Neurosurg Focus 24 (3&4):E19, 2008

Stem cell biology and its therapeutic applications in the

setting of spinal cord injury
NICHOLAS C. BAMBAKIDIS, M.D.,1 JOHN BUTLER, M.D.,1 ERIC M. HORN, M.D.,2
XUKUI WANG, M.D.,1 MARK C. PREUL, M.D.,1 NICHOLAS THEODORE, M.D.,1
ROBERT F. SPETZLER, M.D.,1 AND VOLKER K. H. SONNTAG, M.D.1
Neural Regeneration Laboratory, Barrow Neurological Institute, St. Joseph’s Hospital and Medical
1

Center, Phoenix, Arizona; and 2Indiana University School of Medicine, Indianapolis, Indiana

PThe development of an acute traumatic spinal cord injury (SCI) inevitably leads to a complex cascade of ischemia
and inflammation that results in significant scar tissue formation. The development of such scar tissue provides a
severe impediment to neural regeneration and healing with restoration of function. A multimodal approach to treat-
ment is required because SCIs occur with differing levels of severity and over different lengths of time. To achieve
significant breakthroughs in outcomes, such approaches must combine both neuroprotective and neuroregenerative
treatments. Novel techniques modulating endogenous stem cells demonstrate great promise in promoting neuroregen-
eration and restoring function. (DOI: 10.3171/FOC/2008/24/3-4/E19)

KEY WORDS • neuroregeneration • spinal cord injury • stem cell

N THE UNITED STATES, the effects of SCI on society and
I healthcare are staggering. As many as 300,000 persons
live with chronic disabilities related to SCI, and each
year new SCIs affect 10,000–14,000 people (mean age at
injury, 30 years).14 The annual cost to society is estimated
to reach $8 billion, and the individual costs to patients can
reach $1.35 million over their lifetime. As our ability to
prolong the lives of these patients progresses, these costs
would be expected to increase proportionally. Impressive
strides have been made in the ability to provide disabled
patients societal access. Nonetheless, the ultimate goal of
medical science should be to restore neurological function
corresponding to these patients’ preinjured levels.
The severity of SCI ranges from complete paraplegia to
incomplete myelopathy or paraparesis. Regardless of the
degree of injury, the mechanism of injury is a crucial factor
in determining a patient’s treatment and likelihood of re-
covery. In contusive injuries significant compressive force
is applied to the spinal cord. The initial trauma is related to
destructive shearing and laceration of the nervous tissue.
Progressive disruption of axonal transmission and subse-
quent neuronal and axonal degeneration yield to the forma-
tion of astrocytic scar tissue. In such cases, the treatment
algorithm must include spinal realignment and stabiliza-
Abbreviations used in this paper: bFGF = b-fibroblast growth fac-
tor; CNS = central nervous system; ESC = embryonic stem cell;
FGF = fibroblast growth factor; NSC = neural stem cell; SCI = spinal
cord injury; Shh = sonic hedgehog; SVZ = subventricular zone.
tion, if necessary; prevention of ischemia and demyelina-
tion from the secondary injury cascade; and finally, the pro-
motion of neural regeneration.
In contradistinction to low-impact or slowly progressive
injuries (such as those caused by tumors), the first step in
treatment must be decompression by removal of the of-
fending lesion. Given the chronic ischemic state of injured
tissue in this circumstance, adequate tissue perfusion must
be maintained. In the longer term, regenerative therapies,
including techniques to promote remyelination or prevent
dysmyelination, can be considered.
In 1980 Richardson et al.27 published the first scientific
evidence of the regenerative ability of the injured spinal
cord. This finding led to a shift in thinking away from
merely helping disabled patients integrate into society to
developing research strategies aimed at restoring function.
Although the results in animal models have been promis-
ing, models of treatment translatable to human patients are
still lacking. One limiting factor may be the lack of recog-
nition that SCI represents a complex injury cascade (Fig.
1). It therefore follows that no single “magic bullet” will
address the requirements of a therapeutic modality that
could lead to functional recovery. Instead, a multimodality
approach is needed to make meaningful gains in clinical
treatment. In general, multimodal paradigms can be divid-
ed into those that focus on ameliorating the secondary
injury cascade (neuroprotection), and those that target re-
myelination and axonal and neuronal regeneration (neu-
roregeneration). To date, most work on the potential bene-
fits of stem cell therapy in treating SCI has concentrated on
neuroregeneration, the focus of this article.

Neurosurg. Focus / Volume 24 / March/April 2008 1

 N. C. Bambakidis et al.

FIG. 1. Outline of the primary and secondary injury cascades after acute traumatic SCI. Originally published in Horn
EM, Forage J, Sonntag VKH: Acute treatment of patients with spinal cord injuries, in Herkowitz HN, Gorfin SR, Eismont
FJ, et al (eds): Rothman-Simeone the Spine, ed 5. St. Louis: Elsevier, 2005. Copyright Barrow Neurological Institute.

Progenitor Cells Versus Stem Cells
When considering regenerative therapeutic options for
the treatment of SCI, it is necessary to fully understand the
basics of developmental cell biology. During development,
pluripotent embryonic cells slowly lose their ability to
transdifferentiate. Embryonic stem cells arise from the in-
ner cell mass.31 Previously it was assumed that stem cells
cease to be active in adult mammals. In fact, they are pres-
ent in many different organ systems. Tissue restriction
plays a role in limiting the possible fates of these cells more
than in their embryonic counterparts. Even these cells may
cross fate lines and generate other tissues, although whether
this process represents cell–cell fusion or actual transdiffer-
entiation is controversial. Ideally, there would be no differ-
ence between the fate of a population of adult stem cells in
vitro or in vivo.11
With this understanding, stem cells demonstrate a multi-
potent nature (Fig. 2), and their capacity for self-renewal is
unlimited. They are concentrated in the hippocampus and
SVZ of the CNS (Fig. 3). Similarly, progenitor cells have
multiple potential destinies. However, they have far fewer
potential fates than stem cells. Although the potential of
progenitor cells for further differentiation is much greater
than that of mature cells, their capacity for renewal is more
limited than that of stem cells. This robustness and flexibil-
ity makes stem cells ideally suited for study in the treatment
of SCI and concomitantly avoids the ethical dilemmas asso-
ciated with embryonically derived progenitor cell lines.
Endogenous Stem Cells
Strategies for cellular replacement have involved both
endogenous and exogenous sources of stem cells. Man-
ipulation of endogenous stem cells offers an inherently at-
tractive advantage: it avoids the need for further trauma
caused by cellular transplantation while obviating the need
for chronic immunosuppressive therapies. Furthermore, the
ethical dilemmas posed by the need for ESCs are eliminat-
ed. Efforts to stimulate endogenous precursor cells to mi-
grate to areas of injury have allowed the regions where
these cells are found to be characterized. Rests of actively
dividing stem cells are located in adult mammals, and these
cells can be activated and remodeled in the event of an in-
jury.15 The remodeling process continues after traumatic
injury.7

2 Neurosurg. Focus / Volume 24 / March/April 2008

Therapeutic applications of stem cell biology
FIG. 2. Diagram demonstrating the pluripotent nature of ESCs.
Pluripotent stem cells develop under the influence of various
growth factors into multipotent progenitors that can then form
either neuronal, oligodendroglial, or astrocytic cells. This process
can be modified in vitro and subsequently result in cells that can be
transplanted at the site of injury with the potential for therapeutic
benefit. Originally published in Gantwerker BP, Hoffer A, Preul
MC, Theodore N: Current concepts in nerve regeneration after
traumatic brain injury. Barrow Quarterly 23:1–19, 2007. With
permission from Barrow Neurological Institute.
Such remodeling primarily occurs in the area of injury
nearest to the site of impact. Based on studies of staining
with glial fibrillary acidic protein, an upregulation of reac-
tive astrocytosis leads to the formation of scar tissue about
3 weeks after injury. Based on staining with neuronal nuclei
(NeuN), adjacent gray matter neurons hypertrophy at the
same time.29 The largest rests of NSCs are found in the
SVZ and dentate gyrus of the hippocampus.24 Rests are also
found in the ependymal lining of the spinal cord, where
they actively divide in the presence of an injury.3,9,13,18
Neurosurg. Focus / Volume 24 / March/April 2008
Neuroregeneration
After secondary injury, the potential for neuroregenera-
tive therapies begins. However, the presence of the as-
trocytic scar is the major impediment to the restoration of
functional electrochemical synapses and axonal recovery.
If these limitations can be attenuated or overcome, it is
hoped that restorative therapies may enable repair at the
site of and downstream from the injury. Several strategies
have been used to help achieve this goal in the injured
spinal cord.
One strategy involves the use of activated macrophages,
which are injected into the site of injury with the hope of
reducing the concentration of postinjury inhibitory factors.
Under normal circumstances, infiltration by macrophages
into the site of injury peaks 5–7 days after injury.5,25 Al-
though uncontrolled inflammation exacerbates neuronal
loss after SCI, a controlled inflammatory response seems to
ameliorate damaging effects.6 Theoretically, these macro-
phages aid in the autophagocytosis of cellular debris and
damaged myelin that contribute to the strong inhibition of
regeneration after injury. After the CNS is injured, these
macrophages may also provide a stimulus for changes in
molecules of the extracellular matrix to remove barriers to
axonal regeneration.10
Activated macrophages coexpress markers associated
with radial glial cells, which possess properties of NSCs
and may contribute to neural repair and regeneration.32 This
therapy has been used in patients with a complete SCI in a
Phase I trial.17 In this study, monocytes were isolated from
patients’ blood and incubated ex vivo with autologous der-
mis. The resulting macrophages were then injected into the
spinal cord of 8 patients within 14 days of a complete SCI.
Of these patients, 3 recovered clinically significant neuro-
logical motor and sensory function (improving from Amer-
ican Spinal Injury Association Grade A to C). No adverse
events were related to the injection of the macrophages.
Phase II trials of this therapy are now underway worldwide.
Direct injection of stem cells in animal models dramati-
cally improves functional recovery. Cellular transplantation
has been used previously in these models, focusing on the
ability of xenografts of pluripotent ESCs to incorporate into
the cellular structure of the injured spinal cord and produce
functional improvements in animal behavior.1,7,8,19,26,28,30,33
The mechanism underlying the improvement is controver-
sial. It may be the result of transplanted ESCs maturing in-
to myelinating oligodendrocytes, even though a sizable
proportion of these cells develop along an astrocytic lin-
eage.1,7,8,19,28,33 Furthermore, the transplantation of xeno-
grafted cells requires the coadministration of corticosteroids
during the experimental period to prevent their rejection,
which can confound the results.19,28 Work in humans has
been limited.12 Compounding the difficulties of experimen-
tation in humans is the ethical dilemma associated with the
use of ESCs. Consequently, other solutions, including the
use of stem cells derived from bone marrow and stimula-
tion of endogenous stem cells, have been investigated.
The activation and promotion of endogenous stem cells
are of particular interest because 500,000–2,000,000 new
cells are produced at the site of the injury within a month.22
These endogenous cells are upregulated after a contusion
injury to the spinal cord.16,20,21 Most of these cells originate
near the ependyma of the central spinal canal (Fig. 4). The
3

FIG. 3. The 2 niches containing NSCs in the CNS of adult mam-
mals. Upper: Adult hippocampus. Lower: Longitudinal section
of the adult mouse brain shows the SVZ and rostral migratory
stream (RMS). Neuronal precursors migrate tangentially along the
RMS from the SVZ to the olfactory bulb. Originally published in
Dashti SR, Wilson J, Hu J, Selman WR, Spetzler RF, Nakaji P:
Neural regenerative options in the management of ischemic brain
injury. Barrow Quarterly 23:4–7, 2007. With permission from
Barrow Neurological Institute.
FIG. 5. A: Bar graph shows elevation in the cell counts of active-
ly dividing cells in the spinal cord sections of adult rats. Rats with
a spinal cord lesion were treated with a low dose (3.0 ml) or high
dose (6.0 ml) of Shh. Rats without lesions were given a high dose
of Shh. The number of proliferating cells significantly increased
after rats with a lesion were exposed to Shh. Data were analyzed
with repeated measures analysis of variance, followed by the Stu-
dent– Newman– Keuls post hoc t-test (* p , 0.01). Originally pub-
lished in Bambakidis NC, Theodore N, Nakaji P, Harvey A,
Sonntag VK, Preul MC, et al: Endogenous stem cell proliferation
after central nervous system injury: alternative therapeutic options.
Neurosurg Focus 19(3):E1, 2005. Used with permission from
Neurosurgical Focus.
4 Neurosurg. Focus / Volume 24 / March/April 2008
N. C. Bambakidis et al.
FIG. 4. Photomicrograph of bromodeoxyuridine-stained cells
dividing around the central canal in the spinal cord of an adult
rodent exposed to recombinant Shh after sustaining a demyelinat-
ing lesion. These cells represent endogenous neural progenitors
stimulated by the combination of demyelination and Shh. Fluo-
rescent stain for bromodeoxyuridine (original magnification 3
200). Originally published in Bambakidis NC, Theodore N, Nakaji
P, Harvey A, Sonntag VK, Preul MC, et al: Endogenous stem cell
proliferation after central nervous system injury: alternative thera-
peutic options. Neurosurg Focus 19(3):E1, 2005. Used with per-
mission from Neurosurgical Focus.
greatest level of cellular induction occurs 3–7 days after
injury. In their unaltered state, these cells develop primari-
ly along astrocytic lineages, thus contributing to scar for-
mation and thereby inhibiting a restorative response. A key
goal of current research efforts is modification of this nor-
mal inhibitory response. Specifically, the goal is to alter the
cellular fate of these endogenous stem cells so that they
develop into cell types that can aid in axonal preservation
and repair.
The process of endogenous stem cell differentiation in
the injured adult nervous system has yet to be fully elucid-
ated. Several agents, however, play important roles in mo-
dulating this process. Such agents have included analogs
that stimulate the Shh pathway, and growth factors such as
bFGF. The Shh protein, which is critical in oligodendrog-
lial development and developmental neurogenesis, dramat-
ically increases the number of neuronal progenitor cells in
Therapeutic applications of stem cell biology

FIG. 6. Photomicrographs demonstrating diffuse positivity for nestin in dorsal regions of hyperproliferation from the
spinal cords of rats treated with Shh after contusive SCI. Nestin is an intermediate filament protein found in CNS pre-
cursors. Left, original magnification 3100;C; center, original magnification 3 50; and right, original magnification 3
200. Characteristically, these primitive-appearing cells demonstrate bipolar morphological characteristics and are highly
motile and proliferative. Originally published in Bambakidis NC, Theodore N, Nakaji P, Harvey A, Sonntag VK, Preul
MC, et al: Endogenous stem cell proliferation after central nervous system injury: alternative therapeutic options.
Neurosurg Focus 19(3):E1, 2005. Used with permission from Neurosurgical Focus.

FIG. 7. Triphasic treatment paradigm for SCI. A: Illustration shows optimal clinical management during early min-
utes after spinal cord injury. Spinal protection during prehospital treatment, surgical stabilization, and spinal cord perfu-
sion and oxygenation are key. B: The second step is neuroprotection from the administration of antiinflammatory
agents such as minocycline, erythropoietin, riluzole, or 4-aminopyridine. C: In the weeks after injury, the last phase
includes neuroregeneration based on promotion of endogenous stem cells, or other modalities to prevent scar formation,
ameliorate axonal loss and demyelination, and restore function. Originally published in Horn EM, Preul MC, Sonntag
VK, Bambakidis NC: Multimodality treatment of spinal cord injury: endogenous stem cells and other magic bullets.
Barrow Quarterly 23:9–13, 2007. Used with permission from Barrow Neurological Institute.

Neurosurg. Focus / Volume 24 / March/April 2008 5


the spinal cord.3,4 A similar phenomenon is observed in rats
with a contusion injury to the spinal cord. After oligoden-
drocytic precursors have been transplanted, markedly
reduced demyelination and increased conduction velocity
are observed across lesions. These findings correspond to
significant preservation of behavioral function correlating
to the reduction of cellular damage observed in treated ani-
mals (Figs. 5 and 6).2,3 The availability of small-molecule
analogs to the Shh pathway that may be administered intra-
venously allows these experimental findings to be repro-
duced while obviating the need for direct injection into the
injured spinal cord. In recent experiments involving contu-
sive SCI, these agents upregulated endogenous cell popu-
lations; further work is ongoing.
Other studies have shown that bFGF expression is up-
regulated in rodents after traumatic injury. These bFGF-
derived cells then differentiate into neuronal phenotypes.34
Additional agents that promote neuronal differentiation in
genetically engineered mice after contusion include growth
factors such as Mash1, epidermal growth factor, FGF2, and
neurogenin2.23
Conclusions
A multimodal approach to the treatment of SCI is need-
ed if significant breakthroughs in treatment and recovery
are to be expected. Such treatment must be thought of as
occurring temporally. During the acute period the best clin-
ical treatment is required. During the subacute period neu-
roprotective treatment may be beneficial. Finally, during
the delayed period neuroregenerative therapy is required
(Fig. 7). As more investigations in the expanding field of
stem cell biology are performed, particularly when applied
to the stimulation and manipulation of endogenous stem
cell populations, significant advances in the treatment of
SCI may be expected.
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Manuscript submitted December 13, 2007.
Accepted January 31, 2008.
Address correspondence to: Nicholas C. Bambakidis, M.D., c/o
Neuroscience Publications, Barrow Neurological Institute, 350
West Thomas Road, Phoenix, Arizona 85013. email: neuropub@
chw.edu.
7