but abnormal,
painful sensitivity developed in the forepaws (which had been
unaffected by the injury). Histology indicated that the transplant
had differentiated in situ to a predominantly astrocytic pheno-
type, and that these astrocytes promoted sprouting of sensory
fibers within the spinal cord that were associated with allodynia.
When the NSCs were directed to produce oligodendrocytes,
further functional gains were attained in the hind limbs and
allodynia was avoided. These results have been verified by other
workers (Hendricks et al., 2006). Careful studies such as these
are critical to the preclinical development of stem cell-based
therapies. Nonetheless, it should be appreciated that unexpected
adverse effects do occur in human clinical trials, despite exhaus-
tive preclinical development.
Differentiation to undesirable cell types is a risk inherent to
all multipotent cells. The NSCs used by Hoffstetter and col-
leagues had the potential to produce neurons or oligodendro-
cytes in vitro, but in the complex environment of the injured
spinal cord they produced mostly astrocytes (Hofstetter et al.,
2005). This underscores the importance of evaluating the phe-
notype of the cells after transplant. In addition to evaluating
phenotype, preclinical studies must also evaluate proliferation
and migration of the transplanted cells. Uncontrolled prolifer-
ation and migration is obviously undesirable; spreading beyond
the implant site would increase the risk of adverse events such
as cerebrospinal fluid occlusion or emboli causing stroke.
The nature of the transplant
Preclinical studies will guide decisions on the best transplant
position within the spinal cord, the number of implantation
sites, the appropriate cell number to transplant, and the optimal
timing of transplants relative to the onset of injury. The optimal
time for transplantation of cells into the injured spinal cord is
likely after acute inflammation and excitotoxicity has subsided,
and prior to the formation of extensive glial scar. Other practical
matters concerning the transplant include efficient and safe
methods to expand and differentiate stem cells. It is imperative
that the cells maintain genetic and epigenetic stability, and do
not become senescent. The methods to induce differentiation
must also be efficient, so a high percentage of cells attain the
desired phenotype.
A central question concerning the transplant is the optimal
degree of differentiation. While a less differentiated cell may
better respond to environmental cues and show enhanced capa-
city for migration and growth, a more differentiated cell may
mitigate the risks of inappropriate differentiation and neoplasia. A
broad review of the literature clearly indicates that lineage
restriction to the progenitor stage is essential to limit tumor
formation and differentiation of undesirable cell types, and
enhance integration. Lineage restriction prior to transplantation
overcomes limitations of the environment, which may not
provide appropriate cues for targeted differentiation (for
example, efficient generation of neurons is limited in non-
 M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377
neurogenic regions of the CNS (Song et al., 2002; Shihabuddin
et al., 2000)). The directed differentiation of stem cells to high
purity lineages has recently been achieved (Nistor et al., 2005;
Keirstead et al., 2005). This is a critical advance in stem cell
biology since it allows researchers to generate a potentially
inexhaustible supply of relatively pure, committed, lineage-
specific cells for transplantation.
Stem cells are particularly amenable to genetic modification,
offering the potential to combine cell replacement therapy with
small molecule approaches to repair. Modifying the transplant
population to secrete growth factors or functional blockers of
endogenous inhibitors may augment the effect of cell replace-
ment on injury pathogenesis or repair. The use of a reporter
genes permit sorting of differentiated cells from residual
undifferentiated ES cells, mitigating the risk of tumor formation
(Tang et al., 2002). However, there are inherently unpredictable
consequences when modifying the genome, and regulatory
agencies are understandably cautious with these methods. A
poignant example of this risk was realized in a gene therapy trial to
treat severe combined immunodeficiency, when the replacement
gene and its vector integrated into the patient genome near a
proto-oncogene, resulting in leukemia (Buckley, 2002).
Evaluating success
Ideally, stem cell-based therapies should be assessed using
cellular, physiological and behavioral assays. Many published
studies document functional improvements, but do not
characterize the cells post-transplant nor demonstrate how the
cells contributed to the outcome. Conversely, some publications
provide evidence of successful differentiation using molecular
and immunological methods, but do not report behavioral out-
come. Safety studies are rarely undertaken by the scientific
community, which prioritizes discovery over development.
While this work is scientifically significant, it will be important
to have a more complete picture before human clinical trials can
begin. Ideally, promising findings should be replicated in
independent laboratories prior to the initiation of clinical trials.
A recent concurrence of data from multiple laboratories has
led to the conclusion that myelinogenic transplants elicit histo-
logic repair and functional recovery following SCI. Findings
from our laboratory indicated that hESCs can be manipulated in
vitro to generate high purity human oligodendrocyte progenitor
cells (OPCs) in large quantities (Nistor et al., 2005), and that
their transplantation into sites of moderate spinal cord contusion
in adult rats results in differentiation to mature oligodendrocytes,
remyelination by transplanted cells and improved locomotion
(Keirstead et al., 2005). Follow-up studies indicated that the
procedure was safe, in that the transplant was not associated with
tumor formation, scarring, tissue pathogenesis, or behavioral
decline (Cloutier et al., 2006). Findings from the laboratory of
Michael Fehlings indicated that multipotent adult neural
precursor cells can be isolated from the subventricular zone of
the forebrain, and that their transplantation into sites of
aneurysm clip-induced spinal cord compression in adult rats
resulted in differentiation to mature oligodendrocytes, remyeli-
nation by transplanted cells and improved locomotion; this study
373
combined cell transplantation with minocycline treatment and
growth factor (PDGF-AA, bFGF and EGF) delivery to enhance
cell survival (Karimi-Abdolrezaee et al., 2006). Findings from
the laboratory of Aileen Anderson indicated that multipotent
human CNS stem cells derived from fetal brain could be
maintained as neurospheres, and that their transplantation into
sites of moderate spinal cord contusion in adult NOD-scid mice
resulted in differentiation to neurons and oligodendrocytes,
remyelination by transplanted cells and improved locomotion
(Cummings et al., 2005, 2006). Findings from the laboratory of
Lars Olson indicated that transduction of neural stem cells
isolated from adult rat spinal cords with neurogenin 2 depressed
astroglial differentiation and increased oligodendroglial differ-
entiation, and that their transplantation into sites of weight-drop
injury in adult rats resulted in remyelination and improved
locomotion; notably, in the absence of neurogenin 2 transduc-
tion, neural stem cells differentiated primarily into astrocytes
after transplantation, and caused aberrant axonal sprouting
associated with allodynia-like hypersensitivity of the forepaws
(Hofstetter et al., 2005). These studies clearly indicate that
myelinogenic transplants elicit functional recovery following
SCI. However, in all instances the mechanism of action is
unknown.
Regulatory concerns
Regulatory bodies such as the Food and Drug Administration
(FDA) and Institutional Review Boards (IRBs) will guide the
translation of stem cell therapies for SCI in animals to human
clinical trials. Researchers, biotechnology companies, patients
and patient advocates should be aware of the regulatory issues
that must be addressed before human trials can begin. These new
experimental therapies will have special concerns. First, the
spinal cord is a particularly sensitive and risky site to treat.
Secondly, stem cell therapy is novel and entails a number of
complex issues that have never been addressed before. The
regulatory issues span categories as diverse as pharmacology
and toxicology, rules concerning the manufacture of cell and
tissue based products, xeno- and allogenenic transplantation,
and surgical methods. At a minimum, a stem cell-based trans-
plant must be well defined in terms of sterility, purity, potency,
identity, stability, safety and efficacy.
One regulatory issue common to many different types of
clinical trials is the concept of “process control”. The stem cells
and all of the materials that come in contact with them through-
out their preparation must have a completely traceable identity
and source. Further, the reagents must have documented quality
assurances and quality controls. The stem cells themselves must
be screened for the presence of retroviruses and infectious
pathogens. Karyotypic analysis must be performed to ensure
there are no gross genetic changes. There can be no undif-
ferentiated embryonic stem cells in the transplant population,
which may have the capacity to form tumors. This last qua-
lification can be accomplished by checking for predetermined
markers such as OCT4, SSEA4, and TRA81.
Other critical considerations include four toxicological
parameters: migration of the cell transplant, inappropriate
374 M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377
cellular differentiation, the possibility of tumor formation, and
host immune responses. Cell migration (bio-distribution) from
the implant site can be evaluated in preclinical testing, using
reverse transcription polymerase chain reaction (RT-PCR) for
specific markers on isolated organs following transplantation.
Inappropriate cellular differentiation must also be defined in
preclinical testing. Clinical deficits could be worsened if, for
example, stem cells transplanted into the spinal cord of a patient
inappropriately differentiated into cartilage-producing cells.
Inappropriate differentiation could also include de-differentiation
to a pluripotent state, which would be a risk for tumor formation.
Tumor formation is of particular concern because the cells
implanted in the spinal cord would be difficult or impossible to
remove. Host immune responses must be evaluated in terms of
potential harm both to the host and to the implant. Since the cells
are foreign to the body, some immune response is expected.
Immunosuppression will probably be required, as will histologic
studies to define any immune activation.
Before clinical trials can begin, standard operating proce-
dures must be compiled to standardize the methods used in
preparing and delivering the cell therapy (including cell
cultivation and differentiation methods, transplantation surgery,
etc.). Surgical teams will need to be assembled and well trained
in every aspect of the transplantation. Equipment may be
subject to additional regulatory requirements prior to conduct-
ing human trials. While much of the equipment is used routinely
for clinical neurosurgical practices (e.g. syringes, infusion
pumps, stereotaxic equipment), the application to stem cell
clinical trials will require additional study. Preclinical studies
using animals have developed standard operating procedures,
however some aspects must be translated to human scale, such
as the maximum volume and number of cells that can be
transplanted, or the number of injection sites.
Conclusions and summary
The literature over the past few decades clearly documents a
growing appreciation of the very complicated pathophysiology
of SCI. During this time, the field of stem cell biology has
emerged. This new field has the potential to bring therapies to
previously untreatable diseases and injuries.
Animal models have shown some positive results, validating
scientific principles and strategies. In addition, progress has been
made with a number of practical concerns. Commercial-scale
production of NSC and ESC cultures is progressing, a necessary
step in creating a sufficient volume of cells for human trials.
Great improvements have been made towards creating “defined”
growth conditions to yield safe, transplantable cells, free from
human and zoonotic pathogens. Differentiation protocols are
improving, yielding cells of higher purity and better function.
Spinal cord researchers are appreciating the complexity of SCI,
and appreciate that a multifaceted approach will be needed (for
example, differentiated stem cells, plus trophic factors and tissue
engineering of a permissive environment). These experiments
will be difficult to design and execute such that meaningful
results can be obtained. Unquestionably, there must be an
accumulation of studies that show functional recovery in animal
models before risking treatments in human patients. Patients,
clinicians, scientists and regulatory agencies must also consider
longer-term safety issues of stem cell therapies, including the
likelihood of life-long immunosuppressive regimes. While
attaining a cure will be difficult, the spinal cord research field
should acknowledge the complexity of the challenge and not be
deterred from pursuing it.
References
Aggarwal, S., Pittenger, M.F., 2005. Human mesenchymal stem cells modulate
allogeneic immune cell responses. Blood 105, 1815–1822.
Akiyama, Y., Radtke, C., Honmou, O., Kocsis, J.D., 2002a. Remyelination of
the spinal cord following intravenous delivery of bone marrow cells. Glia
39, 229–236.
Akiyama, Y., Radtke, C., Kocsis, J.D., 2002b. Remyelination of the rat spinal
cord by transplantation of identified bone marrow stromal cells. J. Neurosci. 22,
6623–6630.
Anderson, A.J., Robert, S., Huang, W., Young, W., Cotman, C.W., 2004.
Activation of complement pathways after contusion-induced spinal cord
injury. J. Neurotrauma 21, 1831–1846.
Baptiste, D.C., Fehlings, M.G., 2007. Update on the treatment of spinal cord
injury. Prog. Brain Res. 161, 217–233.
Ben-Hur, T., Einstein, O., Mizrachi-Kol, R., Ben-Menachem, O., Reinhartz, E.,
Karussis, D., Abramsky, O., 2003. Transplanted multipotential neural
precursor cells migrate into the inflamed white matter in response to
experimental autoimmune encephalomyelitis. Glia 41, 73–80.
Billon, N., Jolicoeur, C., Raff, M., 2006. Generation and characterization of
oligodendrocytes from lineage-selectable embryonic stem cells in vitro.
Methods Mol. Biol. 330, 15–32.
Buckley, R.H., 2002. Gene therapy for SCID—a complication after remarkable
progress. Lancet 360, 1185–1186.
Bulte, J.W., Ben-Hur, T., Miller, B.R., Mizrachi-Kol, R., Einstein, O., Reinhartz, E.,
Zywicke, H.A., Douglas, T., Frank, J.A., 2003. MR microscopy of magnetically
labeled neurospheres transplanted into the Lewis EAE rat brain. Magn. Reson.
Med. 50, 201–205.
Caldwell, M.A., He, X., Wilkie, N., Pollack, S., Marshall, G., Wafford, K.A.,
Svendsen, C.N., 2001. Growth factors regulate the survival and fate of cells
derived from human neurospheres. Nat. Biotechnol. 19, 475–479.
Callera, F., do Nascimento, R.X., 2006. Delivery of autologous bone marrow
precursor cells into the spinal cord via lumbar puncture technique in patients
with spinal cord injury: a preliminary safety study. Exp. Hematol. 34,
130–131.
Capello, E., Voskuhl, R.R., McFarland, H.F., Raine, C.S., 1997. Multiple
sclerosis: re-expression of a developmental gene in chronic lesions correlates
with remyelination. Ann. Neurol. 41, 797–805.
Carpenter, M.K., Inokuma, M.S., Denham, J., Mujtaba, T., Chiu, C.P., Rao, M.S.,
2001. Enrichment of neurons and neural precursors from human embryonic
stem cells. Exp. Neurol. 172, 383–397.
Cibelli, J.B., Grant, K.A., Chapman, K.B., Cunniff, K., Worst, T., Green, H.L.,
Walker, S.J., Gutin, P.H., Vilner, L., Tabar, V., Dominko, T., Kane, J.,
Wettstein, P.J., Lanza, R.P., Studer, L., Vrana, K.E., West, M.D., 2002.
Parthenogenetic stem cells in nonhuman primates. Science 295, 819.
Cloutier, F., Siegenthaler, M.M., Nistor, G., Keirstead, H.S., 2006. Transplantation
of human embryonic stem cell-derived oligodendrocyte progenitors into rat
spinal cord injuries does not cause harm. Regen. Med. 1, 469–479.
Cogle, C.R., Yachnis, A.T., Laywell, E.D., Zander, D.S., Wingard, J.R.,
Steindler, D.A., Scott, E.W., 2004. Bone marrow transdifferentiation in brain
after transplantation: a retrospective study. Lancet 363, 1432–1437.
Crang, A.J., Gilson, J., Blakemore, W.F., 1998. The demonstration by
transplantation of the very restricted remyelinating potential of post-mitotic
oligodendrocytes. J. Neurocytol. 27, 541–553.
Cummings, B.J., Uchida, N., Tamaki, S.J., Salazar, D.L., Hooshmand, M.,
Summers, R., Gage, F.H., Anderson, A.J., 2005. Human neural stem cells
differentiate and promote locomotor recovery in spinal cord-injured mice.
Proc. Natl. Acad. Sci. U. S. A. 102, 14069–14074.
 M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377
Cummings, B.J., Uchida, N., Tamaki, S.J., Anderson, A.J., 2006. Human neural
stem cell differentiation following transplantation into spinal cord injured mice:
association with recovery of locomotor function. Neurol. Res. 28, 474–481.
Deshpande, D.M., Kim, Y.S., Martinez, T., Carmen, J., Dike, S., Shats, I., Rubin,
L.L., Drummond, J., Krishnan, C., Hoke, A., Maragakis, N., Shefner, J.,
Rothstein, J.D., Kerr, D.A., 2006. Recovery from paralysis in adult rats
using embryonic stem cells. Ann. Neurol. 60, 32–44.
Doetsch, F., Caille, I., Lim, D.A., Garcia-Verdugo, J.M., Alvarez-Buylla, A.,
1999. Subventricular zone astrocytes are neural stem cells in the adult
mammalian brain. Cell 97, 703–716.
Donnelly, D.J., Popovich, P.G., 2008. Inflammation and its role in neuroprotec-
tion, axonal regeneration and functional recovery after spinal cord injury.
Exp. Neurol. 209, 378–388.
Drukker, M., Katchman, H., Katz, G., Even-Tov Friedman, S., Shezen, E.,
Hornstein, E., Mandelboim, O., Reisner, Y., Benvenisty, N., 2006. Human
embryonic stem cells and their differentiated derivatives are less susceptible
to immune rejection than adult cells. Stem Cells 24, 221–229.
Einstein, O., Karussis, D., Grigoriadis, N., Mizrachi-Kol, R., Reinhartz, E.,
Abramsky, O., Ben-Hur, T., 2003. Intraventricular transplantation of neural
precursor cell spheres attenuates acute experimental allergic encephalomy-
elitis. Mol. Cell. Neurosci. 24, 1074–1082.
Enzmann, G.U., Benton, R.L., Talbott, J.F., Cao, Q., Whittemore, S.R., 2006.
Functional considerations of stem cell transplantation therapy for spinal cord
repair. J. Neurotrauma 23, 479–495.
Evans, M.J., Kaufman, M.H., 1981. Establishment in culture of pluripotential
cells from mouse embryos. Nature 292, 154–156.
Finley, M.F., Kulkarni, N., Huettner, J.E., 1996. Synapse formation and
establishment of neuronal polarity by P19 embryonic carcinoma cells and
embryonic stem cells. J. Neurosci. 16, 1056–1065.
Fitch, M.T., Silver, J., 2008. CNS injury, glial scars, and inflammation:
inhibitory extracellular matrices and regeneration failure. Exp. Neurol. 209,
294–301.
Fleming, J.C., Norenberg, M.D., Ramsay, D.A., Dekaban, G.A., Marcillo, A.E.,
Saenz, A.D., Pasquale-Styles, M., Dietrich, W.D., Weaver, L.C., 2006. The
cellular inflammatory response in human spinal cords after injury. Brain 129,
3249–3269.
Goolsby, J., Marty, M.C., Heletz, D., Chiappelli, J., Tashko, G., Yarnell, D., Fishman,
P.S., Dhib-Jalbut, S., Bever Jr., C.T., Pessac, B., Trisler, D., 2003. Hematopoietic
progenitors express neural genes. Proc. Natl. Acad. Sci. U. S. A. 100,
14926–14931.
Guest, J.D., Hiester, E.D., Bunge, R.P., 2005. Demyelination and Schwann cell
responses adjacent to injury epicenter cavities following chronic human
spinal cord injury. Exp. Neurol. 192, 384–393.
Habwe, V.Q., 2006. Posttransplantation quality of life: more than graft function.
Am. J. Kidney Dis. 47, S98–S110.
Han, S.S., Kang, D.Y., Mujtaba, T., Rao, M.S., Fischer, I., 2002. Grafted
lineage-restricted precursors differentiate exclusively into neurons in the
adult spinal cord. Exp. Neurol. 177, 360–375.
Hausmann, O.N., 2003. Post-traumatic inflammation following spinal cord
injury. Spinal Cord 41, 369–378.
Hendricks, W.A., Pak, E.S., Owensby, J.P., Menta, K.J., Glazova, M., Moretto,
J., Hollis, S., Brewer, K.L., Murashov, A.K., 2006. Predifferentiated
embryonic stem cells prevent chronic pain behaviors and restore sensory
function following spinal cord injury in mice. Mol. Med. 12, 34–46.
Hofstetter, C.P., Holmstrom, N.A., Lilja, J.A., Schweinhardt, P., Hao, J.,
Spenger, C., Wiesenfeld-Hallin, Z., Kurpad, S.N., Frisen, J., Olson, L., 2005.
Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed
differentiation improves outcome. Nat. Neurosci. 8, 346–353.
Hofstetter, C.P., Schwarz, E.J., Hess, D., Widenfalk, J., El Manira, A., Prockop,
D.J., Olson, L., 2002. Marrow stromal cells form guiding strands in the
injured spinal cord and promote recovery. Proc. Natl. Acad. Sci. U. S. A. 99,
2199–2204.
Ibanez, C., Shields, S.A., El-Etr, M., Leonelli, E., Magnaghi, V., Li, W.W., Sim,
F.J., Baulieu, E.E., Melcangi, R.C., Schumacher, M., Franklin, R.J., 2003.
Steroids and the reversal of age-associated changes in myelination and
remyelination. Prog. Neurobiol. 71, 49–56.
Iwanami, A., Kaneko, S., Nakamura, M., Kanemura, Y., Mori, H., Kobayashi, S.,
Yamasaki, M., Momoshima, S., Ishii, H., Ando, K., Tanioka, Y., Tamaoki, N.,
375
Nomura, T., Toyama, Y., Okano, H., 2005. Transplantation of human
neural stem cells for spinal cord injury in primates. J. Neurosci. Res. 80,
182–190.
John, G.R., Shankar, S.L., Shafit-Zagardo, B., Massimi, A., Lee, S.C., Raine, C.S.,
Brosnan, C.F., 2002. Multiple sclerosis: re-expression of a develop-
mental pathway that restricts oligodendrocyte maturation. Nat. Med. 8,
1115–1121.
Karimi-Abdolrezaee, S., Eftekharpour, E., Wang, J., Morshead, C.M., Fehlings,
M.G., 2006. Delayed transplantation of adult neural precursor cells promotes
remyelination and functional neurological recovery after spinal cord injury.
J. Neurosci. 26, 3377–3389.
Keirstead, H.S., Blakemore, W.F., 1997. Identification of post-mitotic
oligodendrocytes incapable of remyelination within the demyelinated
adult spinal cord. J. Neuropathol. Exp. Neurol. 56, 1191–1201.
Keirstead, H.S., Levine, J.M., Blakemore, W.F., 1998. Response of the
oligodendrocyte progenitor cell population (defined by NG2 labelling) to
demyelination of the adult spinal cord. Glia 22, 161–170.
Keirstead, H.S., Nistor, G., Bernal, G., Totoiu, M., Cloutier, F., Sharp, K.,
Steward, O., 2005. Human embryonic stem cell-derived oligodendrocyte
progenitor cell transplants remyelinate and restore locomotion after spinal
cord injury. J. Neurosci. 25, 4694–4705.
Kleinsmith, L.J., Pierce Jr., G.B., 1964. Multipotentiality of single embryonal
carcinoma cells. Cancer Res. 24, 1544–1551.
Koda, M., Okada, S., Nakayama, T., Koshizuka, S., Kamada, T., Nishio, Y.,
Someya, Y., Yoshinaga, K., Okawa, A., Moriya, H., Yamazaki, M., 2005.
Hematopoietic stem cell and marrow stromal cell for spinal cord injury in
mice. Neuroreport 16, 1763–1767.
Lang, K.J., Rathjen, J., Vassilieva, S., Rathjen, P.D., 2004. Differentiation of
embryonic stem cells to a neural fate: a route to re-building the nervous
system? J. Neurosci. Res. 76, 184–192.
Lee, J.B., Lee, J.E., Park, J.H., Kim, S.J., Kim, M.K., Roh, S.I., Yoon, H.S.,
2005. Establishment and maintenance of human embryonic stem cell lines
on human feeder cells derived from uterine endometrium under serum-free
condition. Biol. Reprod. 72, 42–49.
Lengerke, C., Kim, K., Lerou, P., Daley, G.Q., 2007. Differentiation potential
of histocompatible parthenogenetic embryonic stem cells. Ann. N.Y. Acad.
Sci. 1106, 209–218.
Li, X.J., Du, Z.W., Zarnowska, E.D., Pankratz, M., Hansen, L.O., Pearce, R.A.,
Zhang, S.C., 2005. Specification of motoneurons from human embryonic
stem cells. Nat. Biotechnol. 23, 215–221.
Liu, S., Qu, Y., Stewart, T.J., Howard, M.J., Chakrabortty, S., Holekamp, T.F.,
McDonald, J.W., 2000. Embryonic stem cells differentiate into oligoden-
drocytes and myelinate in culture and after spinal cord transplantation. Proc.
Natl. Acad. Sci. U. S. A. 97, 6126–6131.
Liu, X.Z., Xu, X.M., Hu, R., Du, C., Zhang, S.X., McDonald, J.W., Dong, H.X.,
Wu, Y.J., Fan, G.S., Jacquin, M.F., Hsu, C.Y., Choi, D.W., 1997. Neuronal
and glial apoptosis after traumatic spinal cord injury. J. Neurosci. 17,
5395–5406.
Llado, J., Haenggeli, C., Maragakis, N.J., Snyder, E.Y., Rothstein, J.D., 2004.
Neural stem cells protect against glutamate-induced excitotoxicity and
promote survival of injured motor neurons through the secretion of
neurotrophic factors. Mol. Cell. Neurosci. 27, 322–331.
Lois, C., Alvarez-Buylla, A., 1993. Proliferating subventricular zone cells in the
adult mammalian forebrain can differentiate into neurons and glia. Proc.
Natl. Acad. Sci. U. S. A. 90, 2074–2077.
Lu, P., Jones, L.L., Snyder, E.Y., Tuszynski, M.H., 2003. Neural stem cells
constitutively secrete neurotrophic factors and promote extensive host
axonal growth after spinal cord injury. Exp. Neurol. 181, 115–129.
Lu, P., Yang, H., Jones, L.L., Filbin, M.T., Tuszynski, M.H., 2004.
Combinatorial therapy with neurotrophins and cAMP promotes ax-
onal regeneration beyond sites of spinal cord injury. J. Neurosci. 24,
6402–6409.
Lu, P., Jones, L.L., Tuszynski, M.H., 2005. BDNF-expressing marrow stromal
cells support extensive axonal growth at sites of spinal cord injury. Exp.
Neurol. 191, 344–360.
Martin, G.R., 1981. Isolation of a pluripotent cell line from early mouse embryos
cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl.
Acad. Sci. U. S. A. 78, 7634–7638.
376 M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377
Mason, J.L., Toews, A., Hostettler, J.D., Morell, P., Suzuki, K., Goldman, J.E.,
Matsushima, G.K., 2004. Oligodendrocytes and progenitors become progres-
sively depleted within chronically demyelinated lesions. Am. J. Pathol. 164,
1673–1682.
Mayer-Proschel, M., Kalyani, A.J., Mujtaba, T., Rao, M.S., 1997. Isolation of
lineage-restricted neuronal precursors from multipotent neuroepithelial stem
cells. Neuron 19, 773–785.
McTigue, D.M., Wei, P., Stokes, B.T., 2001. Proliferation of NG2-positive cells
and altered oligodendrocyte numbers in the contused rat spinal cord.
J. Neurosci. 21, 3392–3400.
Mezey, E., Key, S., Vogelsang, G., Szalayova, I., Lange, G.D., Crain, B., 2003.
Transplanted bone marrow generates new neurons in human brains. Proc.
Natl. Acad. Sci. U. S. A. 100, 1364–1369.
Morshead, C.M., Craig, C.G., van der Kooy, D., 1998. In vivo clonal analyses
reveal the properties of endogenous neural stem cell proliferation in the adult
mammalian forebrain. Development 125, 2251–2261.
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J.C., 1993.
Derivation of completely cell culture-derived mice from early-passage
embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 90, 8424–8428.
Nanji, S.A., Shapiro, A.M., 2004. Islet transplantation in patients with diabetes
mellitus: choice of immunosuppression. BioDrugs 18, 315–328.
Nistor, G.I., Totoiu, M.O., Haque, N., Carpenter, M.K., Keirstead, H.S., 2005.
Human embryonic stem cells differentiate into oligodendrocytes in high
purity and myelinate after spinal cord transplantation. Glia 49, 385–396.
Noel, D., Djouad, F., Bouffi, C., Mrugala, D., Jorgensen, C., 2007. Multipotent
mesenchymal stromal cells and immune tolerance. Leuk. Lymphoma 48,
1283–1289.
Nunes, M.C., Roy, N.S., Keyoung, H.M., Goodman, R.R., McKhann II, G.,
Jiang, L., Kang, J., Nedergaard, M., Goldman, S.A., 2003. Identification and
isolation of multipotential neural progenitor cells from the subcortical white
matter of the adult human brain. Nat. Med. 9, 439–447.
Ogawa, Y., Sawamoto, K., Miyata, T., Miyao, S., Watanabe, M., Nakamura, M.,
Bregman, B.S., Koike, M., Uchiyama, Y., Toyama, Y., Okano, H., 2002.
Transplantation of in vitro-expanded fetal neural progenitor cells results in
neurogenesis and functional recovery after spinal cord contusion injury in
adult rats. J. Neurosci. Res. 69, 925–933.
Okano, H., Ogawa, Y., Nakamura, M., Kaneko, S., Iwanami, A., Toyama, Y.,
2003. Transplantation of neural stem cells into the spinal cord after injury.
Semin. Cell Dev. Biol. 14, 191–198.
Parr, A.M., Tator, C.H., Keating, A., 2007. Bone marrow-derived mesenchymal
stromal cells for the repair of central nervous system injury. Bone Marrow
Transplant. 40, 609–619.
Pastrana, E., Moreno-Flores, M.T., Gurzov, E.N., Avila, J., Wandosell, F., Diaz-
Nido, J., 2006. Genes associated with adult axon regeneration promoted by
olfactory ensheathing cells: a new role for matrix metalloproteinase 2.
J. Neurosci. 26, 5347–5359.
Pearse, D.D., Pereira, F.C., Marcillo, A.E., Bates, M.L., Berrocal, Y.A., Filbin,
M.T., Bunge, M.B., 2004. cAMP and Schwann cells promote axonal growth
and functional recovery after spinal cord injury. Nat. Med. 10, 610–616.
Pluchino, S., Quattrini, A., Brambilla, E., Gritti, A., Salani, G., Dina, G., Galli,
R., Del Carro, U., Amadio, S., Bergami, A., Furlan, R., Comi, G., Vescovi,
A.L., Martino, G., 2003. Injection of adult neurospheres induces recovery in
a chronic model of multiple sclerosis. Nature 422, 688–694.
Prineas, J.W., Barnard, R.O., Kwon, E.E., Sharer, L.R., Cho, E.S., 1993. Multiple
sclerosis: remyelination of nascent lesions. Ann. Neurol. 33, 137–151.
Raisman, G., Li, Y., 2007. Repair of neural pathways by olfactory ensheathing
cells. Nat. Rev., Neurosci. 8, 312–319.
Ramer, L.M., Ramer, M.S., Steeves, J.D., 2005. Setting the stage for functional
repair of spinal cord injuries: a cast of thousands. Spinal Cord 43, 134–161.
Reubinoff, B.E., Itsykson, P., Turetsky, T., Pera, M.F., Reinhartz, E., Itzik, A.,
Ben-Hur, T., 2001. Neural progenitors from human embryonic stem cells.
Nat. Biotechnol. 19, 1134–1140.
Reynolds, B.A., Weiss, S., 1992. Generation of neurons and astrocytes from
isolated cells of the adult mammalian central nervous system. Science 255,
1707–1710.
Richards, M., Fong, C.Y., Chan, W.K., Wong, P.C., Bongso, A., 2002. Human
feeders support prolonged undifferentiated growth of human inner cell
masses and embryonic stem cells. Nat. Biotechnol. 20, 933–936.
Richter, M.W., Roskams, A.J., 2008. Olfactory ensheathing cell transplantation
following spinal cord injury: hype or hope? Exp. Neurol. 209, 353–367.
Rosengard, B.R., Turka, L.A., 2001. The tolerant recipient: looking great in
someone else's genes. J. Clin. Invest. 107, 33–34.
Roy, N.S., Nakano, T., Keyoung, H.M., Windrem, M., Rashbaum, W.K., Alonso,
M.L., Kang, J., Peng, W., Carpenter, M.K., Lin, J., Nedergaard, M.,
Goldman, S.A., 2004. Telomerase immortalization of neuronally restricted
progenitor cells derived from the human fetal spinal cord. Nat. Biotechnol. 22,
297–305.
Salama, A.D., Remuzzi, G., Harmon, W.E., Sayegh, M.H., 2001. Challenges to
achieving clinical transplantation tolerance. J. Clin. Invest. 108, 943–948.
Sasaki, M., Honmou, O., Akiyama, Y., Uede, T., Hashi, K., Kocsis, J.D., 2001.
Transplantation of an acutely isolated bone marrow fraction repairs
demyelinated adult rat spinal cord axons. Glia 35, 26–34.
Shi, J., Marinovich, A., Barres, B.A., 1998. Purification and characterization of
adult oligodendrocyte precursor cells from the rat optic nerve. J. Neurosci. 18,
4627–4636.
Shields, S., Gilson, J., Blakemore, W., Franklin, R., 2000. Remyelination occurs
as extensively but more slowly in old rats compared to young rats following
fliotoxin-induced CNS demyelination. Glia 29, 102.
Shihabuddin, L.S., Horner, P.J., Ray, J., Gage, F.H., 2000. Adult spinal cord
stem cells generate neurons after transplantation in the adult dentate gyrus.
J. Neurosci. 20, 8727–8735.
Silver, J., Miller, J.H., 2004. Regeneration beyond the glial scar. Nat. Rev.
Neurosci. 5, 146–151.
Snyder, E.Y., Deitcher, D.L., Walsh, C., Arnold-Aldea, S., Hartwieg, E.A.,
Cepko, C.L., 1992. Multipotent neural cell lines can engraft and participate
in development of mouse cerebellum. Cell 68, 33–51.
Song, H., Stevens, C.F., Gage, F.H., 2002. Astroglia induce neurogenesis from
adult neural stem cells. Nature 417, 39–44.
Song, S., Kamath, S., Mosquera, D., Zigova, T., Sanberg, P., Vesely, D.L.,
Sanchez-Ramos, J., 2004. Expression of brain natriuretic peptide by human
bone marrow stromal cells. Exp. Neurol. 185, 191–197.
Steidl, U., Kronenwett, R., Rohr, U.P., Fenk, R., Kliszewski, S., Maercker, C.,
Neubert, P., Aivado, M., Koch, J., Modlich, O., Bojar, H., Gattermann, N.,
Haas, R., 2002. Gene expression profiling identifies significant dif-
ferences between the molecular phenotypes of bone marrow-derived
and circulating human CD34+ hematopoietic stem cells. Blood 99, 2037–2044.
Tang, F., Shang, K., Wang, X., Gu, J., 2002. Differentiation of embryonic stem
cell to astrocytes visualized by green fluorescent protein. Cell. Mol.
Neurobiol. 22, 95–101.
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J.,
Marshall, V.S., Jones, J.M., 1998. Embryonic stem cell lines derived from
human blastocysts. Science 282, 1145–1147.
Totoiu, M.O., Keirstead, H.S., 2005. Spinal cord injury is accompanied by
chronic progressive demyelination. J. Comp. Neurol. 486, 373–383.
Uchida, N., Buck, D.W., He, D., Reitsma, M.J., Masek, M., Phan, T.V., Tsukamoto,
A.S., Gage, F.H., Weissman, I.L., 2000. Direct isolation of human central
nervous system stem cells. Proc. Natl. Acad. Sci. U. S. A. 97, 14720–14725.
Utermohlen, O., Kronke, M., 2007. Survival of priceless cells: active and
passive protection of embryonic stem cells against immune destruction.
Arch. Biochem. Biophys. 462, 273–277.
Vrana, K.E., Hipp, J.D., Goss, A.M., McCool, B.A., Riddle, D.R., Walker, S.J.,
Wettstein, P.J., Studer, L.P., Tabar, V., Cunniff, K., Chapman, K., Vilner, L.,
West, M.D., Grant, K.A., Cibelli, J.B., 2003. Nonhuman primate partheno-
genetic stem cells. Proc. Natl. Acad. Sci. U. S. A. 100 (Suppl 1), 11911–11916.
Weimann, J.M., Charlton, C.A., Brazelton, T.R., Hackman, R.C., Blau, H.M.,
2003. Contribution of transplanted bone marrow cells to Purkinje neurons in
human adult brains. Proc. Natl. Acad. Sci. U. S. A. 100, 2088–2093.
Wolswijk, G., 2000. Oligodendrocyte survival, loss and birth in lesions of
chronic-stage multiple sclerosis. Brain 123 (Pt 1), 105–115.
Wright, L.S., Prowse, K.R., Wallace, K., Linskens, M.H., Svendsen, C.N., 2006.
Human progenitor cells isolated from the developing cortex undergo
decreased neurogenesis and eventual senescence following expansion in
vitro. Exp. Cell Res. 312, 2107–2120.
Yan, J., Xu, L., Welsh, A.M., Hatfield, G., Hazel, T., Johe, K., Koliatsos, V.E.,
2007. Extensive neuronal differentiation of human neural stem cell grafts in
adult rat spinal cord. PLoS. Med. 4, e39.
 M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377 377

Yoon, S.H., Shim, Y.S., Park, Y.H., Chung, J.K., Nam, J.H., Kim, M.O., Park,
H.C., Park, S.R., Min, B.H., Kim, E.Y., Choi, B.H., Park, H., Ha, Y., 2007.
Complete spinal cord injury treatment using autologous bone marrow cell
transplantation and bone marrow stimulation with granulocyte macro-
phage-colony stimulating factor: phase I/II clinical trial. Stem Cells 25,
2066–2073.
Zeng, X., Rao, M.S., 2007. Human embryonic stem cells: long term stability,
absence of senescence and a potential cell source for neural replacement.
Neuroscience 145, 1348–1358.
Zhang, Y.W., Denham, J., Thies, R.S., 2006. Oligodendrocyte progenitor cells
derived from human embryonic stem cells express neurotrophic factors.
Stem Cells Dev. 15, 943–952.