Complete Spinal Cord Injury Treatment Using Autologous BoneMarrow Cell Transplantation and Bone Marrow Stimulation withGranulocyte Macrophage-Colony Stimulating Factor: Phase I/IIClinical Trial

SEUNG HWAN YOON,a,b YU SHIK SHIM,a,b YONG HOON PARK,a,g JONG KWON CHUNG,a,d JUNG HYUN NAM,e

MYUNG OK KIM,f HYUNG CHUN PARK,a,b SO RA PARK,a,c,h BYOUNG-HYUN MIN,i EUN YOUNG KIM,b

BYUNG HYUNE CHOI,a,g HYEONSEON PARK,a,b YOON HAa,b,h

aInha Neural Repair Center, bDepartments of Neurosurgery, cPhysiology, dAnesthesiology, eDiagnostic Pathology,and fRehabilitation Medicine, gInha Research Institute for Medical Sciences, hCenter for Advanced MedicalEducation by BK21 Project, Inha University College of Medicine, Incheon, Korea; iDepartment of Orthopedics,Molecular Science, and Technology, Ajou University Hospital, Suwon, Korea

Key Words. Human bone marrow cells • Granulocyte macrophage-colony stimulating factor • Spinal cord injury • Transplantation

ABSTRACT

To assess the safety and therapeutic efficacy of autologoushuman bone marrow cell (BMC) transplantation and theadministration of granulocyte macrophage-colony stimu-lating factor (GM-CSF), a phase I/II open-label and non-randomized study was conducted on 35 complete spinalcord injury patients. The BMCs were transplanted byinjection into the surrounding area of the spinal cordinjury site within 14 injury days (n � 17), between 14days and 8 weeks (n � 6), and at more than 8 weeks (n �12) after injury. In the control group, all patients (n � 13)were treated only with conventional decompression andfusion surgery without BMC transplantation. The pa-tients underwent preoperative and follow-up neurologicalassessment using the American Spinal Injury AssociationImpairment Scale (AIS), electrophysiological monitoring,and magnetic resonance imaging (MRI). The mean fol-

low-up period was 10.4 months after injury. At 4 months,the MRI analysis showed the enlargement of spinal cordsand the small enhancement of the cell implantation sites,which were not any adverse lesions such as malignanttransformation, hemorrhage, new cysts, or infections.Furthermore, the BMC transplantation and GM-CSF ad-ministration were not associated with any serious adverseclinical events increasing morbidities. The AIS grade in-creased in 30.4% of the acute and subacute treated pa-tients (AIS A to B or C), whereas no significant improve-ment was observed in the chronic treatment group.Increasing neuropathic pain during the treatment and tu-mor formation at the site of transplantation are still remain-ing to be investigated. Long-term and large scale multicenterclinical study is required to determine its precise therapeuticeffect. STEM CELLS 2007;25:2066–2073

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

It has long been believed that intrinsic repair is quite restrictedafter spinal cord injury (SCI) because neurogenesis rarely oc-curs in the central nervous system (CNS). As a result, celltransplantation has become a promising therapeutic option forSCI patients. Many experimental studies have suggested that thetransplantation of bone marrow cells (BMCs), neural progenitorcells, or olfactory ensheathing cells could promote functionalimprovements after SCI [1–4]. In recent years, some have foundthat BMCs differentiate into mature neurons or glial cells underspecific experimental conditions [5–7]. These findings imply thetherapeutic potential of BMCs in patients with neurologicaldiseases and can obviate ethical problems.

The grafting of BMCs into the SCI models has been activelystudied. Transplanted BMCs were found to improve neurolog-

ical deficits in the CNS injury models by generating neural cellsor myelin producing cells [8, 9]. Furthermore, BMCs can pro-duce neuroprotective cytokines, which rescue the neurons withimpending cell death after injury [10, 11]. Several clinical trialshave explored the hypothesis that cell transplantation may en-hance the recovery of neurologic functions after SCI. However,it was reported that significant functional recovery after celltransplantation was rarely achieved in the human clinical trials[12, 13]. These reports raise a concern that treatment protocolsusing only cell transplantation are not sufficient to achieve therequired therapeutic goals in SCI.

Recently, it has been reported that hematopoietic cyto-kines including granulocyte macrophage-colony stimulatingfactor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), or erythropoietin had neuroprotective effects and im-proved neurologic functions after CNS injury [14 –18]. Thesefindings suggest that GM-CSF, which is popularly used and

Correspondence: Yoon Ha, M.D., Ph.D., Department of Neurosurgery, Inha University College of Medicine, 7-206, Sinheung-dong 3-ga,Jung-gu, Incheon 400-712, Korea. Telephone: �82-32-890-3449; Fax: �82-32-890-3099; e-mail: hayoon@inha.ac.kr Received December29, 2006; accepted for publication April 13, 2007; first published online in STEM CELLS EXPRESS April 26, 2007. ©AlphaMed Press1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2006-0807

TRANSLATIONAL AND CLINICAL RESEARCH

STEM CELLS 2007;25:2066–2073 www.StemCells.com

considered safe for hematologic disease, could be used forSCI treatment.

In our previous study, GM-CSF decreased neuronal apopto-sis and improved the functional outcome in SCI animal models[19]. Preliminary results of our study demonstrated that BMCtransplantation and GM-CSF treatment didn’t increase seriouscomplications [14]. Furthermore, it has been published thatGM-CSF stimulates microglial cells to increase brain-derivedneurotrophic factor (BDNF) synthesis [18].

As a result, it was hypothesized that administration of GM-CSF to SCI patients could be an adjunct measure to improve thetherapeutic effects of BMC transplantation (Fig. 1). In thisstudy, autologous BMCs were transplanted, and the bone mar-row was stimulated with GM-CSF in SCI patients to study thetherapeutic and adverse effects of the treatment.

MATERIALS AND METHODS

Patient SelectionThis phase I/II nonrandomized study was approved by the Institu-tional Review Board at Inha University Hospital, and all procedureswere performed after obtaining written informed consent. Detailedprotocols for patient selection and for BMC transplantation and theGM-CSF administration have been reported previously [14]. Inbrief, patients were eligible when they were admitted to Inha Uni-versity Hospital with complete SCI (American Spinal Injury Asso-ciation [ASIA] Impairment Scale [AIS] grade A). Patients firstunderwent successful spinal cord decompression and stabilizationwith anterior cervical fusion using an autologous iliac bone graftand demonstrated persistent complete paralysis below the level ofinjury. Exclusion criteria were the presence of anatomical transec-tion of the cord, visualized by spinal magnetic resonance imaging(MRI), fever (above 39°C), ventilator-assisted breathing, and seri-ous pre-existing medical diseases. According to the time windowbetween the injury and transplantation, eligible patients were di-vided into three groups: acute (�2 weeks), subacute (2–8 weeks),and chronic (�8 weeks). In the control group, all 14 patients weremanaged with decompression surgery of the spinal canal and thenreferred to the rehabilitation clinic for active rehabilitation. Patientgroups are summarized in Figure 2A and Table 1. Patients in thecontrol group were not treated with BMC transplantation or GM-

CSF administration. All 14 patients in the control group wereselected randomly from complete SCI patients (AIS A) who wereadmitted to Inha University Hospital from January 2001 to May2003. The patients in the treatment groups were selected from thepatients admitted to the hospital after June 2003. Demographic datais summarized in Table 1. This study was designed as a nonrandomand open-label safety trial with observer-blind neurologic evalua-tions of patients.

Separation of Human Bone Marrow CellsBone marrow blood (100–150 ml) was aspirated from the iliac boneand diluted in Hanks’ balanced salt solution (HBSS) at a ratio of1:1. After the samples were centrifuged (1,000g for 30 minutes)through a density gradient (Ficoll-Paque Plus, 1.077 g/l; AmershamBiosciences, Piscataway, NJ, http://www.amersham.com), themononuclear cell layer was recovered from the gradient interfaceand washed with HBSS. The cells were centrifuged at 900g for 15minutes and resuspended in 1.8 ml of phosphate-buffered saline ata density of 1.1 � 108 cells per milliliter. The concentrated BMCswere then transferred to the operation room. All the procedures werecarried out in a sterile environment.

OperationIn acute SCI patients, transplantation was done within 14 days afteradmission. Neurologic examination was performed immediatelybefore operation to confirm complete SCI. Complete laminectomywas performed from one vertebra above to one below in order toprovide sufficient access to the transplantation site. The dura materwas then incised, sparing the arachnoid, which was subsequentlyopened separately with microscissors. The dorsal surface of thecontusion site was located using high-power microscopic magnifi-cation. After exposure of sufficient surface at the contusion site,300-�l aliquots of cell paste (total volume of 1.8 ml) were injectedinto six separate positions surrounding the lesion site with theinjection depth of 5 mm from the dorsal surface and 5 mm lateralfrom the midline. To avoid mechanical injury during injection, 2 �108 cells were injected at a rate of 300 �l/minute using a 21-gaugeneedle attached to a 1-ml syringe. To prevent cell leakage throughthe injection track, the injection needle was left in position for 5minutes more after completing the injection. The dura mater andarachnoid were then closed. The muscle and skin were closed layerby layer.

Figure 1. Therapeutic strategyof BMC transplantation and GM-CSF. GM-CSF stimulates andmobilizes the bone marrow stemcells (direct pathway). In addi-tion, GM-CSF can have intrinsicspinal cord repair mechanisms(indirect pathway), includingneuroprotection from apoptosis,endogenous stem cell activation,inhibition of glial scar formation,and microglial cell activation.Abbreviations: BMCs, bone mar-row cells; GM-CSF, granulocytemacrophage-colony stimulatingfactor.

2067Hwan Yoon, Shik Shim, Hoon Park et al.

www.StemCells.com

GM-CSF Injection ScheduleAfter surgery, a total of five cycles (daily for the first 5 days of eachmonth over 5 months) of GM-CSF (Leucogen; LG Life Science,Seoul, Korea, http://www.lgls.co.kr/eng) were injected subcutane-ously (250 g/m2 of body surface area).

Follow-Up StudiesThe neurological status of the patients was determined in terms ofAIS. Using AIS, a five-scale subdivision was used: A, a completeloss of motor and sensory function; B, sensory but not motorfunction is preserved below the neurological level and includes thesacral segments S4–S5; C, motor function is preserved below theneurological level, and more than half of the key muscles below theneurological level have a muscle grade of less than III; D, motorfunction is preserved below the neurological level, and at least halfof the key muscles below the neurological level have a muscle gradeof III or more; E, motor and sensory function are normal. The meanduration for follow-up was 10 months postoperatively. During theGM-CSF administration, vital signs were checked and completeblood analysis was taken daily.

We performed electrophysiological studies such as motorevoked potentials, somatosensory evoked potentials, and electro-

myography to differentiate voluntary muscle contraction from re-flex or involuntary spontaneous limb movement. The spinal MRIwas also carried out to examine any changes in the spinal cord andsurrounding tissues. Changes in activity patterns in the corticalsensorimotor networks were measured using functional MRI duringthe proprioceptive stimulation with repetitive passive toe move-ment. All preoperative and postoperative follow-up studies and thetreatment schedules for the acute group are summarized in Figure2B.

Statistical AnalysisStatistical analyses included analysis of variance or Mann-WhitneyU for the nonparametric variables (age, follow-up duration, andperipheral blood leukocyte number); �2 test or Fisher’s exact testwere performed to analyze the nominal or ordinal variables (sex,injury level, adverse events, neuropathic pain, AIS improvement,and MRI findings). All tests were considered significant at p valuesless than .05. Statistical comparisons were analyzed with SPSS 12.0(SPSS, Chicago, http://www.spss.com).

Role of the Funding SourcesThe sponsors of the study had no role in the study design, datumcollection, datum analysis, datum interpretation, or the writing ofthis report. The corresponding author had full access to all data inthe study and had final responsibility for the decision to submit forpublication.

RESULTS

Among the 53 patients treated, 48 (90.5%) were included in the10-month follow-up (Fig. 1). Five patients (9.5%) were ex-cluded because two patients did not want BMC transplantation,and three patients were ASIA B in preoperative neurologicstatus. The demographic characteristics of the study populationwere similar across the three treatment groups and the controlgroup. The mean follow-up period varied from 10.1–11.3months.

SafetyNo patients experienced serious complications requiring opensurgery such as sepsis or wound infection. Table 2 outlines theadverse events that occurred during the treatment. A higherdegree of febrile reaction was noted in the GM-CSF treatmentgroup. However, it didn’t increase the meaningful mortality ormorbidity. Serious wound infection or gastrointestinal bleedingwas not observed in this study. Theoretically, mechanical injuryto the spinal cord neurons may occur during cell transplantationdirectly into the spinal cord, resulting in the risk of worseningthe neurologic symptoms postoperatively. In this study, onepatient in the chronic stage group showed a transient deteriora-tion of neurologic symptoms, including a mild loss in thegrasping power of both hands (Table 2). This negative effectwas, however, transient and minimal, with the patient recover-ing his previous neurologic status within 1 month. Three pa-tients showed increased muscle rigidity in the posterior neckmuscles after surgery. One of these patients showed severespastic contraction of the posterior neck muscles with musclepain requiring antispasmodics and pain management. Spasticityresolved at 2 months postoperatively.

Neuropathic PainIt has been reported that neuropathic pain is one of the seriouscomplications after SCI. Furthermore, some studies have re-ported that cell transplantation strategy increased the risk ofneuropathic pain postoperatively [20, 21]. To identify the po-tential risk of neuropathic pain under the current protocol, the

Figure 2. Patient groups and treatment schedule. (A): Patient compli-ance, selection criteria, and follow-up from the beginning of the study tothe 10-month follow-up check. (B): Bone marrow cell transplantationand GM-CSF administration protocol. In the acute stage, cell transplan-tation was done within 14 injury days. Daily administration of GM-CSFwas performed for the first 5 days of each month over a 5-month period.Diagnostic studies, including serial neurologic exams, EMG, spine MRI,functional MRI, and EP, were performed both at the beginning of andduring the treatment. Abbreviations: ASIA A, American Spinal InjuryAssociation Impairment Scale grade A; ASIA B, American SpinalInjury Association Impairment Scale grade B; d, days; EMG, electro-myography; EP, evoked potentials; fMRI, functional magnetic reso-nance imaging; GM-CSF, granulocyte macrophage-colony stimulatingfactor; m, months; MRI, magnetic resonance imaging.

2068 Bone Marrow Cells and GM-CSF for Spinal Cord Injury

incidence of neuropathic pain in each group was investigated(Table 3). It was found that some of the patients in the treatmentgroup and control group developed neuropathic pain requiringtreatment. Overall, 20% of the patients in the treatment groupdeveloped neuropathic pain, whereas only 1 of the 13 (7.7%)patients in the control group complained of it during the fol-low-up period. Interestingly, the neuropathic pain occurredmainly in the subacute or chronic treatment group (33.3% each).In the acute treatment group, however, only 1 of 17 patients(5.9%) experienced it. These results imply that BMC transplan-tation and GM-CSF administration may be associated with agreater risk of developing neuropathic pain, particularly in thesubacute and chronic patients.

Aberrant regeneration of axons in damaged spinal cord isone suggested mechanism of increasing neuropathic pain aftercell transplantation [20, 21]. Currently, we don’t have anymethod to assess aberrant regeneration in the patients directly.However, we could visualize an example of aberrant regenera-tion using functional MRI (fMRI) study (Fig. 3C). The predom-inant activation of the temporal lobe and occipital lobe ratherthan the contralateral sensorimotor cortex was identified inpostoperative fMRI at 4 months.

Neurologic ImprovementsFigure 3A provides histograms of the estimated mean change inthe percentage of patients improving on the AIS scale. Overall,29.5% of patients in the acute treatment group showed neuro-logic improvement from AIS A to B or C. In the subacutetreatment group, 33.3% of patients improved to AIS B or Cduring the follow-up period. However, the patients in thechronic treatment group didn’t show any changes in the neuro-logic status.

GM-CSF Effect on the Leukocytosis andNeurologic OutcomeTo investigate the systemic effect of GM-CSF on the outcomeof SCI, the number of white blood cells in the peripheral bloodwas compared with the neurologic outcomes (Fig. 4). Theadministration of GM-CSF has been used extensively to triggerperipheral leukocytosis and to induce bone marrow hematopoi-etic stem cell mobilization. The total number of recruited whiteblood cells in the peripheral blood was elevated after GM-CSFadministration. The number of white blood cells in patientsshowing improved neurologic function was significantly higherthan that in the patients without neurologic improvement.

Spinal MRI FindingsChanges in the MRI findings for the patients with treatment aregiven in Table 4 and Figure 3B. Overall, 42.9% of patients in thetreatment group showed an increase in the diameter of the spinalcord at the cell transplantation site. However, 33.3% of thepatients showed atrophic changes (a decrease in diameter) at thetransplantation site. Six patients (28.6%) showed evidence ofspinal cord enhancement. Other findings, including spinal cordedema demonstrated by increased T2 weight images, cysticdegeneration, or spinal cord atrophy distal to the lesion, wereobserved. But no other significant changes such as tumor for-mation of the transplanted BMCs were found.

DISCUSSION

The present study has demonstrated that patients at acute stageof complete SCI did not show any serious adverse response afterthe combined therapy of BMC transplantation and GM-CSFadministration. The use of autologous BMCs for stem celltherapy in SCI patients has more advantages. First, one canavoid all problems associated with the immunological rejectionor graft-versus-host reactions [22], which are frequently causedin allogenic cell transplantation. Second, autologous BMC ther-

Table 1. Summary of patient demographics, spinal cord injury level, and duration of the MRI follow-up

VariablesAcute

(<2 weeks)Subacute

(2–8 weeks)Chronic

(>8 weeks) Control p

Number 17 6 12 13Age (years) 40.1 � 12.8 29.4 � 13.5 42 � 15.5 47.8 � 14.6 NSFollow-up (months) 10.4 � 4.1 11.3 � 4.9 10.9 � 4.7 10.1 � 4.9 NSNumber of MRI follow-up patients 13 4 4 11Sex NS

Male 16 3 10 9Female 1 3 2 4

Injury NSCervical 7 6 10 7Thoracic-TL 10 0 2 6

Abbreviations: MRI, magnetic resonance imaging; NS, not significant; TL, thoracolumbar junction.

Table 2. Summary of adverse events

Adverse eventsTreatment group

(%) (n � 35)Control group(%) (n � 13) p

Fever 22 (62.9) 3 (23.1) .022Abdominal discomfort 7 (20) 7 (53.8) .034Numbness or tingling

sensation6 (17.1) 4 (30.8) NS

Facial flushing or rash 5 (14.3) 1 (7.7) NSHeadache 3 (8.6) 1 (7.7) NSConstipation 3 (8.6) 3 (23.1) NSGeneral ache 3 (8.6) NSRigidity 3 (8.6) NSTransient neurologic

deterioration1 (2.9) NS

Spasticity 1 (2.9) NS

Abbreviation: NS, not significant.

Table 3. Incidence of neuropathic pain

Treatment group (%)(n � 35)

Control group(%) (n � 13)

Acute(n � 17)

Subacute(n � 6)

Chronic(n � 12)

Neuropathic pain 1 (5.9) 2 (33.3) 4 (33.3) 1 (7.7)Total 7 (20) 1 (7.7)p NS

Abbreviation: NS, not significant.

2069Hwan Yoon, Shik Shim, Hoon Park et al.

www.StemCells.com

apy is considered safe by not being associated with carcinogen-esis [23]. Third, extensive scientific data on BMCs have beenaccumulated from previous experiences in BMC transplantationfor hematological diseases. These advantages have made celltherapy using BMCs widely applicable and investigated clini-cally in various neurologic diseases. However, it also has somedisadvantages. First, the procedure requires open surgery toapproach the injury area, which results in the increase in the

potential adverse effects. Second, the sorting of BMCs needs tobe done in vitro, thus increasing the risk of contamination.Third, it is still unclear whether some BMC components mayhave a deleterious effect on the functional improvement. In thisstudy, approximately 100–150 ml of bone marrow was aspi-rated from the iliac bone, and mononuclear cells were sorted andconcentrated to a final volume of approximately 1.8 ml. TheseBMCs were then transplanted by injection into the surroundingarea of the injury site. This procedure required approximately3–4 hours to complete. Therefore, some technical modificationsare needed to reduce the operation time and enhance the safetyand therapeutic effects.

BMCs are a mixed cell population including hematopoieticstem cells, mesenchymal stem cells, endothelial progenitor cells,macrophage, and lymphocytes. It has been shown in otherstudies that hematopoietic or mesenchymal stem cells had aneuroprotective effect and increased neurite outgrowth [11, 24].Furthermore, activated macrophages are currently being inves-tigated as a therapeutic target for SCI patients [25, 26]. Incontrast, inflammatory cells in BMCs could have adverse ef-fects, such as increasing the inflammatory response, thus inhib-iting the regeneration process [27, 28]. As a result, further

Figure 3. Neurologic outcome and magnetic resonance imaging (MRI)findings. (A): Change in the neurologic status and spinal MRI findingsproduced by the effect of bone marrow cell (BMC) transplantation andgranulocyte macrophage-colony stimulating factor administration. Neu-rologic improvements in the acute and subacute treatment groups weresignificantly higher than the control group (7.7%) and the naturalrecovery of spinal cord injury reported previously (12.5%) [39]. (B):Follow-up MRI findings. Sagittal T2W (Ba), T1W (Bb), and postcon-trast MRI (Bc) performed at 4 months postoperatively showing a smallpatch enhancement in the BMC transplantation area (arrows) and slightcord expansion. (C): Functional MRI (fMRI) findings. Preoperativeinitial (Ca) and postoperative follow-up (Cb) fMRI at 4 months. ThefMRI was performed following the right great toe proprioceptive stim-ulation. Initial fMRI did not reveal any cortical activities, suggestingcomplete disruption of sensory pathways. The 4-month postoperativefMRI demonstrated increased signal activities in bilateral temporal andipsilateral occipital lobes indicating aberrant regeneration (white circlein [Cb]). Abbreviation: wks, weeks.

Figure 4. Change in the peripheral leukocyte number produced byGM-CSF administration. The mean number of peripheral leukocytessignificantly increased in patients with improving American SpinalInjury Association Impairment Scale grades while showing no signifi-cant increase in patients without improvement (� p � .05). Abbrevia-tions: Conc., concentration; GM-CSF, granulocyte macrophage-colonystimulating factor; WBC, white blood cells.

Table 4. Changes in spinal magnetic resonance imaging findings

MRI findingsTreatment group

(%) (total n � 21)Control (%)

(total n � 11) p

Mean F/U months 3.5 4.3 NSChange in spinal cord 16 (76.2) 5 (45.5) NSDiameter

Increase diameter 9 (42.9) 1 (9.1)No change 5 (23.8) 6 (54.5)Decrease diameter 7 (33.3) 4 (36.4)

Cord atrophy distal tothe lesion

6 (28.6) 4 (36.4) NS

Enhancement 6 (28.6) 0 (0) NSCord edema 3 (14.3) 4 (36.4) NSCystic degeneration 1 (4.8) 2 (18.2) NSSyringomyelia 0 1 (9) NS

Abbreviations: F/U, follow-up; MRI, magnetic resonance imaging;NS, not significant.

2070 Bone Marrow Cells and GM-CSF for Spinal Cord Injury

studies comparing the effects of whole BMCs with those of asubpopulation with inflammatory cells removed are required.

Recombinant human GM-CSF has long been used safely inpatients with bone marrow suppression. It is well known that theclassic role of GM-CSF is hematopoiesis by inducing thegrowth of several different hematopoietic cell lineages. It alsoenhances the functional activities of mature effector cells in-volved in antigen presentation and cell-mediated immunity,including neutrophils, monocytes, macrophages, and dendriticcells. Recently, some studies have reported that GM-CSF pre-vented apoptotic cell death not only in hematologic cells butalso in neuronal cells [18, 29, 30]. From these findings, it wasspeculated that administration of GM-CSF to SCI patientswould result in the improvement of neurologic functions with-out any significant complication. In this study, a hypothesis wasdeveloped that GM-CSF would not only activate patient bonemarrow for stem cell mobilization but would also have a directeffect on the transplanted BMCs by enhancing their survival inthe spinal cord and activating them to excrete neurotrophiccytokines (Fig. 1). Recently, some investigators found that GM-CSF stimulated microglial cells to produce neurotrophic cyto-kines such as BDNF [18]. Furthermore, other hematopoieticcytokines including G-CSF and erythropoietins were also shownto have neuroprotective effects and promote neurologic out-comes in CNS injury models [15–17]. Since the safety of mosthematopoietic cytokines is already verified in the clinical fields,these experimental studies could be rapidly turned into clinicalinvestigation.

Complications such as cerebral and myocardial infarctionafter hematopoietic cytokine administration have been reported,and the primary mechanism involved was presumed to be anacute arterial occlusion by thrombus [31]. The thrombogenicproperties of the peripheral blood result from increased viscositydue to the mobilization of hematopoietic cells. In this study, noserious complications by GM-CSF were observed that caused anincrease in the morbidity or mortality. Most patients sufferedfrom a fever, rash, or leukocytosis. These findings could beconsidered not only as adverse effects but also as indicators forthe systemic effect of GM-CSF. Methylprednisolone therapyhas been considered as a standard therapy for acute SCI. How-ever, recent publications noticed that the methylprednisolonetherapy significantly increased the morbidities and mortalities[32, 33]. It increased serious complications such as woundinfection, gastrointestinal bleeding, and urinary tract infection.In order to avoid the detrimental effects of methylprednisolone,the safety of cell therapy protocols comparing that of methylprednisolone therapy needs to be investigated.

The AIS grading is determined by the clinical neurologicfindings and not by pathologic severity. As a result, AIS gradingdoes not completely reflect pathologic severity. It is possiblethat one AIS grade could include a wide spectrum of severity.Less severely injured patients with an AIS grade of A could,therefore, have a greater potential for improvement. In thisstudy, approximately 30% of the patients in AIS A improved tograde B or C (Fig. 3A). It is believed that this partial effect wascaused by an additive or alleviating role inherent in the protocolrather than the innate ability of natural recovery in the lessseverely injured patients. Interestingly, a relationship betweenleukocytosis after GM-CSF administration and neurological im-provement was identified. The mean number of peripheral leu-kocytes significantly increased in patients with improving AISgrades while showing no significant increase in patients withoutimprovement. These findings imply that patients who are moreresponsive to GM-CSF might have a greater capacity for im-provement. As a result, interpersonal inherited variability inresponse to GM-CSF is one possible explanation for the obser-vation that the protocol appeared to have a therapeutic effect on

only a small population of the patient group. Furthermore, thesefindings also support the idea that the mobilizing role of GM-CSF could be one important mechanism of functional improve-ment. However, it is not clear at this point that the higher levelof leukocytosis is a prerequisite for the GM-CSF effect and/orthe recovery from SCI.

Many experimental studies have been completed to find theoptimal period for cell transplantation. To avoid the destructionof transplanted cells by the inflammatory process in the acutephase (less than 1 week), many researchers consider the sub-acute phase between 10 and 14 days as an optimal period for celltransplantation. Results from animal studies show that signifi-cant gliosis is a major obstacle inhibiting axonal regenerationduring the chronic stage [34, 35]. Interestingly, few patients inthis study showed neurologic improvement even if BMC trans-plantation was performed between 2 and 8 weeks after injury.These results indicate that the optimal period of BMC trans-plantation in SCI patients should not be restricted to patients lessthan 2 weeks postinjury.

However, our patients in the subacute group were relativelyyounger (mean 29.4 years) than the other groups (mean �40years). Therefore, it should be considered that their relativelyyounger age could have influenced their ability to recover betterthan the other groups.

Few studies have been published that identify cell transplan-tation therapy as an ideal option for improving the neurologicfunctions in patients at the chronic stage [36–38]. This studyalso did not show any therapeutic benefits for chronic patients.Even though a few patients experienced the improvement in thesensory or motor functions (3–5 dermatome) immediately aftersurgery, these improvements disappeared in 3–4 months. Be-cause of the sudden and immediate nature of these improve-ments, it cannot be suggested that these short-term improve-ments were due to the effect of the engrafted stem cells, as thisprocess needs an incubation period to expand cell numbers.Furthermore, cytokines released from the engrafted stem cellsalso require some time for signal transduction to show finaleffects. It is known that restoration of cerebrospinal fluid flow orthe microtrauma caused by the opening of the dura and injectionof BMCs could exert transient changes in the environmentaround the SCI lesion site such as the disruption of the glial scarbarrier or the transient excitation of quiescent neurons. In thechronic stage treatment group, some patients demonstrated newor changing clinical features such as transient increasing my-elopathy, ascending neurological level, or pain. In this study,increase in the spinal cord dimension at the site of transplanta-tion was found. However, no tumor infiltrating the surroundingspinal cord tissue was identified. These findings imply thatautologous BMC transplantation doesn’t increase the risk ofmalignant transformation, which is still a field under investiga-tion in adult stem cell transplantation.

Approximately 20% of patients who were treated withBMCs and GM-CSF complained about pain during the fol-low-up period. Furthermore, one patient showed the extensionof the spinal cord edema associated with an increase in neuro-pathic pain (data not shown). These findings reinforce the sug-gestion that cell transplantation, especially in chronic stage,increases adverse effects like neuropathic pain. Therefore, weshould be informed that the stem cell therapy for chronic pa-tients doesn’t guarantee promising results and should be cau-tiously performed in selected cases. Recently, it has been re-ported that stem cell transplantation increases neuropathic painin animal study. Neuropathic pain might be related to the aber-rant regeneration of damaged axons. Our preliminary results offMRI studies also support this idea. Further studies using fMRIto understand the correlation between aberrant regeneration andneuropathic pain would provide us with the objective pain

2071Hwan Yoon, Shik Shim, Hoon Park et al.

www.StemCells.com

monitoring method as well as the information to understand thenature of neuropathic pain in SCI patients.

There are several limitations in this study. First, completedouble-blind case control studies were not possible. Althoughthe examiners were well trained and qualified by the controllededucation system, potential bias produced between examinerscould not be removed completely. Second, neurologic improve-ments were described as advancing AIS grades. Therefore,small improvements without changes in AIS grade could not beidentified. In order to overcome these limitations, it is hoped thisstudy can be repeated using a random sample multicenter dou-ble-blind protocol with specially designed assessment methods.Finally, due to the limited number of complete SCI patients, wecould not understand how each of the BMC transplantation andGM-CSF treatments affected the recovery from SCI. Furtherlarge group studies are required to clarify the effects of eithertherapy.

This study suggests that, for the functional recovery ofdamaged spinal cord, human BMCs can be transplanted into thespinal cord of patients with complete SCI. The BMC transplan-tation and GM-CSF administration caused no new deficit in

patients and appeared to be safe in short- and longer-termassessments. In addition to protocol safety, preliminary evi-dence showing the effect of cell therapy in patients with com-plete SCI was also provided. This study was performed in alimited number of patients. It is recommended that the thera-peutic effects should be established in a more comprehensivemulticenter study.

ACKNOWLEDGMENTS

This study was supported by the Grant of the Korea Health 21R&D Project, Ministry of Health & Welfare, Republic of Korea(A050082).

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

REFERENCES

1 Ramer LM, Au E, Richter MW et al. Peripheral olfactory ensheathingcells reduce scar and cavity formation and promote regeneration afterspinal cord injury. J Comp Neurol 2004;473:1–15.

2 Pearse DD, Marcillo AE, Oudega M et al. Transplantation of Schwanncells and olfactory ensheathing glia after spinal cord injury: Does pre-treatment with methylprednisolone and interleukin-10 enhance recovery?J Neurotrauma 2004;21:1223–1239.

3 Ogawa Y, Sawamoto K, Miyata T et al. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and func-tional recovery after spinal cord contusion injury in adult rats. J NeurosciRes 2002;69:925–933.

4 Koshizuka S, Okada S, Okawa A et al. Transplanted hematopoietic stemcells from bone marrow differentiate into neural lineage cells and pro-mote functional recovery after spinal cord injury in mice. J NeuropatholExp Neurol 2004;63:64–72.

5 Munoz-Elias G, Woodbury D, Black IB. Marrow stromal cells, mitosis,and neuronal differentiation: Stem cell and precursor functions. STEMCELLS 2003;21:437–448.

6 Sanchez-Ramos J, Song S, Cardozo-Pelaez F et al. Adult bone marrowstromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247–256.

7 Ha Y, Yoon DH, Yeon DS et al. Neural antigen expressions in culturedhuman umbilical cord blood stem cells in vitro. J of Kor Neurosurg Soc2001;30:963.

8 Chopp M, Zhang XH, Li Y et al. Spinal cord injury in rat: Treatmentwith bone marrow stromal cell transplantation. Neuroreport 2000;11:3001–3005.

9 Akiyama Y, Radtke C, Kocsis JD. Remyelination of the rat spinal cordby transplantation of identified bone marrow stromal cells. J Neurosci2002;22:6623–6630.

10 Kawada H, Takizawa S, Takanashi T et al. Administration of hemato-poietic cytokines in the subacute phase after cerebral infarction is effec-tive for functional recovery facilitating proliferation of intrinsic neuralstem/progenitor cells and transition of bone marrow-derived neuronalcells. Circulation 2006;113:701–710.

11 Chen Q, Long Y, Yuan X et al. Protective effects of bone marrowstromal cell transplantation in injured rodent brain: synthesis of neuro-trophic factors. J Neurosci Res 2005;80:611–619.

12 Feron F, Perry C, Cochrane J et al. Autologous olfactory ensheathing celltransplantation in human spinal cord injury. Brain 2005;128:2951–2960.

13 Huang H, Chen L, Wang H et al. Safety of fetal olfactory ensheathingcell transplantation in patients with chronic spinal cord injury. A 38-month follow-up with MRI. Zhongguo Xiu Fu Chong Jian Wai Ke ZaZhi 2006;20:439–443.

14 Park HC, Shim YS, Ha Y et al. Treatment of complete spinal cord injurypatients by autologous bone marrow cell transplantation and administra-tion of granulocyte-macrophage colony stimulating factor. Tissue Eng2005;11:913–922.

15 Grasso G, Sfacteria A, Passalacqua M et al. Erythropoietin and erythro-poietin receptor expression after experimental spinal cord injury encour-

ages therapy by exogenous erythropoietin. Neurosurgery 2005;56:821–827.

16 Gibson CL, Jones NC, Prior MJ et al. G-CSF suppresses edema forma-tion and reduces interleukin-1beta expression after cerebral ischemia inmice. J Neuropathol Exp Neurol 2005;64:763–769.

17 Brines M, Grasso G, Fiordaliso F et al. Erythropoietin mediates tissueprotection through an erythropoietin and common beta-subunit hetero-receptor. Proc Natl Acad Sci U S A 2004;101:14907–14912.

18 Bouhy D, Malgrange B, Multon S et al. Delayed GM-CSF treatmentstimulates axonal regeneration and functional recovery in paraplegic ratsvia an increased BDNF expression by endogenous macrophages. FASEBJ 2006;20:1239–1241.

19 Ha Y, Kim YS, Cho JM et al. Granulocyte macrophage colony stimu-lating factor (GM-CSF) prevents apoptosis and improves functionaloutcome in experimental spinal cord contusion injury. J Neurosurg2005;11:55–61.

20 Macias MY, Syring MB, Pizzi MA et al. Pain with no gain: Allodyniafollowing neural stem cell transplantation in spinal cord injury. ExpNeurol 2006;201:335–348.

21 Hofstetter CP, Holmstrom NA, Lilja JA et al. Allodynia limits theusefulness of intraspinal neural stem cell grafts; directed differentiationimproves outcome. Nat Neurosci 2005;8:346–353.

22 Luck R, Klempnauer J, Steiniger B. Simultaneous occurrence of graft-versus-host and host-versus-graft reactions after allogenic MHC class IIdisparate small bowel transplantation in immunocompetent rats. Trans-plant Proc 1991;23:677–678.

23 Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cellrenewal in carcinogenesis. Nature 2004;432:324–331.

24 Ankeny DP, McTigue DM, Jakeman LB. Bone marrow transplantsprovide tissue protection and directional guidance for axons after con-tusive spinal cord injury in rats. Exp Neurol 2004;190:17–31.

25 Prewitt CM, Niesman IR, Kane CJ et al. Activated macrophage/micro-glial cells can promote the regeneration of sensory axons into the injuredspinal cord. Exp Neurol 1997;148:433–443.

26 Knoller N, Auerbach G, Fulga V et al. Clinical experience using incu-bated autologous macrophages as a treatment for complete spinal cordinjury: Phase I study results. J Neurosurg Spine 2005;3:173–181.

27 Beattie MS. Inflammation and apoptosis: Linked therapeutic targets inspinal cord injury. Trends Mol Med 2004;10:580–583.

28 Gonzalez R, Glaser J, Liu MT. Reducing inflammation decreases sec-ondary degeneration and functional deficit after spinal cord injury. ExpNeurol 2003;184:456–463.

29 Dame JB, Chegini N, Christensen RD et al. The effect of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on gran-ulocyte macrophage-colony stimulating factor (GM-CSF) production byneuronal precursor cells. Eur Cytokine Netw 2002;13:128–133.

30 Ha Y, Kim YS, Cho JM et al. Role of granulocyte-macrophage colony-stimulating factor in preventing apoptosis and improving functionaloutcome in experimental spinal cord contusion injury. J Neurosurg Spine2005;2:55–61.

31 Tolcher AW, Giusti RM, O’Shaughnessy J et al. Arterial thrombosisassociated with granulocyte-macrophage colony-stimulating factor (GM-CSF) administration in breast cancer patients treated with dose-intensivechemotherapy: A report of two cases. Cancer Invest 1995;13:188–192.

2072 Bone Marrow Cells and GM-CSF for Spinal Cord Injury

32 Short DJ, El Masry WS, Jones, PW. High dose methylprednisolone in themanagement of acute spinal cord injury - a systematic review from aclinical perspective. Spinal Cord 2000;38:273–286.

33 McCutcheon EP, Selassie AW, Gu JK et al. Acute traumatic spinal cordinjury, 1993–2000. A population-based assessment of methylprednisoloneadministration and hospitalization. J Trauma 2004;56:1076–1083.

34 Okano H, Ogawa Y, Nakamura M et al. Transplantation of neuralstem cells into the spinal cord after injury. Semin Cell Dev Biol2003;14:191–198.

35 Hofstetter CP, Schwarz EJ, Hess D et al. Marrow stromal cells formguiding strands in the injured spinal cord and promote recovery. ProcNatl Acad Sci U S A 2002;99:2199–2204.

36 Lima C, Pratas-Vital J, Escada P et al. Olfactory mucosa autografts inhuman spinal cord injury: A pilot clinical study. J Spinal Cord Med2006;29:191–203.

37 Dobkin BH, Curt A, Guest J. Cellular transplants in China: Observationalstudy from the largest human experiment in chronic spinal cord injury.Neurorehabil Neural Repair 2006;20:5–13.

38 Sykova E, Homola A, Mazanec R et al. Autologous bone marrowtransplantation in patients with subacute and chronic spinal cord injury.Cell Transplant 2006;15:675–687.

39 Marino RJ, Ditunno JFJ, Donovan WH et al. Neurologic recovery aftertraumatic spinal cord injury: Data from the Model Spinal Cord InjurySystems. Arch Phys Med Rehabil 1999;80:1391–1396.

2073Hwan Yoon, Shik Shim, Hoon Park et al.

www.StemCells.com