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Functional recovery after severe CNS trauma: Current perspectives for celltherapy with bone marrow stromal cells

Jesus Vaquero *, Mercedes Zurita

Surgical Research Service, Neurosciences Unit, Hospital Universitario Puerta de Hierro-Majadahonda, Madrid, Spain

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

2. Cell therapy strategies for TBI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

3. Current perspectives for BMSC in the treatment of TBI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

4. Cell therapy strategies for SCI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

5. Current perspectives for BMSC in the treatment of SCI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347

Between these two faults, boldness is preferable to timidity. Dare

wins or is defeated, but excessive prudence leads us to a shameful

inactivity.

Santiago Ramon y Cajal, 1923

1. Introduction

In recent years, numerous research groups have studied on

experimental models of brain or spinal cord trauma the possible

therapeutic effect of neurotrophic factors, able to create in the

central nervous system (CNS) an environment conducive to

regeneration. Among these neurotrophic factors, nerve growth

factor (NGF) (Dixon et al., 1997; Zhou et al., 2003), fibroblastgrowth factor-2 (FGF-2) (Yoshimura et al., 2003), and insulin-

like growth factor-1 (IGF-1) (Saatman et al., 1997) have been

considered. Simultaneously, gene therapy protocols, which are

able to bring these factors to damaged areas, have been studied

(Longhi et al., 2004). Although these new strategies offer

interesting perspectives, the truth is that human clinical

applications still require many studies regarding the routes

and patterns of administration and the use of viral vectors. A

second line of research with an increasing importance in recent

years is the use of cell therapy with stem cells. These cells would

be able firstly, to release neurotrophic factors, and secondly, to

regenerate damaged nerve tissue through differentiation or

transdifferentiation into mature neural cells. The first experi-

mental studies used fetal tissue or embryonic stem cells, but it is

Progress in Neurobiology 93 (2011) 341349

A R T I C L E I N F O

Article history:

Received 4 September 2010

Received in revised form 30 November 2010

Accepted 7 December 2010

Available online 14 December 2010

Keywords:

Bone marrow stromal cells

Cell transplantation

Traumatic brain injury

Traumatic spinal cord injury

Stem cells

A B S T R A C T

The aim of this paper is to identify current perspectives for cell therapy applied to traumatic injuries of

the central nervous system (CNS). After using diverse types of cell therapy, at present there is a growing

experimentalevidence thattransplantation of bone marrow stromalcells (BMSC) can be useful to reverse

the sequels of trauma affecting the brain and spinal cord. Although we still do not know many details

about how these cells achieve their beneficial effects, the application of BMSC in humans, for brain or

spinal cord repair, is beginning. An exquisite caution and strict methodological controls are needed to

determine with certainty whether we can open a door of hope for many patients who currently suffer

severe neurological deficits that are now supposedly irreversible.

2010 Elsevier Ltd. All rights reserved.

Abbreviations: BMSC, bone marrow stromal cells; CNS, central nervous system;

FGF-2, fibroblast growth factor-2; IGF-1, insulin like growth factor; iPS, induced

pluripotent stem cells;NSC, neural stemcells; SCI,spinal cord injury; TBI,traumatic

brain injury.

* Corresponding author.

E-mail address: [email protected] (J. Vaquero).

Contents lists available at ScienceDirect

Progress in Neurobiology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p n e u r o b i o

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doi:10.1016/j.pneurobio.2010.12.002


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difficult for these studies to lead to clinical applications in

patients because of logistical, immunological, and ethical

reasons (Widner and Brundin, 1988; Hoffer and Olson, 1991).

Furthermore, the potential usefulness of embryonic stem cells

for the treatment of human diseases has been hampered by the

risk of tumorigenesis (Wu et al., 2007).

Multipotent neural progenitors and neural stem cells (NSC) canbe isolated from either embryonic or adult brain tissue and,

following engraftmentintothe adultbrain, theydifferentiate mainly

into glial cells, suggesting possible efficacy in cell therapy strategies

applied to traumatic CNS injuries (Park et al., 1999). At present, the

functional effect achieved for these cells in the treatment of

experimental CNS injuries has been very limited, and their clinical

usealso raises many problems becauseof the difficulty of obtaining

NSC (Cao et al., 2002). The use of immortalized neural cell lines

represents an alternative strategy (Whittemore and Onifer, 2000)

but raises the possibility of risks inherent to genetic manipulation,

which in practice means that many issues must be resolved before

considering its clinical application. The same considerations can be

applied to recent studies with the so-called induced pluripotent

stem cells (iPS), which can achieve remyelinization, axonal

regrowth, and functional improvement in spinal cord injury (SCI)

models (Tsuji et al., 2010). Because of these limitations, researchers

have paidattention to adult stemcells generally obtained frombone

marrow stroma (Opydo-Chanek, 2007).

The growing interest in cell therapy has resulted in much

knowledge about the many of the details of bone marrow stromal

cells (BMSC). These cells are isolated from the mononuclear

fraction of bone marrow and represent a population of undiffer-

entiated cells that do not express markers of hematopoietic stem

cells, such as CD14, CD19, CD34, CD45 and CD79, but they are

positive for CD73, CD105, and other adhesion molecules, such as

CD90 and CD106 (Horwitz et al., 2005; Dominici et al., 2006). We

also know that BMSC show a low expression of the major

histocompatibility complex antigens (Class II). Therefore, these

cells have low antigenicity, which is one of the main advantages in

the consideration of cell therapy protocols (Le Blanc and Ringden,

2005). Furthermore, these cells show a high expression of growth

factors, cytokines, and extracellular matrix molecules that, under

normal conditions, can contribute to the formation and function of

the bone marrow stromal microenvironment, inducing regulatory

signals not only for stromal stem cells themselves but also for

hematopoietic stem cells (Garca et al., 2004; Chen et al., 2005; Ye

et al., 2005).

Stromal cell cultures are morphologically heterogeneous and

show different cell types including a population of elongated cells

with low proliferation and differentiation, and a second population

of cells with a high capacity of proliferation and differentiation.

This latter cell population, in turn, can be of two types: (1) some

very small and rounded cells, or (2) cells that are larger withsomewhatstarry elements that appear to be progenitors of stromal

cells in culture. We accept that within the set of stromal stem cells

there is a subpopulation of cells known as multipotent adult

progenitor (MAP) cells, which proliferate indefinitely in vitro. It is

assumed that they are probably a rare form of stem cells that are

maintained with this potential from the embryonic period until

adulthood, and that, under certain circumstances, they can

differentiate into the cells of all three embryonic layers (Jiang

et al., 2002; Zeng et al., 2006; Raedt et al., 2007).

Earlier this decade, a diverse set of studies suggested that BMSC

can undergo in vitro transdifferentiation phenomenon when the

medium is treated with different chemical agents, resulting in cells

withadult neuronal-likemorphology (Woodburyet al.,2000;Mezey

and Chandross, 2000; Sanchez-Ramos et al.,2000; Hung et al.,2002;Dezawa et al., 2004; Hermann et al., 2004; Bossolasco et al., 2005).

Several authors have questioned whether this is true neuronal

transdifferentiation of BMSC, because the transformation into

neuronal-like morphology could be due to nonspecific changes of

the cell cytoskeleton. The truth is that there is increasing evidence,

both in vitro and in vivo, that it is possible to transform BMSC to

neurons and glial cells, thus creating the potential to regenerate

injured CNS tissue (Zurita and Vaquero, 2004, 2006; Zurita et al.,

2005, 2008; Vaquero et al., 2006; Bonilla et al., 2009).Studies done in our lab using BMSC for CNS trauma were

conducted in experimental models of paraplegia and have

reported that BMSC can regenerate the injured spinal cord and

produce newly formed nervoustissuethrough which axons cango

upward and downward in the year after transplantation (Zurita

andVaquero, 2006). In these studies,the labelingof BMSC through

gene transfection showed that neurons and glial cells present in

the regenerated tissue came from the transplanted BMSC. An

important detail was to test how BMSC also differentiate to cells

that form new vessels and promote angiogenesis (Zurita and

Vaquero, 2006; Zuritaet al.,2008). Inthe caseof traumatic injuries

of the spinal cord, it is relatively easy to quantify the functional

results obtained with these techniques and to study the

morphological changes that can occur in previously injured

areas. However, the evaluation of neurological deficits and the

possibility of recovery are more difficult in the case of

experimental traumatic brain injury (TBI). Nevertheless, numer-

ous studies are underway to determine whether the therapeutic

effects that seem to make BMSC useful in the treatment of

experimental spinal cord lesions are also applicable to the

treatment of severe TBI.

2. Cell therapy strategies for TBI

Our understanding of cell therapy for TBI has accrued over the

last decades and has shown promise in the management of this

devastating disease. Sinson et al. (1996) grafted fetal rodent

cortical tissue into the injured brains of adult rats resulting in

significant improvement in motor and cognitive function. The

potential effectiveness of neural stem cells engineered to secrete

neurotrophic factors has been studied by some authors on

experimental models of TBI and usually during the acute period

after trauma. So, Philips et al. (2001) described positive results

after intracerebral transplantation of immortalized neural stem

cells that were retrovirally transduced to produce NGF and Bakshi

et al. (2006b) reported cognitive function improvement after

transplantation into the perilesional brain tissue of neural

progenitor cells engineered to secrete glial cell line-derived

neurotrophic factor (GDNF). However, other studies using neural

stem cells have shown no functional improvement (McMahon et

al., 2010). In any case, when analyzing the literature, we can see

that in contrast to what has happened in SCI research, experimen-

tal studies using cell therapy in TBI are relatively few, and theresults have been contradictory (Harting et al., 2008). Because

severe experimental brain trauma produces significant loss of

brain tissue in rodents andthe difficulty in achievingan acceptable

survival of transplanted cells in cell therapy protocols, the results

are difficult to evaluate.

Nevertheless in recent years, Mahmood et al. reported

numerous experimental studies using BMSC for the treatment of

TBI (Mahmood et al., 2001a,b, 2002, 2003, 2004a,b, 2005, 2006,

2007; Lu et al., 2001). In these studies, cell therapy was used in the

early phases after trauma and a significant reduction of

neurological deficits was observed. Because of these and other

observations, BMSC appear to be highly promising for the

treatment of severe TBI to obtain neuroprotection or functional

recovery. In parallel, clinical trials in children using an intravenousadministration of autologous bone marrow-derived mononuclear

cells have been initiated (Harting et al., 2008).

J. Vaquero, M. Zurita / Progress in Neurobiology 93 (2011) 341349342


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3. Current perspectives for BMSC in the treatment of TBI

At present, the experimental studies by Mahmood et al. have

provided further evidence about the possibility of reversing

functional deficits in adult rats subjected to traumatic brain injury

through the administration of adult mesenchymal stem cells

obtained from bone marrow stroma. This group has reported inrecent years that BMSC reverse functional deficits when cells were

administered locally, intravenously, or intra-arterially (Mahmood

et al., 2001a,b, 2002, 2003, 2004a,b, 2005, 2006, 2007; Lu et al.,

2001). This effect is accompanied by identification of these cells in

areas of brain damage, irrespective of the route of administration,

and morphological characteristics suggesting their differentiation

into neural cells (Lu et al., 2001, 2006). However, these studies

generally show a discrepancy between the positive results

obtained and the low rate of cell transdifferentiation, suggesting

that the effect of stromal cells, at least in part, may be due to the

release of neurotrophic factors that induce regeneration in the host

tissue.

Conclusion from these experiences is that beneficial effect can

be achieved when human stromal cells are administered

intravenously to rat TBI models, and without signs of immune

rejection (Mahmood et al., 2003). This finding supports the low

antigenicity of BMSC, which is an important argument when

clinical applications are considered. Perhaps the biggest criticism

of these experiences lies in the fact that in almost all of the studies,

BMSC are administeredat an early stage after TBI, whichis far from

the actual conditions in which these new therapies could be

applied in humans; it seems reasonable that patients would only

use this technique during a chronic phase after having exhausted

the possibilities of rehabilitation treatment. Moreover, the

variability of brain lesions after an experimental trauma, and

the variable functional recovery of animals subjected to similar

parameters of traumatic injury, makes it difficult to evaluate theactual effectiveness of cell therapy.

We are currently developing experimental studies trying to

confirm the therapeutic efficacy of the intracerebral administra-

tion of BMSC during the chronic phase of severe TBI where there is

an established neurological deficit. Our results in rodents have

shownthat thelocal administration of 10 106 BMSCin the areaof

brain injury two months after trauma achieved a clear recovery

according to a functional assessment using a modified sensory-

motor neurological deficit (mNSS) scale (see Fig. 1 and Supple-

mental video 1). This improvement was morphologically correlat-

ed with the presence of transplanted BMSC in the injured area,

some of which showed transdifferentiation to neurons and glial

cells together with an increase of endogenous neurogenesis

(Bonilla et al., 2009).

Although these initial findings appear promising, further

studies are required. The size of the brain lesions and their

variability appear to be determinants of the need to find biological

matrices allowing cell survival and differentiation of the trans-

planted stem cells. Similarly, the critical number of BMSC that are

necessary to restore the functional deficits after experimental

Fig. 1. BMSC can improve established neurological sequels after severe TBI in rodents (see Bonilla et al., 2009). (A) Macroscopical view of the brain two months after

experimental TBI by weight-drop impact in Wistar rat. (B and C) Immunohistochemical studies in injured brain, two months after intracerebral administration of BMSC.

Donor cells were obtained from male-donor rats, and they were transplanted into female rats subjected to TBI. Transplanted BMSC cells showing the SrY gene of Y-

chromosome can be seen into the injury zone by means of in situ hybridization. In (B), double immunostain permits the identification of neurons showing SrY gene (arrows,

SrY gene immunostained in red), and suggesting a neuronal transdifferentiation of the transplanted BMSC (green: neuron nuclei, showing expression of neuron-specific

nuclear antigen (NeuN). Original magnification: 1000. In (C), cells with nuclear presence of SrY gene (arrows) and cytoplasmic expression of gliofibrillary acidic protein

(GFAP, in green) can be seen,suggesting astroglial transdifferentiationof the transplanted BMSC.Original magnification:400. (DF)Histologicalfields in BMSC-treated rats,

four months after severe TBI, and two months after local BMSC transplantation. (D) A stream of cells, identified as migratory neuroblasts due to doublecortin-positivity,

extends from the subventricular zone (SVZ). Original magnification: 40. (E) Injury zone, showing doublecortin-positive cells at the edges (top). Original magnification:

200. (F) Doublecortin-positive cells filling the injury zone can be found. Original magnification: 200. The associated graph shows the temporal profile of functional

recovery in a seriesof brain-injured ratsthat received intracerebral administrationof saline(n: 1 0 )or 5 106 BMSC (n: 10)in saline, twomonths after severeTBI.UsingmNSS

test, significant differences were detected starting 6 weeks after intracerebral BMSC transplantation; p < 0.05). See Supplemental video 1.

J. Vaquero, M. Zurita / Progress in Neurobiology 93 (2011) 341349 343


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brain trauma represents one of the main issues to be resolved. In

one of the experiments reported by Mahmood et al. (2005),

different doses of BMSC were intravenously administered in adult

rats one week after TBI. Three months later, the rats that had

received more than 4 106 BMSC showed some functional

recovery, but rats that received only 2 106 BMSC did not,

suggesting that the number of administered stem cells is adetermining factor for achievement of the therapeutic effect. If this

hypothesis is correct, strategies should aim at making large

quantities of adult stem cells that can reach areas of brain injury.

Nevertheless at this point, one consideration must be born in mind.

Although the number of administered stem cells is important, we

believe that the future research in this field should aim to ensure

that the cells survive a long-term in the brain. Regardless of the

number of cells administered various factors are present in the

host tissue, most of which are related to the pathophysiology of

traumatic injury, that decreases the survival of the transplanted

cells.

However, the experiments conducted in our lab using BMSC

labeled with Indium-111 suggest that, after the systemic

administration of these cells, they preferentially colonize in

infusion-rich organs, such as the liver and spleen, at least when

the BMSC administration is performed during the chronic phase of

CNS trauma (De Haro et al., 2005; Vaquero et al., 2006). Therefore,

it is necessary to know the best route for the administration of cell

therapy to achieve a better survival of the transplanted cells after

administration and the best time for the application of cell therapy

after trauma. Even though there are unresolved issues, it is obvious

that in recentyears newtechniques of cell therapy with adult stem

cells, together with the new concepts about the possibility of

regeneration of the adult nervous system, have opened an avenue

of hope for the treatment of the sequels of TBI. Any advance in this

field, as in many other areas of neurobiology, will require close

collaboration between basic and clinical researchers.

4. Cell therapy strategies for SCI

Although it has been classically considered that traumatic

paraplegia is irreversible, over the last century, numerous

experimental models have been designed to investigate if, in

some cases, the limited regenerative activity following SCI as

described by Ramon y Cajal (1914) can be increased. Around 1940,

transplants were performed into injured spinal cord tissue with

variable results (Brown and Mc Cough, 1947; Barnard and

Carpenter, 1950). Subsequently, Kao (1974) tried to interpose

various neural tissues, such as peripheral nerve, ganglion

nodosum, or cultured cerebellar brain tissue, between the ends

of a transected spinal cord in an attempt to restore its continuity.

As a result of these studies, new evidence showed that the

peripheral nerve, implanted between the ends of a transectedspinal cord, achieved a bridge while reducingthe local mesodermic

scar. By 1980, the group of Aguayo, discussed both the anatomical

and functional possibilities, of the bridges of peripheral nerve

placed between the ends of a transected spinal cord ( David and

Aguayo, 1981). Through axonal labeling techniques using peroxi-

dase, these authors showed that spinal neurons projected their

axons along these transplants and also provided evidence that, at

least in part, the axons that invaded the transplants represented

the regeneration of the previously damaged axons. These

experiences, and those of Wrathall et al. (1982) or Fernandez et

al. (1986), showed that peripheral nerve grafts may constitute a

valid support for axonal growth perhaps due to trophic factors

produced by Schwann cells. Despite this evidence, the literature

contains few attempts at transplanting nervous tissue in theinjured spinal cord. The explanation appears to lie in the technical

difficulty in performing these transplants and from the poor

functional results obtained in the few experiences (Nygren et al.,

1977; Das, 1983a,b, 1987).

In association with these procedures, the placement of tubes

of biological or synthetic materials surrounding the area of SCI has

been studied. These tubes might act as a guide for regenerative

axons especially when various neurotrophic substances are

injected in the interior. The most commonly used materialsinclude biodegradable polymers of a polyester type that carry

Schwann cells or brain derived neurotrophic factor (BDNF). The

demonstration that olfactory bulb ensheathing cells are capable of

achieving the myelination of CNS axons has also led to the use of

these cells,togetherwithSchwann cells and the tube systems, with

promising results in rats subjected to transection (Ramon-Cueto et

al., 1998, 2000).

However, the first clinical studies in human paraplegia using

olfactory ensheathing cell transplantation have not demonstrated

functional improvement (Mackay-Sim et al., 2008).

Fetal neural cells have been used by a diverse set of researchers

in an attempt to regain function after spinal cord trauma with

experimental results that suggest that age may be a determining

factor for functional recovery (Bregman et al., 1993; Bregman,

1994; Diener and Bregman, 1998; Reier, 2004). These studies have

been generally performed on incomplete SCI models during acute

stages after injury. However, when looking for donor nervous

tissue capable of reconstructing the injured spinal cord, it seems

logical to consider the use of spinal cord tissue and specifically,

fetal spinal cord tissue. Although the first experimental transplants

of fetal spinal cord to a sectioned adult spinal cord showed low

survival (Das, 1983a) compared with other types of transplants

such as fetal brain tissue, the fetal spinal cord is seen as the type of

donor tissue most suitable for these experimental studies. The

experience using fetal spinal cord transplantation showed that the

survival of the graft within the previously injured spinal cord

depended on the age of the donor, and it is best when the fetus

corresponded to a period between 13 and 15 days of gestation in

the rat. We also know that such transplants can survive long time

and experience changes that include the formation of unmyelin-

ated areas that resemble the gelatinous substance of dorsal horn.

Immunofluorescence studies indicate that neurons in the trans-

plants may project axons to the host tissue (Reier, 1985). Pallini et

al. (1989) reported their experience grafting fetal spinal cord (13

14 days of gestation) into the spinal cord of adult rats. The

transplant was done immediately after transection and achieved a

survival rate of 55% with good morphological integration, but the

clinical and electrophysiological assessment of the animals did not

show any kind of functional recovery. However, there are other

publications that show functional recovery of animals after

transection and subsequent reconstruction with fetal spinal cord

transplants (Zompa et al., 1997). In studies using fetal brain tissue

to reconstruct the injured spinal cord, the first experimentsindicated a low viability of the grafts, perhaps as a result of a

deficient surgical technique. At least theoretically, the use of fetal

brain tissue may have the advantage of a high proliferative

capacity, which helps to restore the injured tissue (Das, 1987).

Although at present limited experience exists about the useof fetal

cortex transplants in the intact or injured spinal cord (Bernstein et

al., 1984; Das, 1987), the grafted tissue can survive in the adult

spinal cord using an appropriate surgical technique, with easier

integration at the level of the gray matter. However, most

experimental models of SCI in which neural transplants have

been performed have used spinal cord transection, which may

differ significantly from what occurs in humans, where traumatic

paraplegia is usually the result of a contusion injury. In addition,

the neural transplants in these experimental models wereperformed almost always immediately after transection in models

of acute paraplegia, such as the studies by Ramon-Cueto et al.



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(2000) using olfactory bulb ensheathingcells. It must be taken into

account that performing the transplant in an acute phase may be

unfavorable to the survival of the transplanted tissue. Further-

more, with the logical objective of a possible clinical application of

these techniques, it is obvious that their use in humans should be

done in phases of chronic paraplegia, where the chances of

spontaneous functional recovery have already been dismissed.Bearing in mind all these considerations, by 1990 we aimed to

study the functional and morphological results of delayed

transplantation of fetal neocortical tissue in a model of spinal

cord contusion causing chronic paraplegia. The use of neocortical

tissue was based on the finding that this tissue shows a high

mitotic capacity, which theoretically may lead to fill the area of

posttraumatic intramedullary necrosis. As a result of these pilot

studies (Vaquero et al., 1992a,b), we obtained evidence that

transplantationof fetal brain tissuein the injured spinal cord in the

adult rat achieved a survival rate greater than 80% if donor tissue

was obtained from fetuses at 18 days of gestation and transplan-

tation was performed at least one week after the traumatic injury.

These results were similar if the transplantation was performed at

one week or at 34 months after SCI, in a situation of chronically

established paraplegia. Furthermore, grafted tissue showed

integration with the host tissue and neurons within the

transplanted tissue underwent maturational changes, showing

the morphological appearance of cerebral neurons. At this point,

the discrepancy between the good histological features (e.g., the

integration of transplanted fetal brain tissue into the spinal cord)

andthe absence of significant motor recovery led us to believe that

the transplanted animals were followed for a sufficiently long

evolutionary time, especially given the fact that experience of

literature is limited to a few months of follow-up. A proper

microsurgical technique, the prevention of infections, bladder

emptying in the early stages after SCI, healing of pressure ulcers,

and the presence of a single caregiver for our animals, were the

factors that allowed us to maintain paraplegic animals for more

than one year after transplantation. This achievement led us to the

observation that six months after transplantation, the animals

showedsigns of motorand sensory recoverythat was initiatedby a

series of spontaneous movements of thetail and hind legs, together

with a response to pain stimulus applied at these loci. However,

animals that survive long-term after transplantation often have

significant rigidities that prevent any joint movement. Thus by

1995, we started a new experimental phase, incorporating

intensive rehabilitation in the paraplegic animals that allowed

us to keep them alive and free of joint stiffness and avoided, as in

humans, most of the complications of chronically established

paraplegia. At the same time, diverse donor neural tissues were

studied, and the best tissue regeneration rates wereobtainedwhen

the centromedullary posttraumatic cavities were filled with fetal

brain tissue and peripheral nerve, possibly because of the knownneurotrophic effect of Schwann cells (Zurita et al., 2000, 2001).

With this experimental model, we kept paraplegic rats more than

two years after intralesional transplantation of fetal cerebral

cortex with peripheral nerve fragments. A larger follow-up was

limited by the age of the animal itself, which hardly reached the 2-

year survival in Wistar rats. This technique allowed us to confirm

that transplanted rats showed a progressive recovery of sensory

and motor function that became very evident 68 months after

transplantation. Furthermore, the transplanted rats showed

recovery of muscle atrophy, which was clearly significant one

year of transplantation (Zurita et al., 2001). Histological examina-

tion of animals after more than one year of evolution showed a

seamless integration of the transplanted tissue with identifiable

adult brain tissue at the level of what was once an intramedullarycavity. Parallel to these experimental studies, some authors (Falci

et al., 1997; Wirth et al., 2001) marked the starting point for the

implementation of these new techniques of cell therapy in patients

with established paraplegia. In these early clinical trials, fetal

spinal cord tissue was used and, although some patients described

recovery of sensation in the lower limbs, there was no clear motor

recovery. In our opinion, these poor results could be due to the use

of fetal spinal cord tissue rather than brain tissue because in our

experience, fetal cerebral cortex tissue has a higher proliferativecapacity than fetal spinal cord tissue and the transplanted spinal

cord tissue failed to completely fill the posttraumatic cavities

(Wirth et al., 2001). According to our experimental studies, delayed

transplantation of fetal spinal cord in the spinal cord of paraplegic

adult rats has never achieved motor recovery, at least in the 10

months following the transplantation procedure. However, based

on similar parameters of neurological injury, the use of fetal brain

tissue in co-transplantation with peripheral nerve provided

acceptable functional results (Zurita et al., 2001). Regardless of

these observations, it is obvious that the experimental animal and

man represent different biological systems, making it difficult to

predict that the results obtained in rodents can be successfully

applied to the treatment of paraplegic patients. We must consider

that studies with higher numbers of animals are problematic

because of the difficulty in the long-term maintenance of the

paraplegic animals and the difficulty of their rehabilitation.

By 2000, the use of fetal brain tissue for transplantation into

the posttraumatic spinal cord cavities in paraplegic patients

appeared to be a reasonable option. However, this approach was

faced with enormous technical difficulties because it was

extremely difficult to obtain human brain tissue with good cell

viability from either spontaneous or therapeutic abortions. In

addition, the need for immunosuppression added a new difficulty

to the technique, especially considering that paraplegic patients

often have urinary tract infections. Given these difficulties, we

considered alternatives in paraplegicpatients and fundamentally,

we explored the possibilities for CNS regeneration provided from

so-called adult stemcells. Among thedifferent types of adult stem

cells, BMSC were soon regarded as potentially useful, in the same

way that these cells had been considered for the treatment of TBI

because of their easy availability and the possibility of autologous

transplantation.

5. Current perspectives for BMSC in the treatment of SCI

After the first descriptions that BMSC may be transformed in

vitro into neuronal-like cells with the addition of specific chemical

agents to the culture medium (Woodbury et al., 2000; Mezey and

Chandross, 2000; Sanchez-Ramos et al., 2000; Hung et al., 2002;

Dezawa et al., 2004; Hermann et al., 2004; Bossolasco et al., 2005),

several research groups studied the possibility of applying this to

the clinic, specifically to the treatment of traumaticparaplegia. As a

result, comments on the usefulness of BMSC to restore functionaldeficits in experimental models of acute or incomplete SCI were

soon published (Chopp et al., 2000; Chopp and Li, 2002; Hofstetter

et al., 2002; Lee et al., 2003; Ankeny et al., 2004 ). In 2004, we

demonstrated in our laboratory that this form of cell therapy may

also be usefulif applied to complete SCI in a situationof chronically

established paraplegia (Zurita and Vaquero, 2004). In these

experimental conditions, intralesional transplantation of BMSC

was followed by clear signs of neurological recovery. At one year,

an almost complete motor recovery was observed in over 60% of

cases that was associated with an apparent regeneration of nerve

tissue at the previously injured area (Vaquero et al., 2006; Zurita

and Vaquero, 2006).

Once we confirmed the efficacy of BMSC transplantation in the

regeneration of damaged spinal tissue to achieve the motorrecovery of paraplegic animals, and keeping in mind that some

publications indicated that intravenous administration of BMSC



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reversed functional deficits in rats subjected to TBI (Mahmood et

al., 2001a) we asked whether the systemic administration of these

cells could also be effective. As a first step, we labeled BMSC with

bisbenzimide or with Indium-111 and verified the distribution of

labeled cells following intravenous administration. The results

showed that after intravenous administration, labeled BMSC

preferentially colonized the spleen, liver, and kidneys but didnot significantly reach the area of the SCI (De Haro et al., 2005).

These studies were the first to report the usefulness of Indium-111

to the in vivo study of the distribution of BMSC after their

administration in cell therapy procedures. However, it is obvious

that systemic administration of BMSC prevents these cells from

arriving in significant number to areas of traumatic SCI, at least

during the chronic phase after SCI. In another study in chronic

paraplegic rats, we compared the usefulness of systemic adminis-

tration of BMSC with local administration, and a clear difference in

favor of the local administration was found (Vaquero et al., 2006).

These good results, after more than 150 transplants in rodents,

have been reproduced in adult pigs with chronically established

paraplegia, which suggests that this technique may also be helpful

in humans (Zurita et al., 2008) (see Fig. 2 and Supplemental videos

2 and 3).

As a result of these and other experimental studies, the

implementation of this type of cell therapy in patients is beginning.

Early clinical trials have used stem cells that include both stromal

cells andhematopoieticstem cells and have confirmed theabsence

of sideeffects (Park et al., 2005; Sykova et al., 2006; Chernykhet al.,

2007; Yoon et al., 2007; Saito et al., 2008; Deda et al., 2008; Pal et

al., 2009). Recently, Geffner et al. (2008) reported a preliminary

study in a series of paraplegic patients treated with the local

transplantation of stem cells derived from bone marrow. The

patients have been evaluated for one year after transplantation and

show recovery in sensitivity, motility, and bowel control. These

early results seem to justify the initiation of clinical trials in

patients to evaluate a sufficient number of cases to obtain reliable

conclusions.

In the present state of our knowledge, it is difficult to argue

about the advantages of using only BMSC for these transplants, as

diverse groups have done in preclinical or clinical studies (Chopp

et al., 2000; Hofstetter et al., 2002; Ohta et al., 2004; Zurita and

Vaquero, 2004, 2006; Bakshi et al., 2006a; Vaquero et al., 2006;Himes et al., 2006; Parr et al., 2007; Saito et al., 2008; Pal et al.,

2009) or either use, as has been done in the first human clinical

experiences, a mixture of bone marrow mononuclear cells (Park

et al., 2005; Sykova et al., 2006; Chernykh et al., 2007; Yoon et al.,

2007; Deda et al., 2008; Geffner et al., 2008). Given that stromal

cells represent less than 0.1% of bone marrowcells, their use in cell

therapy protocol requires manipulation of the cells in a high

biosecurity environment to expandthe cells andobtain a sufficient

number before transplantation. It involves technical complexity

and this is possibly the main argument for the use of bone marrow

stem cells without excluding the fraction of hematopoietic stem

cells. However, stromal cells have the advantage of their great

specificity to achieve neural transdifferentiation within the host

tissue, ease of expansion, taking into account that the effectiveness

of transplantation appears to be dose-dependent, and finally, low

antigenicity, which could eventually lead to the use of allogeneic

stromal cells in humans.

It is obvious that we have yet to gain a better understanding of

the mechanisms by which such cell therapy achieves neurological

recovery. In experimental studies, it is noteworthy that the

functional recovery of animals begins before the establishment of a

tissue bridge suitable for thepassage of axons (Zurita and Vaquero,

2004, 2006; Vaquero et al., 2006). Therefore, it is obvious that after

transplantation, at least at an early stage, different regenerative

processes, including release of neurotrophic factors by the

transplanted cells or activation of endogenous mechanisms, can

work together to restore neurological functions.

Fig. 2. Experimental studies show the effectiveness of cell therapy with BMSC in adult pigs (AC) and rodents (D and E) suffering established paraplegia. (A and B)

Somatosensory-evoked potentials (SSEP) in our experimental model of SCI in minipig (for technical details, see Zurita et al., 2008). (A) Absence of SSEP, three months after

severe SCI.(B) Recovery of SSEP,three months afterlocal BMSCtransplantation(70 106 autologousBMSC). (C)Two months afterlocal BMSCtransplantation, motorrecovery

canbe seen in previouslyparaplegic pigs. At this time, some treatedanimalsget up spontaneously. See Supplemental videos2 and3. (Dand E)SCI model inadult Wistarrats.

(D) Postraumatic cavities were filled, eight months after intralesional administration of 5 106 BMSC. A clear difference, compared with controls, can be seen. (E) One yearafterlocal administration of 5 106 BMSC,a newnervoustissue, fillingthe previous traumaticcentral spinalcordcavity, canbe seen.H.E.,original magnification:40.At this

time, a clear functional recovery is observed in transplanted animals. See Zurita and Vaquero (2006).



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Based on experimental studies, cell therapy strategies for

repair of SCI should be addressed to bring the greatest possible

number of stem cells to the injured area. This strategy increases

cell survival and facilitates neural differentiation. The adminis-

tration of stem cells in the subarachnoid space can be a

complementary approach to local administration (Ohta et al.,

2004; Sateke et al., 2004; Bakshi et al., 2004, 2006a; Himes et al.,2006), and it is necessary to optimize transplantation procedures

using biological scaffolds. Therecentfinding in ourlaboratorythat

plasma-derived scaffolds are extremely useful in achieving this

aim (Zurita et al., 2010) offers new perspectives in this exciting

field of research.

6. Conclusions

In recent decades, the development of new techniques for cell

therapy allowed to consider its possible application to the

treatment of traumatic injuries of the CNS. After several years of

experimental studies, there is growing evidence in favor that

intralesional transplantation of adult stem cells obtained from

bone marrow stroma can be useful to reverse the sequels oftraumatic injuries affecting the brain and spinal cord. The

application of these techniques to patients is beginning, but it is

obvious that we still do not know many details about how these

cells operate and how we canoptimize the seemingly good results.

Prudence and submission to ethical and methodological standards

are basic premises in bringing these studies to patients.

Conflict of interest

The authors have no relevant affiliations or financial involve-

ment with any organization or entity with a financial interest in or

financial conflict with the subject matter or material discussed in

the manuscript. This includes employment, consultancies, hono-

raria, stock ownership or options, expert testimony, grants or

patents received or pending, or royalties.

Acknowledgements

Most of the experimental work by the authors cited in this

review has been possible by grants received from the Mapfre

Foundation, Mutua Madrilena Foundation, and the Institute of

Health Carlos III (Fondo de Investigacion Sanitaria, FIS 07/0621 and

PS09/01105).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.pneurobio.2010.12.002.

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