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Abstract

Interruption of spinal cord (SC) continuity leads to functional loss below the lesion level. The purpose of this study was to evaluate the safety and efficacy of bone marrow nucleated cell (BMNC) and multiple mesenchymal stem cell (MSC) transplantations in spinal cord injury (SCI). A patient with total SC interruption at the Th2-3 level underwent experimental therapy with BMNC and MSC transplantations followed with intensive neurorehabilitation treatment. At admission, 6 h after SCI, the patient was scored ASIA A, had a Th1 sensation level, paraplegia with sphincter palsy, and was without the ability to control trunk movement. Neurophysiology examination showed bilateral axonal damage to the motor and sensory neural fibers with no motor unit potentials or peripheral motor nerve conduction in the lower extremities. The standard therapy had been applied and had not produced any positive results. The patient was treated with autologous BMNCs injected intravenously (3.2 × 10⁹) and intrathecally (0.5 × 10⁹) 10 weeks after the SCI and with five rounds of MSCs every 3-4 months (1.3-3.65 × 10⁷) administered via lumbar puncture. Total number of transplanted MSC cells during the course of treatment was 1.54 × 10⁸. There were no complications related to transplantations and no side effects related to the therapy during 2 years of treatment. The ASIA score improved from A to C/D (from 112 to 231 points). The sensation level expanded from Th1 to L3-4, and the patient's ability to control the body trunk was fully restored. Bladder filling sensation, bladder control, and anal sensation were also restored. Muscle strength in the left lower extremities improved from plegia to deep paresis (1 on the Lovett scale). The patient's ability to move lower extremities against gravity supported by the movements in quadriceps was restored. The patient gained the ability to stand in a standing frame and was able to walk with the support of hip and knee ortheses. Magnetic resonance imaging (MRI) revealed that at the Th2/Th3 level, where the hemorrhagic necrosis was initially observed, small tissue structures appeared. Our results suggest that repeated intrathecal infusions of MSCs might have the potential to produce clinically meaningful improvements for SCI patients.

Keywords

Mesenchymal stem cells (MSCs)Spinal cord injury (SCI)Transplantation Cell therapy

Introduction

Spinal cord injury (SCI) results in an irreversible loss of neuronal junctions leading to SC dysfunction, profound disability that significantly impacts the quality of life, and even death (18, 48). Every year, approximately 130,000 people worldwide, predominantly young males (82%), sustain new SCIs. Progress in emergency medicine, nursing care, and long-term care of SCI patients have resulted in increased survival. However, survival decreases with age, and it is dependent on the injury level (15).

Standard management after a SCI includes stabilization of the injured region and subsequent long-term conservative rehabilitation treatment (67). Patients with SCI develop several long-term complications, such as pneumonia, atelectasis, deep venous thrombosis (DVT), pressure ulcers, fracture spasticity (up to 70%), and neuropathic pain (40-50%) (38, 44, 54). The complications are a frequent cause of morbidity and mortality and lead to an increased rate of rehospitalizations (38). Thus, modern conservative treatment involves prevention of post-traumatic complications, analgesic treatment, as well as rehabilitation. Nevertheless, we still lack standard treatment methods to achieve regeneration of sensory and motor neurons, which would enable total or partial functional recovery of the patient.

Experimental approaches of SCI treatment using cellular delivery of growth factors demonstrated that IGF-1 supports corticospinal axon survival (23); NT-3 causes partial functional recovery (20); and placement of molecular, cellular, or “synthetic ” bridges in the lesion cavity supports axonal regeneration across the lesion site (45). Sciatic nerve conditioning lesion resulted in regeneration of the injured SC (43), and stimulating cAMP signaling increased the intrinsic growth capacity of injured sensory axons (42) overcoming myelin inhibition (52).

Sensory and functional recovery was also demonstrated using the strategy of glial scar inhibition using chondroitinase ABC (ChABC) (7). Neutralization of an important neurite outgrowth inhibitor, Nogo-A was reported to stimulate axonal sprouting (17).

Bone marrow (BM) was demonstrated to be a source of different stem and progenitor cells, including hematopoietic, endothelial, epithelial (13, 32), and mesenchymal (6), and also to contain neural tissue-committed stem cells, which can be mobilized into a peripheral blood during injury, such as cerebral stroke (30). Moreover, BM cells were shown to produce numerous trophic growth factors (36). Intravenous administration of bone marrow CD34⁺ cells was demonstrated to promote neovascularization of ischemic brain, enabling neuronal regeneration (66). Intracerebral CD34⁺ and/or CD133⁺ cell transplantations improved neurological functions in animal models (61). Bone marrow MSCs (BMMSCs) administered intracerebrally (70) or intracaratoidally (60) into ischemic rats' brains caused significant improvement of the functional performance. Remyelination of the SC axons following intravenous delivery of BM cells (31, 58), and functional recovery after intraspinal administration of BM stromal cells was also demonstrated (12).

Since 2005, numerous clinical trials with over 400 patients evaluated safety and efficacy of autologous BM cell transplantations alone or in combination with GM-CSF for acute (4, 14, 18, 48, 63, 69), subacute (63, 69), and chronic (8, 10, 18, 33, 63, 69) adult SCI. Safety and feasibility of single intravenous and intraspinal single implantations were demonstrated by all groups with only minor adverse events in some patients, like fever, increased leukocytosis, skin rash, dizziness, itching, headache, rigidity, or abdominal discomfort (33, 48, 69). These studies provide evidence of the possibility of neurological, sensory, and functional improvement, better quality of life, and partial restoration of the neural connections in acute, subacute, and chronic SC patients. However, more significant improvements were seen in acute patients, and the time interval from the injury to cell therapy negatively correlated with the outcome (18, 63). Three reports demonstrated the recovery in chronic patients transplanted with BM cells with intrathecal administration (10, 18, 33), while one study revealed no change (69). Regardless of the time from injury, intramedullary rather than intravenous administration was more likely to induce neurological improvement (63). Cell number and dosing seemed to influence the appearance and the degree of the improvement (4). Application of a single implantation of BMMSCs caused moderate improvement in acute (57), subacute, and chronic SCI patients (47, 49) although considerable improvement in subacute patients was also reported (29). Allogeneic umbilical cord MSCs were demonstrated to cause moderate recovery in incomplete SCI (35) and cord blood MSCs in complete chronic SCI (28). Considerable improvement was reported in incomplete SCI after multiple transplantations of cord blood CD34⁺ in combination with umbilical cord-derived MSCs (25). No improvement and some side effects were also reported after multiple MSC transplantations in chronic patients (30).

Single use of olfactory ensheathing cells (OECs) demonstrated moderate (24) and multiple use of OEC considerable improvement (53) in chronic complete SCI patients. Transplantation of olfactory mucosa in complete chronic SCI patients caused modest (11) or considerable improvement in some patients (34). Recently, olfactory bulb cells transplanted intrathecally in multiple sites were reported to bring considerable recovery in a chronic patient (65). Administration of Schwann cells was demonstrated to be safe and cause moderate patient improvement (56, 71). Similarly, correction in sensation was seen in some patients after single administration of a combination of MSCs and Schwann cells (68). Considerable improvement for chronic patients was obtained using combination of MSCs with patient's autoimmune T-cells (39). The use of neuronal stem/progenitor cells was also shown to be very promising in brain regeneration (21, 59).

Based on our previous experience of multiple bone marrow nucleated cell (BMNC) transplantations (27) and building suggestions and reports from animal and human studies that MSCs have potential to support regeneration after SCI, we set up this study to assess the safety and efficacy of combined intravenous/intrathecal BMNCs and multiple lumbar puncture MSC administrations to treat subacute complete SCI.

Materials and Methods

Patient

We present a case report of a 15-year-old girl with complete SCI. The patient had been involved in a moving vehicle accident (MVA) resulting in a vertebral column fracture at the Th2-Th3 level. The patient did not receive methylprednisolone treatment in the first 8 h after MVA. Standard therapy, including anti-inflammatory therapy, stabilization of the destroyed level, and neurorehabilitation (including kinezy therapy; manual therapy: stretching, massage, hands-on strengthening exercises; ESTIM: functional electrical stimulation of lower limb muscle groups and use of Lokomat Gait Orthosis; 5 days a week, 6 h per day), which started 21 days after injury, did not produce any improvement. However, the patient did not develop any signs of autonomic dysreflexia.

At admission, 3 months after SCI, the patient was paraplegic (0 on the Lovett scale) with the Th1 sensory level. She was able to breathe independently and eat using both hands but was unable to control the body trunk. The patient couldn't control her bladder and bowels.

The study was performed at University Children's Hospital (UCH) of Cracow, Poland, with the approval of the Jagiellonian University Medical College Bioethical Committee (KBET/31/L/2012). The girl's parents, as well as the girl herself, provided written informed consent.

Bone Marrow Aspiration

Autologous BM was harvested from the posterior superior iliac crest under general anesthesia using aspiration from multiple punctures of the iliac ala. BM was collected in a medium consisting of 0.9% NaCl (B. Braun, Melsungen, Germany), heparin (Polfa Warsaw, Warsaw, Poland), and gentamicin (PAA Laboratories, GmbH, Goetzis, Austria).

Bone Marrow Nuclear Cell Isolation, Evaluation, and Preparation for Transplantation

Isolation of BMNCs from the BM suspension was performed in the Department of Transplantation of Jagiellonian University Medical College using 10% hydroxyethyl starch solution (HES) (B. Braun). HES had been added to BM until final 3% concentration was reached. Afterward, the obtained solution was mixed and left in a sterile bottle for 40-50 min for distribution. After all erythrocytes had sedimented at the bottom of the bottle, upper cell suspension was collected to 50-ml tubes and centrifuged. The supernatant was discarded and the cell suspension was washed with phosphate-buffered saline (PBS) solution (PAA Laboratories) and suspended in PBS. Cell number was evaluated using a hematocytometer, and cells were divided into two parts.

Viability of the cells was checked in trypan blue exclusion assay (0.4% solution; Sigma Chemicals, Seelze, Germany). Percentage of CD34⁺ and CD133⁺ cells was assessed according to ISHAGE protocol (19) using 20 μl of monoclonal anti-CD34-PE and anti-CD133-PE antibodies per 100 μl of the cell suspension and cytofluorymetric evaluation on a FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Sterility of the material was confirmed via microbiological evaluation.

The first part of BMNCs, comprising 0.5 × 10⁹ cells, suspended in 5 ml of 5% glucose solution (FreseniusKabi, Kutno, Poland) was injected intrathecally. The second part, comprising the rest of the cells, was prepared in 30 ml of NaCl (B. Braun) for intravenous injection.

Isolation and Culture of the Mesenchymal Stem Cells

MSCs were isolated and cultured according to the protocol established by our group previously (26) with a slight improvement. Mononuclear cells (MNCs) were isolated from the BM sample using density-gradient centrifugation. The CD271⁺ population was isolated according to the manufacturer's instructions (Stem Cell Technologies, Vancouver, Canada). MNCs were incubated with antihuman CD32 blocker (25 μl per 1 ml of cells) and anti-CD271 monoclonal antibodies (50 μl per 1 ml of cells) labeling the LNGF receptor (or p75 NTR) (Stem Cell Technologies) for 15 min at room temperature; then nanoparticles (50 μl per 1 ml of cells) were added and incubated for another 15 min. Isolation was performed using a magnet. Isolated cells were plated into 75-cm² tissue culture flasks (Sarstedt Stare Babice, Poland) in Dulbecco's modified Eagle's medium (StemCell Technologies) supplemented with 10% fetal bovine serum (FBS) (Stem Cell Technologies), platelet-derived growth factor (PDGF) (PeproTech Inc., Rocky Hill, NJ, USA), and epidermal growth factor (EGF) (PeproTech). Flasks were incubated at 37°C in a humidified atmosphere containing 5% CO₂, and after 7 days, the medium was changed. At confluence, the adherent cells were detached using 0.25% trypsin (PAA Laboratories) and reseeded at 7.5 × 10⁴ cells/75 cm² flask and incubated again until confluence with a weekly medium change.

Phenotypic Evaluation of Cultured Mesenchymal Stem Cells

The MSC phenotype was analyzed with 20 μl of antibodies specific for MSCs (CD73, CD90, and CD105) and hematopoietic cells (CD45) (all antibodies from BD Biosciences, San Jose, CA, USA) per 100 μl of cell suspension, using a FACSCalibur cytometer with Cellquest software (BD Biosciences). Expanded MSCs were used for transplantation only if they were ≥95% positive for CD73, CD90, and CD105, and <3% positive for CD45.

Evaluation of the Cytogenetic Stability of Cultured Mesenchymal Stem Cells

Cytogenetic stability of cultured MSCs was confirmed via GTG banding. Metaphases were analyzed under an Olympus BX51 microscope with a camera (Olympus Corporation, Tokyo, Japan) to document photomicrographs. A Cytovision program (Leica Microsystem, Inc., Buffalo Grove, IL, USA) was used to arrange chromosomes into a karyogram.

Surgical Procedure and Cell Transplantation Procedure

The surgical procedure was performed by the team of specialists from the Department of Pediatric Neurosurgery, University Children's Hospital of Cracow. During the surgical procedure, the SC was exposed using a laminectomy, the injury was verified under a microscope, and the necrotic tissue was resected. BMNCs (0.5 × 10⁹) were administered to the SCI cavity and intravenously (3.22 × 10⁹) via cubital vein over 10 min using a drip infusion set (Table 1). The wound was closed in a typical manner, the meninx was closed with a watertight suture, and the resected lamella of the vertebral arch was restored. The patient was subsequently transplanted with five rounds of cultured MSCs (dose range: 13.0-36.5 × 10⁶) every 3-1 months via lumbar puncture (LP) into cerebral-spinal fluid (CSF) (Table 1). The patient underwent intensive neurorehabilitation after the BMNC transplantations.

Table 1.

Numbers of Injected Cells and Routes of Their Delivery; WBC and CRP Postinjection Data

	Number of Cells Administered Intrathecally			Number of Cells Administered Intravenously			Number of Cells Administered via Lumbar Puncture	Inflammatory Parameters After Each Cell Administration
Number of Implantations	BMNC (×l0⁹)	CD34⁺ (×10⁶)	CD133⁺ (×10⁶)	BMNC (×10⁹)	CD34⁺ (×10⁶)	CD133⁺ (×10⁶)	MSC (×10⁶)	WBC (10³/μl)	CRP (mg/ml)
1st	0.50	2.65	0.75	3.22	17.1	4.8	-	5.1	<5.0
2nd	-	-	-	-	-	-	13.0	4.7	<5.0
3rd	-	-	-	-	-	-	36.5	5.0	<5.0
4th	-	-	-	-	-	-	36.0	7.6	<5.0
5th	-	-	-	-	-	-	36.0	5.3	<5.0
6th	-	-	-	-	-	-	33.0	5.0	<5.0

Pretreatment and Follow-up Efficacy Assessment

Pretreatment and follow-up neurological examinations were performed according to the guidelines for the conduct of clinical trials for SCI developed by the ICCP panel (62). They included American Spinal Injury Association (ASIA) impairment scale and the Frankel grading system; sensory deficits were evaluated using dermatome sensory maps. Posttraumatic spasticity was assessed using the Ashworth scale, and bladder function evaluation was based on the patient's reports. Muscle strength was evaluated based on the Lovett scale (0-6 terms). Quality of live evaluation was assessed based on the functional recovery estimated using the SCIM (Spinal Cord Independence Measure) method. When the first improvement appeared, additional objective tests were included in the evaluation. EMG and ENG (Neurowerk Sigma MedizinTechnik, Gelenau, Germany) were used to test peripheral conductivity, and the motor-evoked potentials (EP) method was used to test the conductivity from the cerebral cortex and spine to the motor units and back. MRI (Siemens Healthcare Poland, Warszawa, Poland) was used to assess the structural changes in the SC.

Safety Evaluation Criteria

The safety criteria of the transplantation procedure included the following: infection, fever, headache, pain, increased level of C-reactive protein (CRP), increased leukocytosis, allergic reaction (presenting as shock), and perioperative complications (anesthesia and analgesia complications, infections of the wound). The safety criteria of the therapy included cancer development, deterioration of the neurological status, and development of neuropathic pain, secondary infections, urinary tract infections, and pressure ulcers.

Results

MSCs used for transplantations had fibroblast-like morphology (Fig. 1A) and expressed key MSC markers, according to the International Society for Cellular Therapy (16), CD73, CD90, and CD105 (Fig. 1B). They did not express hematopoietic marker CD45 (Fig. 1B). The cytogenetic analysis did not show any chromosomal aberrations (Fig. 1C).

Figure 1.

Characteristics and quality control of transplanted MSC cells. (A) Phase-contrast morphology. (B) Surface markers. (C) Karyotype.

There were neither early adverse events like infection, fever, pain, headache, increased level of CRP, increased leukocytosis, allergic reaction shock. nor other perioperative complications that could be related to the transplantation procedures (Tables 1 and 2). We did not observe any late adverse events related to therapy like aggravation of neurological status or cancer development observed during 2 years of treatment (Table 2).

Table 2.

Summary of Early and Late Complications Over 2-Year Follow-Up

Complications
	Early
Infection	-
Fever	-
Pain	-
Headache	-
Increased level of C reactive protein (CRP)	-
Increased leukocytosis	-
Allergic reaction - shock	-
Perioperative complications	-
	Late
Secondary infections	-
Urinary tract infections	-
Deterioration of neurosical status	-
Neuropathic pain	-
Carcinogenesis	-

Throughout the treatment, the patient demonstrated continuous improvement (Tables 3-5). Among the first signs of improvement, occurring 2-3 weeks after the first MSC administration, was a decrease in sensation level to Th5-6 and partial restoration of bladder sensation. Hip muscle strength slightly increased (1 to 1+), and quality of life increased to 50 on the SCIM scale. In addition, the patient gained the ability to control her trunk. The next improvement, which occurred after the second MSC administration, included an additional sensation level decrease to Th8, the ability to stand in a standing frame, further hip muscle strength increased (1+ to 3), and a quality of life improvement to 54. The ASIA score advanced from A to B/C. After the third MSC implantation, the sensation level decreased to L1, bladder control was partly restored, further hip muscle strength increased (2 to 3), and the quality of life increased to 61. The ASIA score changed to C, and the plegia improved to deep paresis. The patient gained the ability to walk supported by a hip orthesis. The fourth MSC administration produced a further sensation improvement to L2/3, bladder control was fully restored, and anal sensation developed. The patient's quality of life increased to 67. The fifth MSC administration resulted in further sensation improvement to L3/4 and a further hip muscle strength increase. The patient gained the ability to walk supported by a knee orthesis. The changes in ASIA, Frankel, sensation level, functional improvement, and conductivity are summarized in Table 3. The muscle strength improvement results are presented in Table 4, and life quality improvement changes are summarized in Table 5.

Table 3.

Change in ASIA, Frankel, Sensation Level, Functional Improvement, and Conductivity

		At Admission	BMNC Injection	I MSC Injection	II MSC Injection	III MSC Injection	IV MSC Injection	V MSC Injection
ASIA scale score		A	A	A	B/C	C	C/D	C/D
Motor points		50	50	50	53	55	57	59
Light touch points		32	32	52	68	77	80	87
Pin prick points		30	30	50	66	78	79	85
Frankel scale score		A	A	B	C	C/D	D	D
Sensation level		Thl	Thl	Th5-6	Th8	LI	L2-3	L3-4
Superficial sensation - evoked potentials		nd	nd	nd	C5-C7 normal Th8-Th9 significantly decreased	nd	Th9 decreased	Th10 normal Th11 no potential
Motor fiber activity - evoked potentials	Abdominal muscles	nd	nd	nd	Significantly reduced	nd	Reduced	Normal
	Lower extremities				Infinitesimal (left > right)		Infinitesimal (left > right)	Reduced (left > right)
Peripheral motor fiber conductivity - evoked potentials		nd	nd	nd	Reduced	nd	Slightly reduced	Normal
Peripheral motor fiber conductivity (abdominal roots) - evoked potentials		nd	nd	nd	L5-S1 preserved with minimal reduction	nd	L5-S1 preserved right side normal	L5-S1 preserved normal
Efferent conductivity	Trunk muscles	nd	nd	nd	Preserved	nd	Preserved	Normal
	Lower extremities				Observed, reduced only on the left side		Observed, slightly -reduced on both sides	Normal both sides
Bladder function/sensation		-/-	-/-	-/+	-/+	+/+	+/+	+/+
Anal function/sensation		-/-	-/-	-/-	-/-	-/-	-/+	-/+
Ability to control the trunk		-	-	+	+	+	+	+
Ability to stand in a standing frame		-	-	-	+	+	+	+
Ability to supported walk in hip orthesis		-	-	-	-	+	+	+
Ability to supported walk in knee orthesis		-	-	-	-	-	+	+

Evaluation was performed 3 months after each implantation. nd, not done.

Table 4.

Changes in Muscle Strength After MSC Implantations

	At Admission		BMNC Injection		I MSC Injection		II MSC Injection		III MSC Injection		IV MSC Injection		V MSC Injection
	R	L	R	L	R	L	R	L	R	L	R	L	R	L
Hips
Flexors	0	0	0	0	1	1+	1+	2	2	3	2	3	2	3
Extensors	0	0	0	0	1	1+	2	3	2	3	2	3	2	3
Abductors	0	0	0	0	1	1+	1+	2+	2	2+	2	2+	2+	3
Adductors	0	0	0	0	1	1+	1+	2+	2	2+	2	2+	2+	2+
Internal rotators	0	0	0	0	1+	1+	1+	3	2	3	2	3	3	3
External rotators	0	0	0	0	1	1	2	2+	2+	2+	2+	2+	2+	2+
Knee
Flexors	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Extensors	0	0	0	0	0	0	0	1	0	1	0	1	0	1
Ankle
Dorsiflexors	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Plantarflexors	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Eversion	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Inversion	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Toes
Flexors	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Extensors	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Evaluation was performed 3 months after each implantation.

Table 5.

Quality of Life Improvement Evaluated Using the SCIM Scale

		Quality of Life Improvement: SCIM Scale Appendix A
		At Admission	BMNC Injection	I MSC Injection	II MSC Injection	III MSC Injection	IV MSC Injection	V MSC Injection
Part 1	Self care	11	11	13	15	16	16	16
Part 2	Respiratory/sphincter	16	16	27	28	29	29	29
	Mobility (room toilet)	3	3	4	7	8	8	8
Part 3	Mobility (indoors/outdoors)	3	3	6	6	10	14	14

Evaluation was performed 3 months after each implantation.

MRI revealed that in the place of destroyed SC structures at level Th2/Th3, where the hemorrhagic necrosis was initially observed, small tissue structures appeared, which may represent a zone of regenerated neural tissue (Fig. 2). Additionally, near-normal appearance of neural tissue surrounding the degeneration zone at Th3/Th4 was detected by MRI (Fig. 2). The MRI signal in the injured area of the spine improved. EP evaluation of the superficial sensations demonstrated restoration of normal potentials down to Th10 and restoration of normal abdominal muscle activity, improved activity of the lower extremities, and restoration of normal conductivity of the tibialis anterior and peroneus longus nerves. Efferent conductivity from the cortex to the abdominal and lower extremity muscles was restored. EMG, ENG, and EP evaluations objectively confirmed trend of improvement.

Figure 2.

MRI evaluation of the SC structure. (A) At admission (coordinates ET: 21, TR: 3128, TE: 60, DFOV: 26 × 26cm, W: 2348, L: 1350). (B) After 2 years of treatment (coordinates ET: 27, TR: 2980, TE: 112.8, DOV: 36 × 36cm, W: 2582, L: 1290).

Discussion

In the past two decades, reports have demonstrated the safety and potential efficacy of cell therapy using cells from various adult tissues for SCI adult patients, including BM cells (4, 8, 10, 14, 18, 27, 33, 48, 63, 69), MSCs (3, 25, 28, 29, 35, 47, 49, 57), OECs (11, 24), olfactory mucosa (11, 34), olfactory bulb cells (65), Schwann cells (56, 71), or cell combinations (2, 39). More considerable improvements have been observed for acute patients, with multiple transplantations, or when treatment strategies combined different cell types.

Our therapy approach for a 15-year-old girl with complete SCI (verified by MRI and clinical examination) with vertebral column fracture at the level Th2/Th3 included single intravenous administration of BMNCs and multiple intrathecal MSC implantations.

The transplanted BMNCs and MSCs were obtained according to established protocols (26). Quality of MSC preparations was verified according to the International Society for Cellular Therapy (16). Microbiological and cytogenetic safety was always checked. The transplantation regimen was developed according to our previous studies (27). Safety and efficacy evaluation was performed following ASIA scale and the Frankel grading system, Ashworth scale and Lovett scale, SCIM method, EMG, ENG, MEP, SSEP, and MRI (2, 59).

Within 2 years of treatment, the ASIA score improved gradually from A to C/D (from 112 to 231 points), and it reflected the sensation level change from Th1 to L3-4 and the patient's ability to control the body trunk. The patient regained bladder control, and the muscle strength in lower extremities improved from plegia to deep paresis. Most importantly, the patient recovered some of the movement activities in lower parts of the body and gained the ability to stand in a standing frame and was able to walk with the support of hip and knee ortheses. Regeneration of neural tissue was demonstrated in macroscale by the only possible way: MRI evaluation. Although histological confirmation could not be performed, the electrophysiological results and clinical improvement stands for the MRI results.

The safety and feasibility of cell therapies have already been demonstrated through intrathecal and LP implantations in adult patients (8, 10, 11, 14, 18, 24, 25, 28, 29, 33 -35, 39, 47 -49, 53, 56, 57, 63, 65, 68, 69, 71) and multiple BMNC implantations in pediatric patients (26), with only minor side effects observed in some patients (30, 33, 48, 69). Given that our patient showed neither early nor late complications, our results confirm and expand existing data demonstrating the safety and feasibility of multiple, intrathecal MSC implantations for pediatric SCI patients.

In case of CNS injury, cells can be delivered intravenously or intrathecally. The intravenous delivery is easier and was used in one of the first clinical trials but resulted in inferior improvement compared to intrathecal delivery (63). The intravenous delivery limitation is necessity to migrate through the BBB to get to the CNS and possibility to get entrapped into a pulmonary network and lost in the lungs (9, 37, 46). The intrathecal delivery, technically more difficult, allows introducing cells directly into the cerebral fluid where they exert their therapeutic function and can actively migrate toward the inflammation side, further increasing the chance of positive influence. For the above reasons, most clinical studies conducted so far used intrathecal delivery. We assume that the use of the intrathecal route of delivery in our study likely contributed to an improved overall therapeutic effect.

Most published clinical studies used a single injection of cells either intravenously or intrathecally (4, 8, 10, 11, 14, 24, 28, 33 -35, 47 -49, 56, 57, 63, 68, 69, 71). Our approach included single BMNC and multiple MSC implantations every 3-4 months. This approach resulted in considerable, continuous neurological and quality of life improvement confirmed by objective MRI, EP, EMG, and ENG assessments. Interestingly, the recovery of bladder sensation and control indicates sequential regeneration of the ortho- and antidromic conductivity of the nerve pathways responsible for bladder innervation. Antidromic regeneration was observed first, and orthodromic regeneration was observed 3 months later. Our data may suggest that the denervation process of the lower extremities was stopped and reversed.

Optimally scheduled multiple injections could provide sustained delivery of pro-neurotrophic factors to the place of injury providing continuous stimulation and regeneration of the damaged tissue. The improvement after using the multiple injection therapy approach seems to be higher than in case of single injections for SCI treatment (18, 29, 39, 53, 65) and also for other diseases (22, 55).

However, contradictory results were also published from animal (3) and human (30) studies showing that multiple injections do not provide additional benefit over single implantation or can even worsen the clinical status of the patients (30). The differences observed might be due to the different cell types, transplantation schedule, and number of cells used in our study versus the other.

The multiple intrathecal injections could also lead to the activation of the immune system (5, 51, 52). However, we did not observe any immunological responses likely due to the use of autologous MSCs, which have well-established immunomodulatory potential (1, 40, 50) (Table 1).

According to our experience (we implanted cells intrathecally to the cerebrospinal fluid at the L4 level, low lumbar spine), there is also a minimal possibility of reinjuring the cord during MSC administration. No change to the inflammatory status of the patient after each injection, as measured by CRP level (Table 1) and lack of early side effects (Table 2), confirms the safety of our approach.

The use of MSCs in cell therapy in pediatric patients has never been reported. The obtained improvement is better than the best improvement that has been previously reported for freshly isolated BM cell-based (14, 18, 33, 48, 69) and MSC-based (6, 10) strategies for adults with complete SCI. Considerable improvement (ASIA A to D) was demonstrated using multiple implantations of cord blood CD34⁺ cells combined with Wharton's Jelly MSCs, but only for a patient with incomplete injury (25), and no improvement was reported for patients with complete injury (35). Therapeutic outcomes of our strategy have clear advantage over OEC (53, 64), Schwann cell (71), and olfactory mucosa autografts applied in adult patients (31) or recently published olfactory bulb cells (65). It cannot be ruled out that the younger age of the patient has favored better improvement. Some studies actually suggest that a younger age might be beneficial for the cell-based therapy of SCI (41). However, in our previous study when chronic pediatric SCI patients were transplanted with BMNCs, the younger age of the patient did not seem to correspond with better results (27). Thus, to address the age factor in SCI therapy, large clinical studies that directly compare younger and older patients are necessary.

Interestingly, our report, and most others, showed that the most effective treatments for patients with complete SCI are stem cell therapy strategy combined with either intense rehabilitation/neurorehabilitation alone (14, 18, 39, 53, 65) or combined with electrostimulation (29). Additionally, most of these strategies employed multiple implantations or multiple sites of implantation (18, 29, 39, 53, 65).

Thus, available data, including this report, seem to indicate the usefulness of multiple implantations combined with intense rehabilitation/neurorehabilitation to maximize the cell therapy treatment efficacy in SCI patients. We are aware, however, that our case study observations must be confirmed in a larger group of patients. Based on our experience, the future studies should also include more detailed observations of recovery using more sophisticated imaging systems, combined with a cell tracking. Moreover, cell dose escalation, not feasible in a single patient, should also be considered in the future studies. Nevertheless, we believe the multiple implantation approach is worth considering when designing various cell therapy treatment protocols for patients with complete SCI.

Conclusions

We report the safety and feasibility of multiple BMNC and MSC implantations and of the treatment strategy introduced herein in a pediatric patient. Our preliminary results demonstrate the possibility of obtaining considerable, continuous neurological and quality of life improvement in patients with subacute complete SCI. Despite these very promising results, we acknowledge that case reports have to be confirmed on a larger group of patients in randomized clinical trials.

Footnotes

Acknowledgments

This work was partially financed by Jagiellonian University Medical College Grant K/ZDS/004044, by Leading National Research Center (KNOW) scheme supported by the Ministry of Science and Higher Education and by the “Dar Nadziei ” Foundation. The authors declare no conflict of interest.

References

Abumaree

Al Jumah

Pace

R. A.

Kalionis

Immunosuppressive properties of mesenchymal stem cells. Stem Cell Rev. 8: 375–392; 2012.

Anderson

Aito

Atkins

Otr

Biering-Sørensen

Charlifue

Curt

Ditunno

Glass

Marino

Marshall

Mulcahey

M. J.

Post

Savic

Scivoletto

Catz

Functional Recovery Outcome Measures Work Group. Functional recovery measures for spinal cord injury: An evidence-based review for clinical practice and research. J. Spinal Cord Med. 31: 133–144; 2008.

Ankeny

D. P.

McTigue

D. M.

Jakeman

L. B.

Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats. Exp. Neurol. 190: 17–31; 2004.

Attar

Ayten

Ozdemir

Ozgencil

Bozkurt

Kaptanoglu

Beksac

Kanpolat

An attempt to treat patients who have injured spinal cords with intralesional implantation of concentrated autologous bone marrow cells. Cytotherapy 13: 54–60; 2011.

Beggs

K. J.

Lyubimov

Borneman

J. N.

Bartholomew

Moseley

Dodds

Archambault

M. P.

Smith

A. K.

McIntosh

K. R.

Immunologic consequences of multiple, high-dose administration of allogeneic mesenchymal stem cells to baboons. Cell Transplant. 15: 711–721; 2006.

Bobis

Jarocha

Majka

Mesenchymal stem cells: Characteristics and clinical applications. Folia Histochem. Cytobiol. 44: 215–230; 2006.

Bradbury

E. J.

Moon

L. D. F.

Popat

R. J.

King

V. R.

Bennett

G. S.

Patel

P. N.

Fawcett

J. W.

McMahon

S. B.

Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416: 636–640; 2002.

Callera

Do Nascimento

R. X.

Delivery of autologous bone marrow precursor cells into the spinal cord via lumbar puncture technique in patients with spinal cord injury: A preliminary safety study. Exp. Hematol. 34: 130–131; 2006.

Chen

B. K.

Staff

N. P.

Knight

A. M.

Nesbitt

J. J.

Butler

G. W.

Padley

D. J.

Parisi

J. E.

Dietz

A. B.

Windebank

A. J.

A safety study on intrathecal delivery of autologous mesenchymal stromal cells in rabbits supporting Phase I human trials. Transfusion doi: 10.1111/trf.12938. 2014 [Epub ahead of print].

10.

Chernykh

E. R.

Stupak

V. V.

Muradov

G. M.

Sizikov

M. Y.

Shevela

E. Y.

Leplina

O. Y.

Tikhonova

M. A.

Kulagin

A. D.

Lisukov

I. A.

Ostanin

A. A.

Kozlov

V. A.

Application of autologous bone marrow stem cells in the therapy of spinal cord injury patients. Bull. Exp. Biol. Med. 143: 543–547; 2007.

11.

Chhabra

H. S.

Lima

Sachdeva

Mittal

Nigam

Chaturvedi

Arora

Aggarwal

Kapur

Khan

T. A.

Autologous olfactory [corrected] mucosal transplant in chronic spinal cord injury: An Indian Pilot Study. Spinal Cord 47: 887–895; 2009.

12.

Chopp

Zhang

X. H.

Wang

Chen

Rosenblum

Spinal cord injury in rat: Treatment with bone marrow stromal cell transplantation. Neuroreport 11: 3001–3005; 2000.

13.

Civin

C. I.

Strauss

L. C.

Brovall

Fackler

M. J. O.

Schwartz

J. F.

Shaper

J. H.

Antigenic analysis of hematopoiesis iii. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J. Immunol. 133: 157–165; 1984.

14.

Deda

Inci

M. C.

Kürekçi

A. E.

Kayihan

Ozgün

Ustünsoy

G. E.

Kocabay

Treatment of chronic spinal cord injured patients with autologous bone marrow-derived hematopoietic stem cell transplantation: 1-year follow-up. Cytotherapy 10: 565–574; 2008.

15.

Devivo

M. J.

Epidemiology of traumatic spinal cord injury: Trends and future implications. Spinal Cord 50: 365–372; 2012.

16.

Dominici

Le Blanc

Mueller

Slaper-Cortenbach

Marini

Krause

Deans

Keating

Prockop

D. J.

Horwitz

Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8: 315–317; 2006.

17.

Freund

Wannier

Schmidlin

Bloch

Mir

Schwab

M. E.

Rouiller

E. M.

Anti-Nogo-A antibody treatment enhances sprouting of corticospinal axons rostral to a unilateral cervical spinal cord lesion in adult macaque monkey. J. Comp. Neurol. 659: 644–659; 2007.

18.

Geffner

L. F.

Santacruz

Izurieta

Flor

Maldonado

Auad

A. H.

Montenegro

Gonzalez

Silva

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. 17: 1277–1293; 2008.

19.

Gratama

J. W.

Kraan

Keeney

Sutherland

D. R.

Granger

Barnett

Validation of the single-platform ISHAGE method for CD34(+) hematopoietic stem and progenitor cell enumeration in an international multicenter study. Cytotherapy 5: 55–65; 2003.

20.

Grill

Murai

Blesch

Gage

F. H.

Tuszynski

M. H.

Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury. J. Neurosci. 17: 5560–5572; 1997.

21.

Gupta

Henry

R. G.

Strober

Kang

S. M.

Lim

D. A.

Bucci

Caverzasi

Gaetano

Mandelli

M. L.

Ryan

Perry

Farrell

Jeremy

R. J.

Ulman

Huhn

S. L.

Barkovich

A. J.

Rowitch

D. H.

Neural stem cell engraftment and myelination in the human brain. Sci. Transl. Med. 4: 155ra137; 2012.

22.

J. H.

Tseng

T. C.

W. H.

Ong

W. K.

Chen

Y. F.

Chen

M. H.

Lin

M. W.

Hong

C. Y.

Lee

O. K.

Multiple intravenous transplantations of mesenchymal stem cells effectively restore long-term blood glucose homeostasis by hepatic engraftment and β-cell differentiation in streptozocininduced diabetic mice. Cell Transplant. 21: 997–1009; 2012.

23.

HolIis

E. R.

2nd Lu

Blesch

Tuszynski

M. H.

IGF-I gene delivery promotes corticospinal neuronal survival but not regeneration after adult CNS injury. Exp. Neurol. 215: 53–59; 2009.

24.

Huang

Chen

Zhang

Liu

Long-term outcome of olfactory ensheathing cell therapy for patients with complete chronic spinal cord injury. Cell Transplant. 21: S23–S31; 2012.

25.

Ichim

T. E.

Solano

Lara

Paris

Ugalde

Rodriguez

J. P.

Minev

Bogin

Ramos

Woods

E. J.

Murphy

M. P.

Patel

A. N.

Harman

R. J.

Riordan

N. H.

Feasibility of combination allogeneic stem cell therapy for spinal cord injury: A case report. Int. Arch. Med. 3: 1–10; 2010.

26.

Jarocha

Lukasiewicz

Majka

Advantage of mesenchymal stem cells (MSC) expansion directly from purified bone marrow CD105+ and CD271+ cells. Folia Histochem. Cytobiol. 46: 307–314; 2008.

27.

Jarocha

Milczarek

Kawecki

Wendrychowicz

Kwiatkowski

Majka

Preliminary study of autologous bone marrow nucleated cells transplantation in children with spinal cord injury. Stem Cells Transl. Med. 3: 395–104; 2014.

28.

Kang

K. S.

Kim

S. W.

Y. H.

J. W.

Kim

K. Y.

Park

H. K.

Song

C. H.

Han

A 37-year-old spinal cord-injured female patient, transplanted of multipotent stem cells from human UC blood, with improved sensory perception and mobility, both functionally and morphologically: A case study. Cytotherapy 7: 368–373; 2005.

29.

Karamouzian

Nematollahi-Mahani

S. N.

Nakhaee

Eskandary

Clinical safety and primary efficacy of bone marrow mesenchymal cell transplantation in subacute spinal cord injured patients. Clin. Neurol. Neurosurg. 114: 935–939; 2012.

30.

Kishk

N. A.

Gabr

Hamdy

Afifi

Abokresha

Mahmoud

Wafaie

Bilal

Case control series of intrathecal autologous bone marrow mesenchymal stem cell therapy for chronic spinal cord injury. Pulmonary network entrapment. Neurorehabil. Neural Repair 24: 702–708; 2010.

31.

Kiyama

Radtke

Honmou

Kocsis

J. D.

Remyelination of the spinal cord following intravenous delivery of bone marrow cells. Glia 39: 229–236; 2002.

32.

Kucia

Zhang

Y. P.

Reca

Wysoczynski

Machalinski

Majka

Ildstad

S. T.

Ratajczak

Shields

C. B.

Ratajczak

M. Z.

Cells enriched in markers of neural tissue-committed stem cells reside in the bone marrow and are mobilized into the peripheral blood following stroke. Leukemia 20: 18–28; 2006.

33.

Kumar

A. A.

Kumar

S. R.

Narayanan

Arul

Baskaran

Autologous bone marrow derived mononuclear cell therapy for spinal cord injury: A phase I/II clinical safety and primary efficacy data. Exp. Clin. Transplant. 7: 241–248; 2009.

34.

Lima

Escada

Pratas-Vital

Branco

Arcangeli

C. A.

Lazzeri

Maia

C. A.

Capucho

Hasse-Ferreira

Peduzzi

J. D.

Olfactory mucosal autografts and rehabilitation for chronic traumatic spinal cord injury. Neurorehabil. Neural Repair 24: 10–22; 2010.

35.

Liu

Han

Wang

Xue

Zhu

Yan

Zheng

Guo

Wang

Clinical analysis of the treatment of spinal cord injury with umbilical cord mesenchymal stem cells. Cytotherapy 15: 185–191; 2013.

36.

Majka

Janowska-Wieczorek

Ratajczak

Ehrenman

Pietrzkowski

Kowalska

M. A.

Gewirtz

A. M.

Emerson

S. G.

Ratajczak

M. Z.

Numerous growth factors, cytokines, and chemokines are secreted by human CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner. Blood 97: 3075–3085; 2001.

37.

Mäkelä

Takalo

Arvola

Haapanen

Yannopoulos

Blanco

Ahvenjärvi

Kiviluoma

Kerkelä

Nystedt

Juvonen

Lehenkari

Safety and biodistribution study of bone marrow-derived mesenchymal stromal cells and mononuclear cells and the impact of the administration route in an intact porcine model. Cytotherapy S1465-3249(14)00884–6; 2015.

38.

McKinley

W. O.

Jackson

A. B.

Cardenas

D. D.

DeVivo

M. J.

Long-term medical complications after traumatic spinal cord injury: A regional model systems analysis. Arch. Phys. Med. Rehabil. 80: 1402–1410; 1999.

39.

Moviglia

G. A.

Fernandez Viña

Brizuela

J. A.

Saslavsky

Vrsalovic

Varela

Bastos

Farina

Etchegaray

Barbieri

Martinez

Picasso

Schmidt

Brizuela

Gaeta

C. A.

Costanzo

MovigliaBrandolino

M. T.

Merino

Pes

M. E.

Veloso

M. J.

Rugilo

Tamer

Shuster

G. S.

Combined protocol of cell therapy for chronic spinal cord injury. Report on the electrical and functional recovery of two patients. Cytotherapy 8: 202–209; 2006.

40.

Nakajima

Uchida

Guerrero

A. R.

Watanabe

Sugita

Takeura

Yoshida

Long

Wright

K. T.

Johnson

W. E.

Baba

Transplantation of mesenchymal stem cells promotes an alternative pathway of macrophage activation and functional recovery after spinal cord injury. J. Neurotrauma 29: 1614–1625; 2012.

41.

Neuhuber

Himes

T. B.

Shumsky

J. S.

Gallo

Fischer

Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations. Brain Res. 1035: 73–85; 2005.

42.

Neumann

Bradke

Tessier-Lavigne

Basbaum

A. I.

Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation. Neuron 34: 885–893; 2002.

43.

Neumann

Woolf

C. J.

Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron 23: 83–91; 1999.

44.

Norrbrink

Löfgren

Hunter

J. P.

Ellis

Patients' perspectives on pain. Top Spinal Cord Inj. Rehabil. 18: 50–56; 2012.

45.

Novikova

L. N.

Novikov

L. N.

Kellerth

J. O.

Biopolymers and biodegradable smart implants for tissue regeneration after spinal cord injury. Curr. Opin. Neurol. 16: 711–715; 2003.

46.

Nystedt

Anderson

Tikkanen

Pietilä

Hirvonen

Takalo

Heiskanen

Satomaa

Natunen

Lehtonen

Hakkarainen

Korhonen

Laitinen

Valmu

Lehenkari

Cell surface structures influence lung clearance rate of systemically infused mesenchymal stromal cells. Stem Cells 31: 317–326; 2013.

47.

Pal

Venkataramana

N. K.

Bansal

Balaraju

Jan

Chandra

Dixit

Rauthan

Murgod

Totey

Ex vivo-expanded autologous bone marrow-derived mesenchymal stromal cells in human spinal cord injury/paraplegia: A pilot clinical study. Cytotherapy 11: 897–911; 2009.

48.

Park

H. C.

Shim

Y. S.

Yoon

S. H.

Park

S. R.

Choi

B. H.

Park

H. S.

Treatment of complete spinal cord injury patients by autologous bone marrow cell transplantation and administration of granulocyte-macrophage colony stimulating factor. Tissue Eng. 11: 913–922; 2005.

49.

Park

J. H.

Kim

D. Y.

Sung

I. Y.

Choi

G. H.

Jeon

M. H.

Kim

K. K.

Jeon

S. R.

Long-term results of spinal cord injury therapy using mesenchymal stem cells derived from bone marrow in humans. Neurosurgery 70: 1238–1247; discussion 1247; 2012.

50.

Payne

N. L.

Sun

McDonald

Layton

Moussa

Emerson-Webber

Veron

Siatskas

Herszfeld

Price

Bernard

C. C.

Distinct immunomodulatory and migratory mechanisms underpin the therapeutic potential of human mesenchymal stem cells in autoimmune demyelination. Cell Transplant. 22: 1409–1425; 2013.

51.

Poh

K. K.

Sperry

Young

R. G.

Freyman

Barringhaus

K. G.

Thompson

C. A.

Repeated direct endomyocardial transplantation of allogeneic mesenchymal stem cells: Safety of a high dose, “off-the- shelf,” cellular cardiomyoplasty strategy. Int. J. Cardiol. 117: 360–364; 2007.

52.

Qiu

Cai

Dai

Mcatee

Hoffman

P. N.

Bregman

B. S.

Filbin

M. T.

Spinal axon regeneration induced by elevation of cyclic AMP. Neuron. 34: 895–903; 2002.

53.

Rao

Zhu

Liu

Jia

Zhao

Wang

Clinical application of olfactory ensheathing cells in the treatment of spinal cord injury. J. Int. Med. Res. 41: 473–481; 2013.

54.

Rekand

Hagen

E. M.

Grønning

Spasticity following spinal cord injury. Tidsskr. Nor. Legeforen. 132: 970–973; 2012.

55.

Richardson

J. D.

Psaltis

P. J.

Frost

Paton

Carbone

Bertaso

A. G.

Nelson

A. J.

Wong

D. T.

Worthley

M. I.

Gronthos

Zannettino

A. C.

Worthley

S. G.

Incremental benefits of repeated mesenchymal stromal cell administration compared with solitary intervention after myocardial infarction. Cytotherapy 16: 460–470; 2014.

56.

Saberi

Firouzi

Habibi

Moshayedi

Aghayan

H. R.

Arjmand

Hosseini

Razavi

H. E.

Yekaninejad

M. S.

Safety of intramedullary Schwann cell transplantation for postrehabilitation spinal cord injuries: 2-year follow-up of 33 cases. J. Neurosurg. Spine 15: 515–525; 2011.

57.

Saito

Nakatani

Iwase

Maeda

Hirakawa

Murao

Suzuki

Onodera

Fukushima

Ide

Spinal cord injury treatment with intrathecal autologous bone marrow stromal cell transplantation: The first clinical trial case report. J. Trauma 64: 53–59; 2008.

58.

Sasaki

Honmou

Akiyama

Uede

Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons. Glia 35: 26–34; 2008.

59.

Selden

N. R.

Al-Uzri

Huhn

S. L.

Koch

T. K.

Sikora

D. M.

Nguyen-Driver

M. D.

Guillaume

D. J.

Koh

J. L.

Gultekin

S. H.

Anderson

J. C.

Vogel

Sutcliffe

T. L.

Jacobs

Steiner

R. D.

Central nervous system stem cell transplantation for children with neuronal ceroid lipofuscinosis. J. Neurosurg. Pediatr. 11: 643–652; 2013.

60.

Shen

L. H.

Chen

Zhang

Vanguri

Borneman

Chopp

Intracarotid transplantation of bone marrow stromal cells increases axon-myelin remodeling after stroke. Neuroscience 137: 393–399; 2006.

61.

Shyu

W. C.

Lin

S. Z.

Chiang

M. F.

C. Y.

Intracerebral peripheral blood stem cell (CD34+) implantation induces neuroplasticity by enhancing beta1 integrinmediated angiogenesis in chronic stroke rats. J. Neurosci. 26: 3444–3453; 2006.

62.

Steeves

J. D.

Lammertse

Curt

Fawcett

J. W.

Tuszynski

M. H.

Ditunno

J. F.

Ellaway

P. H.

Fehlings

M. G.

Guest

J. D.

Kleitman

Bartlett

P. F.

Blight

A. R.

Dietz

Dobkin

B. H.

Grossman

Short

Nakamura

Coleman

W. P.

Gaviria

Privat

Guidelines for the conduct of clinical trials for spinal cord injury (SCI) as developed by the ICCP panel: Clinical trial outcome measures. Spinal Cord 45, 206–221; 2007.

63.

Syková

Homola

Mazanec

Lachmann

Konrádová

S. L.

Kobylka

Pádr

Neuwirth

Komrska

Vávra

Stulík

Bojar

Autologous bone marrow transplantation in patients with subacute and chronic spinal cord injury. Cell Transplant. 15: 675–687; 2006.

64.

Tabakow

Jarmundowicz

Czapiga

Fortuna

Miedzybrodzki

Czyz

Huber

Szarek

Okurowski

Szewczyk

Gorski

Raisman

Transplantation of autologous olfactory ensheathing cells in complete human spinal cord injury. Cell Transplant. 22: 1591–1612; 2013.

65.

Tabakow

Raisman

Fortuna

Czyz

Huber

Szewczyk

Okurowski

Miedzybrodzki

Czapiga

Salomon

Halon

Lipiec

Kulczyk

Jarmundowicz

Functional regeneration of supraspinal connections in a patient with transected spinal cord following transplantation of bulbar olfactory ensheathing cells with peripheral nerve bridging. Cell Transplant. 23: 1631–1655; 2014.

66.

Taguchi

Soma

Tanaka

Kanda

Nishimura

Yoshikawa

Tsukamoto

Iso

Fujimori

Stern

D. M.

Naritomi

Matsuyama T. Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model. J. Clin. Invest. 114: 330–338; 2004.

67.

Thuret

Moon

L. D.

Gage

F. H.

Therapeutic interventions after spinal cord injury. Nat. Rev. Neurosci. 7: 628–643; 2006.

68.

Yazdani

S. O.

Hafizi

Zali

A. R.

Atashi

Ashrafi

Seddighi

A. S.

Soleimani

Safety and possible outcome assessment of autologous Schwann cell and bone marrow mesenchymal stromal cell co-transplantation for treatment of patients with chronic spinal cord injury. Cytotherapy 15: 782–791; 2013.

69.

Yoon

S. H.

Shim

Y. S.

Park

Y. H.

Chung

J. K.

Nam

J. H.

Kim

M. O.

Park

H. C.

Park

S. R.

Min

B. H.

Kim

E. Y.

Choi

B. H.

Park

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 25: 2066–2073; 2007.

70.

Zhao

L. R.

Duan

W. M.

Reyes

Keene

C. D.

Verfaillie

C. M.

Low

W. C.

Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting in the ischemic brain of rats. Exp. Neurol. 174: 11–20; 2002.

71.

Zhou

X. H.

Ning

G. Z.

Feng

S. Q.

Kong

X. H.

Chen

J. T.

Zheng

Y. F.

Ban

D. X.

Liu

Wang

Transplantation of autologous activated Schwann cells in the treatment of spinal cord injury: Six cases, more than five years of follow-up. Cell Transplant. 21: 39–18, 2012.

Continuous Improvement after Multiple Mesenchymal Stem Cell Transplantations in a Patient with Complete Spinal Cord Injury

Abstract

Keywords

Introduction

Materials and Methods

Patient

Bone Marrow Aspiration

Bone Marrow Nuclear Cell Isolation, Evaluation, and Preparation for Transplantation

Isolation and Culture of the Mesenchymal Stem Cells

Phenotypic Evaluation of Cultured Mesenchymal Stem Cells

Evaluation of the Cytogenetic Stability of Cultured Mesenchymal Stem Cells

Surgical Procedure and Cell Transplantation Procedure

Pretreatment and Follow-up Efficacy Assessment

Safety Evaluation Criteria

Results

Discussion

Conclusions

Footnotes

Acknowledgments

References