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Repeated subarachnoid administrations of autologous mesenchymal stromal cells supported in autologous plasma improve quality of life in patients suffering incomplete spinal cord injury

Open AccessPublished:January 06, 2017DOI:https://doi.org/10.1016/j.jcyt.2016.12.002

      Abstract

      Background aims

      Cell therapy with mesenchymal stromal cells (MSCs) offers new hope for patients suffering from spinal cord injury (SCI).

      Methods

      Ten patients with established incomplete SCI received four subarachnoid administrations of 30 × 106 autologous bone marrow MSCs, supported in autologous plasma, at months 1, 4, 7 and 10 of the study, and were followed until the month 12. Urodynamic, neurophysiological and neuroimaging studies were performed at months 6 and 12, and compared with basal studies.

      Results

      Variable improvement was found in the patients of the series. All of them showed some degree of improvement in sensitivity and motor function. Sexual function improved in two of the eight male patients. Neuropathic pain was present in four patients before treatment; it disappeared in two of them and decreased in another. Clear improvement in bladder and bowel control were found in all patients suffering previous dysfunction. Before treatment, seven patients suffered spasms, and two improved. Before cell therapy, nine patients suffered variable degree of spasticity, and 3 of them showed clear decrease at the end of follow-up. At this time, nine patients showed infra-lesional electromyographic recordings suggesting active muscle reinnervation, and eight patients showed improvement in bladder compliance. After three administrations of MSCs, mean values of brain-derived neurotrophic factor, glial-derived neurotrophic factor, ciliary neurotrophic factor, and neurotrophin 3 and 4 showed slight increases compared with basal levels, but without statistically significant difference.

      Conclusions

      Administration of repeated doses of MSCs by subarachnoid route is a well-tolerated procedure that is able to achieve progressive and significant improvement in the quality of life of patients suffering incomplete SCI.

      Key Words

      Introduction

      As a result of the experience provided in literature, in recent years various techniques of cell therapy have been implemented, mainly using mesenchymal stromal cells (MSCs) in patients with traumatic spinal cord injury (SCI), and early clinical trials have confirmed the absence of significant side effects [
      • Park J.H.
      • Kim D.Y.
      • Sung I.
      • Choi G.H.
      • Jeon M.H.
      • Kim K.K.
      • et al.
      Long-term results of spinal cord injury therapy using mesenchymal stem cells derived from bone marrow in humans.
      ,
      • Syková E.
      • Homola A.
      • Mazanec R.
      • Lachmann H.
      • Konrádová S.L.
      • Kobylka P.
      • et al.
      Autologous bone marrow transplantation in patients with subacute and chronic spinal cord injury.
      ,
      • Yoon S.H.
      • Shim Y.S.
      • Park Y.H.
      • Chung J.K.
      • Nam J.H.
      • Kim M.O.
      • et al.
      Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: phase I/II clinical trial.
      ].
      However, at present the advantages of using exclusively adult MSCs or a mixture of MSCs and other bone marrow mononuclear cells for these transplants are not clear [
      • Park J.H.
      • Kim D.Y.
      • Sung I.
      • Choi G.H.
      • Jeon M.H.
      • Kim K.K.
      • et al.
      Long-term results of spinal cord injury therapy using mesenchymal stem cells derived from bone marrow in humans.
      ,
      • Syková E.
      • Homola A.
      • Mazanec R.
      • Lachmann H.
      • Konrádová S.L.
      • Kobylka P.
      • et al.
      Autologous bone marrow transplantation in patients with subacute and chronic spinal cord injury.
      ,
      • Yoon S.H.
      • Shim Y.S.
      • Park Y.H.
      • Chung J.K.
      • Nam J.H.
      • Kim M.O.
      • et al.
      Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: phase I/II clinical trial.
      ,
      • Chopp M.
      • Zhang X.H.
      • Li Y.
      • Wang L.
      • Chen J.
      • Lu D.
      • et al.
      Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation.
      ,
      • Hofstetter C.P.
      • Schwarz E.J.
      • Hess D.
      • Widenfalk J.
      • El Manira A.
      • Prockop D.J.
      • et al.
      Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery.
      ,
      • Zurita M.
      • Vaquero J.
      Functional recovery in chronic paraplegia after bone marrow stromal cells transplantation.
      ,
      • Zurita M.
      • Vaquero J.
      Bone marrow stromal cells can achieve cure of chronic paraplegic rats: functional and morphological outcome one year after transplantation.
      ,
      • Vaquero J.
      • Zurita M.
      Bone marrow stromal cells for spinal cord repair: a challenge for contemporary neurobiology.
      ,
      • Vaquero J.
      • Zurita M.
      • Oya S.
      • Santos M.
      Cell therapy using bone marrow stromal cells in chronic paraplegic rats: systemic or local administration?.
      ,
      • Ohta M.
      • Suzuki Y.
      • Noda T.
      • Ejiri Y.
      • Dezawa M.
      • Kataoka K.
      • et al.
      Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation.
      ,
      • Bakshi A.
      • Hunter C.
      • Swanger S.
      • Lepore A.
      • Fischer I.
      Minimally invasive delivery of stem cells for spinal cord injury: advantages of the lumbar puncture technique.
      ,
      • Bakshi A.
      • Barshinger A.L.
      • Swanger S.A.
      • Madhavani V.
      • Shumsky J.S.
      • Neuhuber B.
      • et al.
      Lumbar puncture delivery of bone marrow stromal cells in spinal cord contusion: a novel method for minimally invasive cell transplantation.
      ,
      • Himes B.T.
      • Neuhuber B.
      • Coleman C.
      • Kushner R.
      • Swanger S.A.
      • Kopen G.C.
      • et al.
      Recovery of function following grafting of human bone marrow-derived stromal cells into the injured spinal cord.
      ,
      • Parr A.M.
      • Tator C.H.
      • Keating A.
      Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury.
      ,
      • Deda H.
      • Inci M.C.
      • Kürekçi A.E.
      • Kayihan K.
      • Ozgün E.
      • Ustünsoy G.E.
      • et al.
      Treatment of chronic spinal cord injured patients with autologous bone marrow-derived hematopoietic stem cell transplantation: 1-year follow-up.
      ] and the advantages of either of the two options have been discussed extensively in recent publications from our own research group [
      • Vaquero J.
      • Zurita M.
      Bone marrow stromal cells for spinal cord repair: a challenge for contemporary neurobiology.
      ,
      • Vaquero J.
      • Zurita M.
      Functional recovery after severe central nervous system trauma: current perspectives for cell therapy with bone marrow stromal cells.
      ].
      Cell therapy is clearly a current therapeutic promise in this field of research [
      • Park J.H.
      • Kim D.Y.
      • Sung I.
      • Choi G.H.
      • Jeon M.H.
      • Kim K.K.
      • et al.
      Long-term results of spinal cord injury therapy using mesenchymal stem cells derived from bone marrow in humans.
      ,
      • Syková E.
      • Homola A.
      • Mazanec R.
      • Lachmann H.
      • Konrádová S.L.
      • Kobylka P.
      • et al.
      Autologous bone marrow transplantation in patients with subacute and chronic spinal cord injury.
      ,
      • Yoon S.H.
      • Shim Y.S.
      • Park Y.H.
      • Chung J.K.
      • Nam J.H.
      • Kim M.O.
      • et al.
      Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: phase I/II clinical trial.
      ,
      • Parr A.M.
      • Tator C.H.
      • Keating A.
      Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury.
      ,
      • Deda H.
      • Inci M.C.
      • Kürekçi A.E.
      • Kayihan K.
      • Ozgün E.
      • Ustünsoy G.E.
      • et al.
      Treatment of chronic spinal cord injured patients with autologous bone marrow-derived hematopoietic stem cell transplantation: 1-year follow-up.
      ,
      • Vaquero J.
      • Zurita M.
      Functional recovery after severe central nervous system trauma: current perspectives for cell therapy with bone marrow stromal cells.
      ,
      • Saito F.
      • Nakatani T.
      • Iwase M.
      • Maeda Y.
      • Hirakawa A.
      • Murao Y.
      • et al.
      Spinal cord injury treatment with intrathecal autologous bone marrow stromal cell transplantation: the first clinical trial case report.
      ,
      • Pal R.
      • Venkataramana N.K.
      • Bansai A.
      • Balaraju S.
      • Jan M.
      • Chandra R.
      • et al.
      Ex vivo-expanded autologous bone marrow-derived mesenchymal stromal cells in human spinal cord injury/paraplegia: a pilot clinical study.
      ,
      • Saito F.
      • Nakatani T.
      • Iwase M.
      • Maeda Y.
      • Murao Y.
      • Suzuki Y.
      • et al.
      Administration of cultured autologous bone marrow stromal cells into cerebrospinal fluid in spinal injury patients: a pilot study.
      ,
      • Jiang P.C.
      • Xiong W.P.
      • Wang G.
      • Ma C.
      • Yao W.Q.
      • Kendell S.F.
      • et al.
      A clinical trial report of autologous bone marrow-derived mesenchymal stem cell transplantation in patients with spinal cord injury.
      ,
      • Mendonça M.V.P.
      • Larocca T.F.
      • Souza B.S.
      • de Freitas Souza B.S.
      • Villarreal C.F.
      • Silva L.F.
      • et al.
      Safety and neurological assessments after autologous transplantation of bone marrow mesenchymal stem cells in subjects with chronic spinal cord injury.
      ,
      • Zurita M.
      • Vaquero J.
      • Bonilla C.
      • Santos M.
      • De Haro J.
      • Oya S.
      • et al.
      Functional recovery of chronic paraplegic pigs after autologous transplantation of bone marrow stromal cells.
      ,
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ] but is still subject to many uncertainties, with significant confusion due to the disparity of protocols, subject selection, cell type, doses and routes of administration used.
      MSCs have the advantage of easy expansion and low antigenicity, which may allow, at least theoretically, the use of allogeneic MSCs in human clinical practice, but there are still evident uncertainties about the mechanisms through which this type of cell therapy achieves neurological recovery, both in experimental animals and in the few patients treated so far. In experimental studies carried out, it is noteworthy that the functional recovery of paraplegic animals after MSC transplantation starts before tissue regeneration occurs, allowing the passage of ascending and descending axons [
      • Zurita M.
      • Vaquero J.
      Functional recovery in chronic paraplegia after bone marrow stromal cells transplantation.
      ,
      • Zurita M.
      • Vaquero J.
      Bone marrow stromal cells can achieve cure of chronic paraplegic rats: functional and morphological outcome one year after transplantation.
      ,
      • Pal R.
      • Venkataramana N.K.
      • Bansai A.
      • Balaraju S.
      • Jan M.
      • Chandra R.
      • et al.
      Ex vivo-expanded autologous bone marrow-derived mesenchymal stromal cells in human spinal cord injury/paraplegia: a pilot clinical study.
      ,
      • Vaquero J.
      • Zurita M.
      Functional recovery after severe central nervous system trauma: current perspectives for cell therapy with bone marrow stromal cells.
      ], a finding that has also been discussed in clinical trials [
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ].
      Therefore, it is obvious that after MSC transplantation, various repair processes must exist, including the release of neurotrophic factors by the transplanted stem cells [
      • Chen Q.
      • Long Y.
      • Yuan X.
      • Zou L.
      • Sun J.
      • Chen S.
      • et al.
      Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors.
      ,
      • Paradisi M.
      • Alviano F.
      • Pirondi S.
      • Lanzoni G.
      • Fernandez M.
      • Lizzo G.
      • et al.
      Human mesenchymal stem cells produce bioactive neurotrophic factors: source, individual variability and differentiation issues.
      ,
      • Wang L.J.
      • Zhang R.P.
      • Li J.D.
      Transplantation of neurotrophin-3-expressing bone mesenchymal stem cells improves recovery in a rat model of spinal cord injury.
      ,
      • Crigler L.
      • Robey R.C.
      • Asawachaicharn A.
      • Gaupp D.
      • Phinney D.G.
      Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis.
      ,
      • Wilkins A.
      • Kemp K.
      • Ginty M.
      • Hares K.
      • Mallam E.
      • Scolding N.
      Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro.
      ], or the activation of endogenous mechanisms of the spinal cord, able to partially restore neurological functions previously abolished, as has been suggested in experimental models of brain damage [
      • Bonilla C.
      • Zurita M.
      • Otero L.
      • Aguayo C.
      • Vaquero J.
      Delayed intralesional transplantation of bone marrow stromal cells increases endogenous neurogenesis and promotes functional improvement after severe traumatic brain injury.
      ,
      • Otero L.
      • Zurita M.
      • Bonilla C.
      • Aguayo C.
      • Rico M.A.
      • Rodriguez A.
      • et al.
      Allogeneic bone marrow stromal cell transplantation after cerebral hemorrhage achieves cell transdifferentiation and modulates endogenous neurogenesis.
      ].
      On the other hand, various experimental studies have shown that MSCs can reach areas of SCI after being deposited in the subarachnoid space, providing a safe method for minimally invasive cell transplantation [
      • Vaquero J.
      • Zurita M.
      Bone marrow stromal cells for spinal cord repair: a challenge for contemporary neurobiology.
      ,
      • Ohta M.
      • Suzuki Y.
      • Noda T.
      • Ejiri Y.
      • Dezawa M.
      • Kataoka K.
      • et al.
      Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation.
      ,
      • Bakshi A.
      • Hunter C.
      • Swanger S.
      • Lepore A.
      • Fischer I.
      Minimally invasive delivery of stem cells for spinal cord injury: advantages of the lumbar puncture technique.
      ,
      • Bakshi A.
      • Barshinger A.L.
      • Swanger S.A.
      • Madhavani V.
      • Shumsky J.S.
      • Neuhuber B.
      • et al.
      Lumbar puncture delivery of bone marrow stromal cells in spinal cord contusion: a novel method for minimally invasive cell transplantation.
      ,
      • Nakano N.
      • Nakai Y.
      • Seo T.B.
      • Homma T.
      • Yamada Y.
      • Ohta M.
      • et al.
      Effects of bone marrow stromal cell transplantation through CSF on the subacute and chronic spinal cord injury in rats.
      ,
      • Satake K.
      • Lou J.
      • Lenke L.G.
      Migration of mesenchymal stem cells through cerebrospinal fluid into injured spinal cord tissue.
      ], and this finding has been confirmed in patients [
      • Chotivichit A.
      • Ruangchainikom M.
      • Chiewvit P.
      • Wongkajornsilp A.
      • Sujirattanawimol K.
      Chronic spinal cord injury treated with transplanted autologous bone marrow-derived mesenchymal stem cells tracked by magnetic resonance imaging: a case report.
      ].
      In humans, the first subarachnoid administration of MSCs for the treatment of SCI was described in 2008, as the first pilot case of a clinical trial in which cell therapy was administered early after SCI [
      • Saito F.
      • Nakatani T.
      • Iwase M.
      • Maeda Y.
      • Hirakawa A.
      • Murao Y.
      • et al.
      Spinal cord injury treatment with intrathecal autologous bone marrow stromal cell transplantation: the first clinical trial case report.
      ]. Since then, the intrathecal route has been generally used in human clinical trials [
      • Karamouzian S.
      • Nematollahi-Mahani S.N.
      • Nakhaee N.
      • Eskandary H.
      Clinical safety and primary efficacy of bone marrow mesenchymal cell transplantation in subacute spinal cord injured patients.
      ,
      • Satti H.S.
      • Waheed A.
      • Ahmed P.
      • Ahmed K.
      • Akram Z.
      • Aziz T.
      • et al.
      Autologous mesenchymal stromal cell transplantation for spinal cord injury: a Phase I pilot study.
      ] with variable results.
      Our preclinical experience using a paraplegic mini pig model [
      • Zurita M.
      • Aguayo C.
      • Bonilla C.
      • Otero L.
      • Rico M.
      • Rodriguez A.
      • et al.
      The pig model of chronic paraplegia: a challenge for experimental studies in spinal cord injury.
      ] showed that direct intralesional administration of MSCs is the most effective route to allow a large number of cells in areas of the SCI, but because the subarachnoid route is a safe method for minimally invasive cell transplantation, it should clearly be considered in patients with incomplete SCI to avoid the possibility of any surgical complication that could cause a loss of residual neurological function. However, the analysis of the reported clinical trials using subarachnoid injections of MSCs reveals a great variability in the dose and timing of administration, with a number of cells being scarce. Our previous studies suggest that transplanting a great number of cells is advisable because cell therapy seems to show a dose-dependent effect and that repeated cell therapy administration could be beneficial [
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ].
      Here we present the results of a phase II clinical trial (ClinicalTrials.gov NCT02165904, EudraCT 2011-005684-24) that studied the efficacy and safety of four doses of 30 × 106 MSCs in 10 patients suffering chronically established neurological dysfunction secondary to an incomplete SCI.

      Methods

      Study design and treatment

      The present clinical trial included 10 patients (male/female: 8/2) suffering chronic and incomplete SCI (American Spinal Injury Association [ASIA] classification B, C or D). The mean age was 42.20 years (SD: 9.30 years), and time from SCI to treatment ranged from 2.43 to 34.59 years (mean: 14.21 years, SD: 9.88 years). Table I shows the main clinical and demographic data of the patients.
      Table IClinical data of patients in our series.
      PatientSexAgeLevelASIAYears since SCI
      1M37L1B6.00
      2M34L1C8.17
      3M41L1-L2C13.06
      4M56D7-D8C34.59
      5M38D2B2.43
      8F37C5-C6C20.90
      9M34C5-C6C14.31
      10M36C5-C5D17.76
      11M59C3-C4B3.60
      12F50C5-C6B21.32
      Age ranged between 34 and 59 years (mean: 42.20, SD: 9.30 years), and time from SCI to treatment ranged from 2.43 to 34.59 years (mean: 14.21, SD: 9.88 years).
      The clinical trial protocol was approved by the ethic committee of Puerta de Hierro-Majadahonda Hospital and by the Spanish Agency of Medicament and Health Products and conducted in accordance with the principles of the Declaration of Helsinki [
      • World Medical Association
      World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects.
      ] and good clinical practice guidelines [
      International Conference on Harmonisation Expert Working Group
      ICH 21armonized tripartite guideline: guideline for good clinical practice.
      ]. A flow chart of the patients can be seen in the supplementary Figure S1. Adverse events were collected throughout the follow-up and classified according to the Medical Dictionary for Regulatory Activities (MedDRA v. 18.1).
      Treatment consisted of subarachnoid administration, by lumbar puncture, of 30 × 106 autologous MSCs obtained from bone marrow and supported in autologous plasma. It was repeated at months 4, 7 and 10, reaching a total administration of 120 × 106 MSCs for each patient. The patients were followed monthly, from the first administration of MSCs (month 1) through month 12.
      Clinical scores were obtained from each patient by means of the following scales: The ASIA scale [
      • Kirshblum S.C.
      • Burns S.P.
      • Biering-Sorensen F.
      • Donovan W.
      • Graves D.E.
      • Jha A.
      • et al.
      International standards for neurological classification of spinal cord injury (revised 2011).
      ]; the SCI functional rating scale of the International Association of Neurorestoratology (IANR-SCIFRS scale) [
      • International Association of Neurorestoratology
      Spinal cord injury functional rating scale.
      ]; the Functional Independence Measure (FIM) scale [
      • Research Foundation State University of New York
      Guide for use of the uniform data set for rehabilitation.
      ] and the Barthel scale [
      • Mahoney F.
      • Barthel D.
      Functional evaluation: the Barthel Index.
      ] for the study of functional independence in the activities of daily life (ADLs); the Visual Analog Scale (VAS) [
      • Woodforde J.M.
      • Merskey H.
      Some relationship between subjective measures of pain.
      ] for the evaluation of neuropathic pain; the Penn [
      • Penn R.D.
      • Savoy S.M.
      • Corcos D.
      • Latash M.
      • Gottlieb G.
      • Parke B.
      • et al.
      Intrathecal baclofen for severe spinal spasticity.
      ] and the modified Ashworth [
      • Bohannon R.W.
      • Smith M.B.
      Interrater reliability of a modified Ashworth scale of muscle spasticity.
      ] scales for the evaluation of spasms and spasticity, respectively; the Geffner scale [
      • Geffner L.F.
      • Santacruz P.
      • Izurieta M.
      • Flor L.
      • Maldonado B.
      • Auad A.H.
      • et al.
      Administration of autologous bone marrow stem cells into spinal cord injury patients via multiple routes is safe and improves their quality of life: comprehensive case studies.
      ] for the study of bladder function; and the Neurogenic Bowel Dysfunction (NBD) scale [
      • Krogh K.
      • Christensen P.
      • Sabroe S.
      • Laurberg S.
      Neurogenic bowel dysfunction score.
      ] for the evaluation of symptoms related to neurogenic bowel dysfunction. Neurophysiological, urodynamic and magnetic resonance studies were also performed before and after treatment. Furthermore, the enzyme-linked immunosorbent assay technique was used to measure the neurotrophins brain-derived neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor, neurotrophin 3 and 4, in cerebrospinal fluid samples obtained before each administration of MSCs, at months 1, 4, 7 and 10 of the study. Technical details on the neurophysiological and urodynamic studies, and data about our cell therapy medicament, including genetic studies, culture, formulation, packaging and phenotypic characterization of the MSCs (supplementary Figure S2) are provided in the supplementary material.

      Statistical analysis

      To study the differences between the scores of the clinical scales, parameters of urodynamic studies, and changes in neurotrophic factors, the nonparametric Wilcoxon rank test was used, comparing the result of each time period with results at baseline. In the results deemed statistically significant, the size of the effect was calculated using Cohen's d, and the cutoffs proposed by Cohen [
      • Cohen J.
      A power primer.
      ] were used for the general interpretation of the cutoffs of this statistic. For the analysis of the section of neurophysiology, the chi-square test was used to study whether there were differences in the frequency distribution of each variable at each time point, and the McNemar test to study whether there were changes in each of the parameters evaluated between 6 and 12 months. Correlations were obtained using Spearman's rank correlation coefficient. Statistical analysis was performed using SPSS software (v. 21.0, IBM). The graphs were made with the GraphPad Prism program for Windows (v. 5.04, GraphPad Software). All inferential procedures used α = 0.05 as the level of risk. The treatment of missing values in the neurotrophic factors section was done by listwise.

      Results

      Two patients initially selected to form part of the clinical trial (patients 06 and 07) were eliminated due to alterations in the genetic study and replaced by two other patients to make up the 10 patients of the present study. In our present clinical trial, the cell expansion process did not involve any alteration to the genome of the cells in any of the cases, according to the results obtained after analysis by the Array CGH platform.

      Adverse events

      During the study, 20 adverse events (AEs) were seen; and 8 (40%) were probably related to the administration of cell therapy. They generally consisted of headaches and pain in the area of the lumbar puncture. Regarding the degree of these AEs, 17 (84.21%) were considered mild and 3 (15.79%) moderate. There was one severe AE, which was not related to the administration of cell therapy (acute bronchitis). Details of collected AEs are provided in the supplementary material (supplementary Table SI).

      Sensitivity and motor improvement

      Sensitivity improvement according to the ASIA scale was already evident in the first assessment after the first administration of cells (at month 2 of the study) with a mean score of sensitivity in the patients that improved at this time from a basal value of 135.2 ± 42.79 points to 144.5 ± 47.90 points (P = 0.03). In 60% of cases, significant motor improvement was also found at an early stage after the first administration of cell therapy, which was confirmed by a mean motor score in the series, at month 2, of 55.10 ± 21.62 points, compared to the baseline 53 ± 20.45 points (P = 0.027). Throughout the follow-up period, progressive improvement was observed in both sensitivity and motor scores, reaching, at month 12, an improvement in the ASIA total score that ranged between 13 and 85 points from the baseline score, with a mean of 47.30 ± 28.81 points, and with a P value of 0.005 (effect size [ES]: 0.886) when the ASIA total score of the series, obtained at the end of the study, was compared with the basal ASIA total score. Figure 1 shows the progressive improvement obtained in the different scores of the ASIA scale.
      Figure 1
      Figure 1Graphs showing the progressive improvement in the different ASIA scores of the series, at different time points. PPS, Pin Prick Score; LTS, Light Touch Score; MS, Motor Score.
      Motor score (MS) improved in the entire series between 0 and 12 points (mean: 6.20 ± 4.15 points) but did not correlate with the ASIA grade or chronicity of SCI. Nevertheless, when the level of SCI was analyzed, we found that higher levels of SCI correlated with greater improvement in ASIA total scores at the end of the follow-up (P = 0.036; r = 0.6775) due to the points added by the greater infralesional sensitivity improvement. On the other hand, MS improvement showed no significant correlation with respect to SCI level (P = 0.240; r = 0.4078).
      In the entire series, the MS of the lower extremities improved during the study, in comparison with basal values, reaching an early statistical significance in the ASIA assessment. At month 3, after the first administration of MSCs, statistical analysis showed a P value of 0.028 (ES: 0.696), and at the end of the study, the p-value was 0.012 (ES: 0.798). This improvement supported the observation, in most of our patients, of a clear and progressive improvement in walking (supplementary Video S1).
      In the ASIA assessment, the five tetraplegic patients in our series (patients 08, 09, 10, 11 and 12) showed variable degrees of improvement in muscle power of the upper extremities, and all except one showed motor improvement in muscle power of their lower extremities as well. The improvement in motor power of the upper extremities ranged between 1 to 5 points (mean ± SD: 2.4 ± 1.67 points) and the motor power of the lower extremities ranged between 0 to 7 points (mean ± SD: 5 ± 2.9 points). Table II shows the evolution of ASIA scores at different time points and the statistical analysis performed. Additional information is provided in the supplementary material (supplementary Tables SII–SIV and supplementary Figures S3–S9).
      Table IIASIA scores at different time points.
      Score subjectTimeMeanSDP valueES
      Motor ScoreBefore treatment53.0020.45
      At 3 months FU55.6021.450.028*0.700
      At 6 months FU57.7021.150.008**0.840
      At 9 months FU58.6020.830.008**0.850
      At 12 months FU59.2021.150.008**0.840
      Pin Prick ScoreBefore treatment54.5034.36
      At 3 months FU61.2037.420.028*0.696
      At 6 months FU71.6031.960.008**0.844
      At 9 months FU78.3027.330.008**0.844
      At 12 months FU82.8024.690.005**0.886
      Light Touch ScoreBefore treatment80.7011.70
      At 3 months FU85.4014.080.010*0.811
      At 6 months FU89.1013.190.005**0.890
      At 9 months FU92.0013.260.002**0.886
      At 12 months FU93.5012.890.005**0.887
      ASIA total ScoreBefore treatment188.2060.00
      At 3 months FU202.2063.670.005**0.887
      At 6 months FU218.4057.500.005**0.886
      At 9 months FU228.9051.840.005**0.886
      At 12 months FU235.5049.350.005**0.886
      Bold values indicate statistical significance. Statistical analysis showed early and progressive improvement in sensitivity and muscle power.
      FU, follow-up.

      Overall spinal cord function

      The IANR-SCIFRS scale evaluates spinal cord function through nine sections, with a final section that only applies to men and assesses sexual function.
      In our patients, the mean score in overall IANR-SCIFRS before treatment was 29.10 points (SD: 9.96), and at end of the study it was 36.90 points (SD: 8.21), showing a clear and statistically significant improvement (P = 0.005, ES: 0.889). The mean improvement during the follow-up ranged between 4 and 19 points, with a mean of 8.80 ± 4.96 points) (Table III). Additional information is provided in the Supplementary Appendix (Figures S10 and S11).
      Table IIIScores in overall IANR-SCIFRS scale, at different time points, with statistical analysis.
      TimeMeanSDP valueES
      Before treatment29.109.96
      At 3 months FU31.508.890.017*0.755
      At 6 months FU33.909.730.005*0.890
      At 9 months FU35.909.010.005*0.890
      At 12 months FU36.908.210.005*0.889
      Bold values indicate statistical significance.
      FU, follow-up.
      According to the IANR-SCIFRS scale, before treatment, five patients of the series showed a “slight degree of functional disability,” three patients showed a “medium degree of functional disability” and two patients showed a “severe degree of functional disability,” while at the end of the follow-up, six patients showed a “slight degree of functional disability,” and the four remaining patients showed a “medium degree of functional disability” (Figure 2).
      Figure 2
      Figure 2Evolution of the functional rating score of the patients, according to the IANR-SCIFRS scale. On this scale, a global score that ranged between 34 and 47 represents a slight handicap, between 17 and 33 represents a medium handicap and between 0 and 16, a severe handicap.

      Sexual function

      Sexual function was evaluated in the eight male patients of the series, according to the IANR-SCIFRS scale. In two of them (25%) sexual function improved, mainly as a consequence of improved sensitivity in the genital area. See supplementary Table SV and Supplementary Figure S12.

      Activities of daily living

      The FIM and Barthel scales studied ADL in our study. Both scales showed significant improvement at 12 months of follow-up. At this time point, the difference from the baseline overall score showed a P value of 0.027 (ES: 0.700) for the FIM scale, and a P value of 0.039 (ES: 0.651) for the Barthel scale. See supplementary Tables SVI and SVII and supplementary Figures S13 and S14.

      Neuropathic pain

      Neuropathic pain was studied using the VAS scale. Only 4 patients in our series (40%) suffered neuropathic pain (patients 01, 03, 04 and 05). Patients 01 and 03 showed clear improvement after the first administration of cell therapy, with the disappearance of neuropathic pain at months 7 and 2, respectively. Patient 04 showed no improvement, and patient 05 improved slightly as of month 2 (supplementary Table SVIII and supplementary Figure S15).

      Spasms and spasticity

      The evolution of spasms and spasticity was studied by the Penn and modified Ashworth scales, respectively. Although our patients generally described improvement in spasms and spasticity throughout the study, the low number of patients showing these symptoms precludes obtaining conclusions from a statistical point of view. Only seven patients in our series suffered spasms before treatment, and in two of them (28.57%), the spasms reduced over the course of follow-up, according to the scores in the Penn scale. See supplementary Table SIX and supplementary Figure S16.
      Nine patients of the series showed variable degrees of spasticity, according to the modified Ashworth scale, and three of them (33.3%) showed improvement over the course of follow-up (patients 02, 03 and 04). One of them (patient 04) was carrying a baclofen pump, the administration of which was gradually reduced during follow-up, with no increase in spasticity. See supplementary Table SX and supplementary Figure S17.

      Sphincter function

      Sphincter function was studied using the Geffner scale (bladder dysfunction) and the NBD scale, for the study of bowel control. All patients except one (90%), suffered bladder dysfunction before treatment, and eight of them (88.8%) improved over the follow-up period. The statistical study showed a significant difference between the baseline score of the Geffner scale and the score at the end of follow-up (P = 0.024, ES: 0.712) (see Figure 3, supplementary Figure S18 and supplementary Table SXI).
      Figure 3
      Figure 3Evolution of the progressive improvements observed in the Geffner (bladder dysfunction) and NBD (bowel dysfunction) scales, at different time points. At the end of the study (month 12) statistical differences with basal scores were found. For the Geffner scale, P = 0.024 and ES was 0.712. For the NBD scale, P = 0.018, and ES was 0.750.
      The analysis of the NBD scale showed an early and progressive improvement in NBD symptoms of our patients, with a P value, at the end of the study, of 0.018 (ES: 0.750) (Table IV). In the series, all patients except one (90%) showed clear symptoms of bowel dysfunction, and seven of them (77.7%) showed clear improvement over the follow-up period (see Figure 3, Table IV and supplementary Figure S19).
      Table IVScores in NBD scale, at different time points, with statistical analysis.
      TimeMeanSDP valueES
      Before treatment10.606.64
      At 3 months FU6.104.150.042*0.643
      At 6 months FU5.704.350.018*0.748
      At 9 months FU4.403.860.018*0.751
      At 12 months FU4.203.880.018*0.750
      Bold values indicate statistical significance.
      FU, follow-up.
      According to the rating score of the NBD scale, before cell therapy, two patients had severe neurogenic bowel dysfunction, five had moderate dysfunction, one had mild dysfunction and two had minimal dysfunction. At the end of the follow-up, six patients had absent or minimal dysfunction, three patients had mild dysfunction and one patient had moderate dysfunction (Figure 4).
      Figure 4
      Figure 4Evolution of the functional rating score of our patients, according to the NBD scale. On this scale, a global score between 0 and 6 represents a minimal NBD dysfunction, between 7 and 9 mild dysfunction, between 10 and 13 moderate dysfunction and 14 or more severe NBD dysfunction.

      Neurophysiological studies

      All patients showed neurophysiological improvement during the follow-up period. In eight patients, somatosensory evoked potentials showed progressive improvement in parameters of latency and/or amplitude in comparison with the basal study. Improvement in motor evoked potentials was seen in four patients at month 6 and in five patients at month 12 of follow-up. With respect to basal recordings, improvement in sensitive nerve conduction, in terms of conduction velocity and amplitude, was only recorded in two patients at month 6. They showed progressive improvement in the study performed at month 12, and at this time point, another patient showed improvement with respect to the basal study. Similarly, five patients showed improvement in motor nerve conduction at month 6 compared with baseline, and seven patients at month 12. In comparison with basal studies, improvement in electromyography parameters showing voluntary muscle contraction was recorded in four patients of the series at month 6, and in six patients at the end of the follow-up (see supplementary Video S2). Moreover, infra-lesional polyphasic motor potentials, considered typical of active muscle reinnervation, were recorded in seven patients at month 6, and in all patients except one at the end of the follow-up (P = 0.011). Additional information is provided in supplementary Tables S12 and S13).

      Urodynamic studies

      Supplementary Table S14 shows the improvement in urodynamic parameters obtained for each patient of the series when compared with baseline. The possibility of voluntary micturition, which was not present at the basal study, was recorded in five patients (50%) at the end of the follow-up. Compared with baseline, at this time point, 60% of patients improved in first sensation at filling, 50% improved in maximum cystometric capacity and 60% improved in the parameter of detrusor pressure. Furthermore, at the end of the study, 80% of our patients showed significant improvement in bladder compliance (P = 0.037, ES: 0.661). Additional information is provided in the supplementary material (supplementary Tables S15–S17 and supplementary Figures S20–S22).

      Neuroimaging studies

      Neuroimaging studies (conventional magnetic resonance imaging and myelography) were performed before cell therapy and at the end of the follow-up (at month 12) and failed to show changes in the morphology of SCI zones compared with basal images.

      Neurotrophins in CSF

      CSF samples obtained before each administration of cell therapy showed great variability in the expression of neurotrophins. In samples of CSF obtained at month 10 (after 3 administrations of MSCs), mean values of brain-derived neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor and neurotrophin 3 and 4 showed slight increases in comparison with basal levels. Statistical analysis failed to obtain statistical significance, except for the finding of a P value of 0.011 (ES: 0.850) for ciliary neurotrophic factor levels at month 7 of follow-up, but this statistical significance was not maintained in the CSF samples obtained at month 10 (see supplementary Table S18 and supplementary Figure S23).

      Discussion

      In this clinical trial, and as a result of our experience gained using animal models [
      • Zurita M.
      • Vaquero J.
      Functional recovery in chronic paraplegia after bone marrow stromal cells transplantation.
      ,
      • Zurita M.
      • Vaquero J.
      Bone marrow stromal cells can achieve cure of chronic paraplegic rats: functional and morphological outcome one year after transplantation.
      ,
      • Vaquero J.
      • Zurita M.
      Bone marrow stromal cells for spinal cord repair: a challenge for contemporary neurobiology.
      ,
      • Vaquero J.
      • Zurita M.
      • Oya S.
      • Santos M.
      Cell therapy using bone marrow stromal cells in chronic paraplegic rats: systemic or local administration?.
      ,
      • Vaquero J.
      • Zurita M.
      Functional recovery after severe central nervous system trauma: current perspectives for cell therapy with bone marrow stromal cells.
      ,
      • Zurita M.
      • Vaquero J.
      • Bonilla C.
      • Santos M.
      • De Haro J.
      • Oya S.
      • et al.
      Functional recovery of chronic paraplegic pigs after autologous transplantation of bone marrow stromal cells.
      ,
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ,
      • Bonilla C.
      • Zurita M.
      • Otero L.
      • Aguayo C.
      • Vaquero J.
      Delayed intralesional transplantation of bone marrow stromal cells increases endogenous neurogenesis and promotes functional improvement after severe traumatic brain injury.
      ,
      • Otero L.
      • Zurita M.
      • Bonilla C.
      • Aguayo C.
      • Rico M.A.
      • Rodriguez A.
      • et al.
      Allogeneic bone marrow stromal cell transplantation after cerebral hemorrhage achieves cell transdifferentiation and modulates endogenous neurogenesis.
      ,
      • Zurita M.
      • Aguayo C.
      • Bonilla C.
      • Otero L.
      • Rico M.
      • Rodriguez A.
      • et al.
      The pig model of chronic paraplegia: a challenge for experimental studies in spinal cord injury.
      ,
      • Zurita M.
      • Aguayo C.
      • Bonilla C.
      • Rodríguez A.
      • Vaquero J.
      Perilesional intrathecal administration of autologous bone marrow stromal cells achieves functional improvement in pigs with chronic paraplegia.
      ] and in humans [
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ], we administered a cell therapy medicament consisting of autologous MSCs supported by autologous plasma to patients suffering incomplete SCI, and assuming that these patients might show improvement after injury, we only included patients with long-standing SCI and with established neurological dysfunction. With regard to the dose of MSCs used, at present, clinical experience with cell therapy in SCI is limited, and there are no clear criteria in the literature to recommend dosage or administration intervals. Doses of 30 × 106 MSCs were already used by us in intrathecal administration in a previous clinical trial with perfect tolerance [
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ]. The hypothesis that injected MSCs can die after administration is also valid. Because of these considerations, we repeated administrations to a total dose of 120 × 106 MSCs.
      In the ASIA scale assessment, scores showed progressive improvement during the study, including improvement in the motor power of the upper extremities in tetraplegic patients, a finding supported by neurophysiological studies, suggesting that motor benefit can be obtained in cervical SCI after intrathecal administration of MSCs in the lumbar region. Although tetraplegic patients improved their motor power in the upper extremities, the improvement was scarce, and, at least in our present study, in no case did we obtain complete muscle recovery. This observation requires further study with a greater number of patients suffering cervical SCI.
      Our results showed that all our patients experienced gradual improvement in clinical parameters without reaching a plateau at the end of the follow-up period. Recovery of infra-lesional sensitivity occurred early after the first administration of cell therapy, a finding we recently described after the intralesional administration of MSCs in complete chronic paraplegia [
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ], suggesting a possible effect through the cytokines released by the transplanted cells that activate preserved but non-functional circuits, rather than a mechanism of nerve pathway regeneration.
      On the other hand, in the present clinical trial, the patients showed progressive improvement in scores of the IANR-SCIFRS scale, with a clear parallel between this improvement and that obtained from the ASIA scale, a finding we previously described when our cell therapy medicament was applied to patients with complete SCI [
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ]. The important improvement obtained in sphincter dysfunction supports our previously reported findings in patients suffering chronic complete paraplegia [
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ] and its obvious impact on quality of life.
      Scales evaluating ADLs (MIF and Barthel scales) are not useful for the assessment of patients with chronic SCI because they have generally adapted to the dysfunction and are able to perform most activities without assistance [
      • Vaquero J.
      • Zurita M.
      • Rico M.A.
      • Bonilla C.
      • Aguayo C.
      • Montilla J.
      • et al.
      An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
      ], but we found significant improvement in our series at the end of the follow-up, supporting the effectiveness of the treatment.
      Improvement in neuropathic pain was difficult to ascertain in our present study because only four patients had significant neuropathic pain before treatment. However, we did observe a tendency for neuropathic pain to decrease as of the first administrations of cell therapy, with one patient (patient 01) showing an important decrease after the first MSC administration and a complete disappearance of neuropathic pain at month 6 of follow-up.
      Furthermore, patients with spasms and spasticity improved, but conclusions could not be drawn because of the limited number of patients suffering these symptoms in the present study.
      In neurophysiological studies, although the sample size prevents obtaining statistically significant results in most of the parameters studied, all patients showed improvement during the follow-up period, mainly in somatosensory evoked potentials and motor nerve conduction. Electromyography recordings showing progressive improvement in voluntary muscle contraction with signs of infra-lesional active muscle reinnervation represent an objective finding supporting the efficacy of the treatment.
      Urodynamic studies showed variability between patients, but 80% of them showed improvement in bladder compliance, reflecting the improvement in bladder function after cell therapy.
      In our study, magnetic resonance imaging studies failed to show changes in the morphology of SCI after cell therapy, suggesting that subarachnoid administration of MSCs is not able to modify the morphology of established spinal cord lesions and that improvement may be mainly due to the release of neurotrophic factors without changing the neuroimage associated with SCI.
      With regard to the values of neurotrophins, it is difficult to obtain conclusions in the present study because of limitations due to the number of patients studied, the low expression of these factors in CSF and its variability. Despite the great variability among patients that prevented our obtaining statistically significant results, our findings show slight increases in some neurotrophic factors when the average values were compared with those obtained before MSC administration. It is well known that neurotrophic factors can be secreted by MSCs, and they have been linked to their beneficial effects [
      • Chen Q.
      • Long Y.
      • Yuan X.
      • Zou L.
      • Sun J.
      • Chen S.
      • et al.
      Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors.
      ,
      • Paradisi M.
      • Alviano F.
      • Pirondi S.
      • Lanzoni G.
      • Fernandez M.
      • Lizzo G.
      • et al.
      Human mesenchymal stem cells produce bioactive neurotrophic factors: source, individual variability and differentiation issues.
      ,
      • Wang L.J.
      • Zhang R.P.
      • Li J.D.
      Transplantation of neurotrophin-3-expressing bone mesenchymal stem cells improves recovery in a rat model of spinal cord injury.
      ,
      • Crigler L.
      • Robey R.C.
      • Asawachaicharn A.
      • Gaupp D.
      • Phinney D.G.
      Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis.
      ,
      • Wilkins A.
      • Kemp K.
      • Ginty M.
      • Hares K.
      • Mallam E.
      • Scolding N.
      Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro.
      ]. In the present study the increase of ciliary neurotrophic factor with respect to baseline seems to be greater than other neurotrophic factors that we have studied. It is a protein that promotes neurotransmitter synthesis and survival and/or differentiation of a variety of neuronal cell types [
      • Abbaszadeh H.A.
      • Tiraihi T.
      • Noori-Zadeh A.
      • Delshad A.R.
      • Sadeghizade M.
      • Taheri T.
      Human ciliary neurotrophic factor-overexpressing stable bone marrow stromal cells in the treatment of a rat model of traumatic spinal cord injury.
      ], and its possible role in the functional recovery of patients subjected to cell therapy requires further study. On the other hand, the possibility that other neurotrophic factors released by MSCs may play a role in the functional recovery of our patients must be taken into account.

      Conclusions

      In conclusion, our cell therapy treatment is a safe procedure that significantly improves neurological dysfunction and increases the quality of life of patients suffering incomplete SCI. The experience obtained from the present clinical trial shows the benefit of this simple procedure in patients with incomplete SCI and suggests the desirability of studying whether this form of cell therapy may be useful in other diseases with similar clinical features, such as severe spondylotic myelopathy.

      Acknowledgments

      We thank the institutions supporting the development of our cell therapy program, in particular, Mapfre and Rafael del Pino Foundations . The present clinical trial was mainly supported by Carlos III Institute (expedient EC11-089 ). Additional support was obtained from the Sermes Foundation , Atresmedia Foundation , Mutua Madrileña Foundation and APINME Association . We extend special thanks to Paula Campello and Carmen Calabia, from Sermes CRO for help during the development and analysis of the present study. For clinical assistance, we especially appreciate the cooperation of the Neurological Cell Therapy Group from the Puerta de Hierro-Majadahonda-Hospital (listed in the supplementary material) and external rehabilitation teams from the Lesionado Medular Foundation, Lescer, NeuroFis and Crene centers.
      Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

      Appendix. Supplementary material

      The following is the supplementary data to this article:

      References

        • Park J.H.
        • Kim D.Y.
        • Sung I.
        • Choi G.H.
        • Jeon M.H.
        • Kim K.K.
        • et al.
        Long-term results of spinal cord injury therapy using mesenchymal stem cells derived from bone marrow in humans.
        Neurosurgery. 2012; 70: 1238-1247
        • Syková E.
        • Homola A.
        • Mazanec R.
        • Lachmann H.
        • Konrádová S.L.
        • Kobylka P.
        • et al.
        Autologous bone marrow transplantation in patients with subacute and chronic spinal cord injury.
        Cell Transplant. 2006; 15: 675-687
        • Yoon S.H.
        • Shim Y.S.
        • Park Y.H.
        • Chung J.K.
        • Nam J.H.
        • Kim M.O.
        • et al.
        Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: phase I/II clinical trial.
        Stem Cells. 2007; 25: 2066-2073
        • Chopp M.
        • Zhang X.H.
        • Li Y.
        • Wang L.
        • Chen J.
        • Lu D.
        • et al.
        Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation.
        Neuroreport. 2000; 11: 3001-3005
        • Hofstetter C.P.
        • Schwarz E.J.
        • Hess D.
        • Widenfalk J.
        • El Manira A.
        • Prockop D.J.
        • et al.
        Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery.
        Proc Natl Acad Sci USA. 2002; 99: 2199-2204
        • Zurita M.
        • Vaquero J.
        Functional recovery in chronic paraplegia after bone marrow stromal cells transplantation.
        Neuroreport. 2004; 15: 1105-1108
        • Zurita M.
        • Vaquero J.
        Bone marrow stromal cells can achieve cure of chronic paraplegic rats: functional and morphological outcome one year after transplantation.
        Neurosci Lett. 2006; 402: 51-56
        • Vaquero J.
        • Zurita M.
        Bone marrow stromal cells for spinal cord repair: a challenge for contemporary neurobiology.
        Histol Histopathol. 2009; 24: 107-116
        • Vaquero J.
        • Zurita M.
        • Oya S.
        • Santos M.
        Cell therapy using bone marrow stromal cells in chronic paraplegic rats: systemic or local administration?.
        Neurosci Lett. 2006; 398: 129-134
        • Ohta M.
        • Suzuki Y.
        • Noda T.
        • Ejiri Y.
        • Dezawa M.
        • Kataoka K.
        • et al.
        Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation.
        Exp Neurol. 2004; 187: 266-278
        • Bakshi A.
        • Hunter C.
        • Swanger S.
        • Lepore A.
        • Fischer I.
        Minimally invasive delivery of stem cells for spinal cord injury: advantages of the lumbar puncture technique.
        J Neurosurg Spine. 2004; 1: 330-337
        • Bakshi A.
        • Barshinger A.L.
        • Swanger S.A.
        • Madhavani V.
        • Shumsky J.S.
        • Neuhuber B.
        • et al.
        Lumbar puncture delivery of bone marrow stromal cells in spinal cord contusion: a novel method for minimally invasive cell transplantation.
        J Neurotrauma. 2006; 23: 55-65
        • Himes B.T.
        • Neuhuber B.
        • Coleman C.
        • Kushner R.
        • Swanger S.A.
        • Kopen G.C.
        • et al.
        Recovery of function following grafting of human bone marrow-derived stromal cells into the injured spinal cord.
        Neurorehabil Neural Repair. 2006; 20: 278-296
        • Parr A.M.
        • Tator C.H.
        • Keating A.
        Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury.
        Bone Marrow Transplant. 2007; 40: 609-619
        • Deda H.
        • Inci M.C.
        • Kürekçi A.E.
        • Kayihan K.
        • Ozgün E.
        • Ustünsoy G.E.
        • et al.
        Treatment of chronic spinal cord injured patients with autologous bone marrow-derived hematopoietic stem cell transplantation: 1-year follow-up.
        Cytotherapy. 2008; 10: 565-574
        • Vaquero J.
        • Zurita M.
        Functional recovery after severe central nervous system trauma: current perspectives for cell therapy with bone marrow stromal cells.
        Prog Neurobiol. 2011; 93: 341-349
        • Saito F.
        • Nakatani T.
        • Iwase M.
        • Maeda Y.
        • Hirakawa A.
        • Murao Y.
        • et al.
        Spinal cord injury treatment with intrathecal autologous bone marrow stromal cell transplantation: the first clinical trial case report.
        J Trauma. 2008; 64: 53-59
        • Pal R.
        • Venkataramana N.K.
        • Bansai A.
        • Balaraju S.
        • Jan M.
        • Chandra R.
        • et al.
        Ex vivo-expanded autologous bone marrow-derived mesenchymal stromal cells in human spinal cord injury/paraplegia: a pilot clinical study.
        Cytotherapy. 2009; 11: 897-911
        • Saito F.
        • Nakatani T.
        • Iwase M.
        • Maeda Y.
        • Murao Y.
        • Suzuki Y.
        • et al.
        Administration of cultured autologous bone marrow stromal cells into cerebrospinal fluid in spinal injury patients: a pilot study.
        Restor Neurol Neurosci. 2012; 30: 127-136
        • Jiang P.C.
        • Xiong W.P.
        • Wang G.
        • Ma C.
        • Yao W.Q.
        • Kendell S.F.
        • et al.
        A clinical trial report of autologous bone marrow-derived mesenchymal stem cell transplantation in patients with spinal cord injury.
        Exp Ther Med. 2013; 6: 140-146
        • Mendonça M.V.P.
        • Larocca T.F.
        • Souza B.S.
        • de Freitas Souza B.S.
        • Villarreal C.F.
        • Silva L.F.
        • et al.
        Safety and neurological assessments after autologous transplantation of bone marrow mesenchymal stem cells in subjects with chronic spinal cord injury.
        Stem Cell Res Ther. 2014; 5: 126
        • Zurita M.
        • Vaquero J.
        • Bonilla C.
        • Santos M.
        • De Haro J.
        • Oya S.
        • et al.
        Functional recovery of chronic paraplegic pigs after autologous transplantation of bone marrow stromal cells.
        Transplantation. 2008; 86: 845-853
        • Vaquero J.
        • Zurita M.
        • Rico M.A.
        • Bonilla C.
        • Aguayo C.
        • Montilla J.
        • et al.
        An approach to personalized cell therapy in chronic complete paraplegia: the Puerta de Hierro phase I/II clinical trial.
        Cytotherapy. 2016; 18: 1024-1035
        • Chen Q.
        • Long Y.
        • Yuan X.
        • Zou L.
        • Sun J.
        • Chen S.
        • et al.
        Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors.
        J Neurosci Res. 2005; 80: 611-619
        • Paradisi M.
        • Alviano F.
        • Pirondi S.
        • Lanzoni G.
        • Fernandez M.
        • Lizzo G.
        • et al.
        Human mesenchymal stem cells produce bioactive neurotrophic factors: source, individual variability and differentiation issues.
        Int J Immunopathol Pharmacol. 2014; 27: 391-402
        • Wang L.J.
        • Zhang R.P.
        • Li J.D.
        Transplantation of neurotrophin-3-expressing bone mesenchymal stem cells improves recovery in a rat model of spinal cord injury.
        Acta Neurochir (Wien). 2014; 156: 1409-1418
        • Crigler L.
        • Robey R.C.
        • Asawachaicharn A.
        • Gaupp D.
        • Phinney D.G.
        Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis.
        Exp Neurol. 2006; 198: 54-64
        • Wilkins A.
        • Kemp K.
        • Ginty M.
        • Hares K.
        • Mallam E.
        • Scolding N.
        Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro.
        Stem Cell Res. 2009; 3: 63-70
        • Bonilla C.
        • Zurita M.
        • Otero L.
        • Aguayo C.
        • Vaquero J.
        Delayed intralesional transplantation of bone marrow stromal cells increases endogenous neurogenesis and promotes functional improvement after severe traumatic brain injury.
        Brain Inj. 2009; 23: 760-769
        • Otero L.
        • Zurita M.
        • Bonilla C.
        • Aguayo C.
        • Rico M.A.
        • Rodriguez A.
        • et al.
        Allogeneic bone marrow stromal cell transplantation after cerebral hemorrhage achieves cell transdifferentiation and modulates endogenous neurogenesis.
        Cytotherapy. 2012; 14: 34-44
        • Nakano N.
        • Nakai Y.
        • Seo T.B.
        • Homma T.
        • Yamada Y.
        • Ohta M.
        • et al.
        Effects of bone marrow stromal cell transplantation through CSF on the subacute and chronic spinal cord injury in rats.
        PLoS ONE. 2013; 8 (e73494)
        • Satake K.
        • Lou J.
        • Lenke L.G.
        Migration of mesenchymal stem cells through cerebrospinal fluid into injured spinal cord tissue.
        Spine. 2004; 29: 1971-1979
        • Chotivichit A.
        • Ruangchainikom M.
        • Chiewvit P.
        • Wongkajornsilp A.
        • Sujirattanawimol K.
        Chronic spinal cord injury treated with transplanted autologous bone marrow-derived mesenchymal stem cells tracked by magnetic resonance imaging: a case report.
        J Med Case Rep. 2015; 9: 79
        • Karamouzian S.
        • Nematollahi-Mahani S.N.
        • Nakhaee N.
        • Eskandary H.
        Clinical safety and primary efficacy of bone marrow mesenchymal cell transplantation in subacute spinal cord injured patients.
        Clin Neurol Neurosurg. 2012; 114: 935-939
        • Satti H.S.
        • Waheed A.
        • Ahmed P.
        • Ahmed K.
        • Akram Z.
        • Aziz T.
        • et al.
        Autologous mesenchymal stromal cell transplantation for spinal cord injury: a Phase I pilot study.
        Cytotherapy. 2016; 18: 518-522
        • Zurita M.
        • Aguayo C.
        • Bonilla C.
        • Otero L.
        • Rico M.
        • Rodriguez A.
        • et al.
        The pig model of chronic paraplegia: a challenge for experimental studies in spinal cord injury.
        Prog Neurobiol. 2012; 97: 288-303
        • World Medical Association
        World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects.
        JAMA. 2000; 284: 3043-3045
        • International Conference on Harmonisation Expert Working Group
        ICH 21armonized tripartite guideline: guideline for good clinical practice.
        (Available from:)
        • Kirshblum S.C.
        • Burns S.P.
        • Biering-Sorensen F.
        • Donovan W.
        • Graves D.E.
        • Jha A.
        • et al.
        International standards for neurological classification of spinal cord injury (revised 2011).
        J Spinal Cord Med. 2011; 34: 535-546
        • International Association of Neurorestoratology
        Spinal cord injury functional rating scale.
        Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2008; 22: 1021-1023
        • Research Foundation State University of New York
        Guide for use of the uniform data set for rehabilitation.
        University of New York, New York1991
        • Mahoney F.
        • Barthel D.
        Functional evaluation: the Barthel Index.
        Md Med J. 1965; 14: 61-65
        • Woodforde J.M.
        • Merskey H.
        Some relationship between subjective measures of pain.
        J Psychosom Res. 1972; 16: 173-178
        • Penn R.D.
        • Savoy S.M.
        • Corcos D.
        • Latash M.
        • Gottlieb G.
        • Parke B.
        • et al.
        Intrathecal baclofen for severe spinal spasticity.
        N Engl J Med. 1989; 320: 1517-1521
        • Bohannon R.W.
        • Smith M.B.
        Interrater reliability of a modified Ashworth scale of muscle spasticity.
        Phys Ther. 1987; 67: 206-207
        • Geffner L.F.
        • Santacruz P.
        • Izurieta M.
        • Flor L.
        • Maldonado B.
        • Auad A.H.
        • et al.
        Administration of autologous bone marrow stem cells into spinal cord injury patients via multiple routes is safe and improves their quality of life: comprehensive case studies.
        Cell Transplant. 2008; 17: 1277-1293
        • Krogh K.
        • Christensen P.
        • Sabroe S.
        • Laurberg S.
        Neurogenic bowel dysfunction score.
        Spinal Cord. 2006; 44: 625-631
        • Cohen J.
        A power primer.
        Psychol Bull. 1992; 112: 155-159
        • Zurita M.
        • Aguayo C.
        • Bonilla C.
        • Rodríguez A.
        • Vaquero J.
        Perilesional intrathecal administration of autologous bone marrow stromal cells achieves functional improvement in pigs with chronic paraplegia.
        Cytotherapy. 2013; 15: 1218-1227
        • Abbaszadeh H.A.
        • Tiraihi T.
        • Noori-Zadeh A.
        • Delshad A.R.
        • Sadeghizade M.
        • Taheri T.
        Human ciliary neurotrophic factor-overexpressing stable bone marrow stromal cells in the treatment of a rat model of traumatic spinal cord injury.
        Cytotherapy. 2015; 17: 912-921