INTRODUCTION
Similar to the percentage observed in bone marrow, about 2% the human umbilical cord blood cells (HUCBCs) are positive selection of CD34+-expressing cells (1-4). Human stem cells derived from the umbilical cord are one of strong candidates used in cell therapy for spinal cord injury (SCI) because of its greater availability, weak immunogenicity, and lower risk of mediating viral transmission (5). For example, intraspinal transplantation of CD34+ HUCBCs after spinal cord hemisection (6) or contusion (7) injury improved hindlimb functional recovery in adult rats. Saporta et al. (8) reported that i.v. infusion of unfractionated HUCBCs improved hindlimb function in a rat model of spinal cord compression. However, it remains to be elucidated what factors or mediators secreted from HUCBCs are crucial for restoration of the injured spinal cord.
Glial cell line-derived neurotrophic factor (GDNF), a potent survival factor for motor neurons in vitro and in vivo (9, 10), has been shown to promote axonal growth and cellular protection in injured adult motor neurons (11). Neural stem cells give rise to the differentiated progeny that constitute the neurons, astrocytes, and oligodendrocytes. Neural stem cells, which spontaneously express GDNF, have been shown to provide neuroprotection in a mouse model of Parkinson disease (12, 13). In addition, it has also been demonstrated that neural stem cells protect against glutamate-induced excitotoxicity and promote survival of injured motor neurons through the secretion of GDNF (14). In a rodent model of acute stroke, human umbilical cord blood mannitol treatment significantly increased brain levels of neurotrophic factors that correlated positively with reduced cerebral infarcts and improved behavioral functions (15). In addition, mediators produced by CD34+ cells (16), such as vascular endothelial growth factor (VEGF), have been shown to accelerate endogenous neurogenesis (17).
In this study, we report that systemic administration of 95% pure CD34+ progenitor cells derived from HUCBC is able to attenuate spinal cord infarction and apoptosis (18) and behavioral deficits in a standard rat compression SCI model. In addition, current investigation provides the new evidence to suggest that CD34+ cell therapy may cause restoration of spinal cord function during SCI by stimulating both GDNF and VEGF production in injured spinal cord.
MATERIALS AND METHODS
Animals
Adult male Sprague-Dawley rats (weight, 278 ± 14 g) were obtained from the Animal Resource Center of the National Science Council of the Republic of China (Taipei, Taiwan). The animals were housed in groups of four at an ambient temperature of 22°C ± 1°C, with a 12-h light-dark cycle. Pellet rat chow and tap water were available ad libitum. All protocols were approved by the Animal Ethics Committee of the Chi Mei Medical Center (Tainan, Taiwan) in accordance with the Guide for the Care Use of Laboratory Animals of the National Institutes of Health and the Guidelines of the Animal Welfare Act.
Surgery
A laminectomy, with removal of vertebral peduncle, was performed at T8 or T9 on rats anesthetized with sodium pentobarbital (25 mg/kg, intraperitoneally; Sigma Chemical Co, St Louis, Mo) and a mixture containing ketamine (44 mg/kg, i.m.; Nankang Pharmaceutical, Taipei, Taiwan), atropine (0.02633 mg/kg, i.m.; Sintong Chemical Industrial Co, Taoyuan, Taiwan), and xylazine (6.77 mg/kg, i.m.; Bayer, Leverkusen, Germany). The jaws of a calibrated aneurysm clip with a closing pressure of 55 g were placed between the dorsal and ventral surfaces of the spinal cord and left in place for 1 min (19). The sham-operated controlled animals received the same laminectomy, but were not compressed by placing the clip. All animals were given 0.1 mL of Baytril (Bayer) antibiotic for 3 days after surgery. Animals with SCI were individually housed on special bedding to prevent skin breakdown, and had bowel and bladder manual expressed twice daily. Food and water were freely accessible at a lowered height in their cages.
Human CD34+ cell preparation
Human CD34+ cells were isolated from cord blood using a Direct CD34 Progenitor Cell Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD34 Multisort kit (Miltenyi Biotec) according to the manufacturer's protocol. Immediately after SCI, human CD34+ cells (defined by 5 × 105 human cord blood lymphocytes and monocytes that contained ∼95% CD34+ cells) isolated from human cord blood or CD34− cells (defined by 5 × 105 human cord blood lymphocytes and monocytes that contained <0.2% CD34+ cells) were administered i.v. via the tail vein. The supernatants obtained about 1 h after incubating of CD34+ cells (5 × 105) with phosphate-buffered saline (PBS) by centrifugation were used as the effluent of CD34+ cells.
Experimental groups
Animals were assigned randomly to one of the following four major groups. One group of rats were treated with laminectomy at T8 or T9 and followed immediately by i.v. infusion of CD34− cells or isotonic sodium chloride solution (0.3 mL) per rat. The second group of animals were treated with laminectomy at T8 or T9 and followed immediately by an i.v. infusion of human CD34+ cells isolated from HUCBCs. The third group of animals was used as sham-operated controls. In the fourth group, the spinal cord-injured rats were treated with an i.v. dose of 0.3 mL effluent of CD34+ cells (5 × 105) per rat.
In experiment 1, an i.v. dose of CD34+, CD34− cells (5 × 105), or saline was randomly administered immediately after SCI (n = 32), and their effects on the maximal angle animals could cling to an inclined plane were assessed 1 to 7 days after SCI.
In experiment 2, an i.v. dose of CD34+, CD34− cells (5 × 105), or saline was randomly administered immediately after SCI, and their effects on spinal cord infarction zone were assessed 1 to 7 days after SCI (n = 96).
In experiment 3, an i.v. dose of isotonic sodium chloride solution (0.3 mL), CD34+ cells (5 × 105 in 0.3 mL), or CD34− cells (5 × 105 in 0.3 mL) was randomly administered immediately after SCI, and their effects on the amounts of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive cells in the injured spinal cord were assessed 7 days after SCI (n = 32).
In experiment 4, an i.v. dose of isotonic sodium chloride solution (0.3 mL), CD34+ cells (5 × 105 in 0.3 mL), or CD34− cells (5 × 105 in 0.3 mL) was randomly administered immediately after SCI, and their effects on the amounts of VEGF-positive cells and GDNF concentration in the injured spinal cord were assessed 7 days after SCI (n = 32).
In experiment 5, an i.v. dose of isotonic sodium chloride solution (0.3 mL), CD34+ cells (5 × 105 in 0.3 mL), or CD34− cells (5 × 105 in 0.3 mL) was randomly administered immediately after SCI, and their effects on the expression of human nuclei antibody immunoreactivity in the spinal cord of spinal cord-injured animals (n = 18) were assessed 7 days after SCI.
Inclined plane
The inclined plane was used to measure limb strength. Animals were placed, facing right and then left, perpendicular to the slope of a 20 × 20 cm ruff-ribbed surface of an inclined plane starting at an angle of 55 degrees (20). The angle was increased or decreased in 5°C increments to determine the maximal angle an animal could hold to the plane. Data for each day were the mean of left and right side maximal angles. All behavioral tests were examined and independently scored by two observers who were unaware of prior treatment. These scores were averaged to arrive at one score for each animal for the behavioral session.
Spinal infarction assay
The triphenyltetrazolium chloride (TTC) staining procedures followed those described elsewhere (21). All animals were killed at day 7 after SCI. Under deep anesthesia (sodium pentobarbital, 100 mg/kg, intraperitoneally), animals were perfused intracardially with saline. The spinal cord tissue was then removed, immersed in cold saline for 5 min, and sliced into 2.0-mm sections. The spinal cord slices were incubated in 2% TTC dissolved in PBS for 30 min at 37°C, and then transferred to 5% formaldehyde solution for fixation. The volume of infarction, as revealed by negative TTC stains indicating dehydrogenase-deficient tissue, was measured in each slice and summed using computerized planimetry (PC-based Image Tools Software). The volume of infarction was calculated as 2 mm (thickness of the slice) × (sum of the infarction area in all spinal cord slices [mm2]) (21).
TUNEL assay
The TUNEL assay was performed using the same spinal cord tissues used in histological verification. Color was developed using 3,3′-diaminobenzidine tetrachloride (Sigma Chemical Co). Sections were treated with xylene and ethanol to remove paraffin and for dehydration. They were then washed with PBS and incubated in 3% hydrogen peroxide solution for 20 min. The sections were treated with 5 μg/mL proteinase K for 2 min at room temperature, and rewashed with PBS (0.1 M, pH 7.4, PBS). The sections were then treated with a TUNEL reaction mixture (terminal deoxynucleotidyl transferase, nucleotide mixture, Roche, Mannhiem, Germany) at 37°C for 1 h, and then the sections were washed with distilled water. They were then reincubated in antifluorescein antibody-conjugated with horseradish peroxidase at room temperature for 30 min, rewashed, and then visualized using the ABC technique and 0.05% 3,3′-diaminobenzidine tetrachloride as a chromogen. The numbers of TUNEL-positive cells were counted by a pathologist at ×200 magnification, 30 fields per section. Blinding was performed for the pathologist's grading of results.
Immunohistochemistry
Autofluorescence was first quenched using the method of Vendrame et al. (22), after which the spinal cord sections were incubated with mouse monoclonal antibody against human nuclei (HuNu, Chemicon Inc, Pittsburgh, Pa), followed by fluorescein isothiocyanate-conjugated goat antimouse secondary antibody (Annexin Alexa Molecular Probe). 4′, 6-Diamidino-2-phenylindole (DAPI) staining (Molecular Probes, Eugene, Ore) was performed to visualize nucleated cells. Slides were examined under epifluorescence on an Olympus BX60 microscope. For determination of VEGF expression at the level of T8 to T9 sections, the sections were incubated with PBS containing anti-VEGF mouse antibody in 1:200 dilution and then detected with Alexa-Fluor 568 goat antimouse (IgG) antibody.
Enzyme-linked immunosorbent assay
Supernatants were collected from the tissue homogenates of spinal cord, and enzyme-linked immunosorbent assay (ELISA) measurements were done with the Promega GDNF Immunoassay System following the recommendations of the manufacturer (Promega, Madison, Wis). A standard curve of pure GDNF protein provided in the kit was used to quantify the production of the neurotrophic factors by CD34+ cell therapy. The ELISA kit used is specific for human GDNF. The amounts of GDNF in the supernatants obtained approximately 1 h after incubating of CD34+ cells (5 × 105) with PBS were found to be negligible.
Statistical analysis
Data are presented as the mean ± SEM. Repeated-measures analysis of variance (ANOVA) was conducted to test the treatment-by-time interactions and the effect of treatment over time on each score. The Duncan multiple range test was used for post hoc multiple comparisons among means. P < 0.05 was considered evidence of statistical significance.
RESULTS
CD34+ cell grafts improve motor performance
Behavioral tests of motor function were conducted at days 1, 4, and 7 after SCI to determine whether CD34+ cell therapy adopted immediately after SCI would produce immediate and robust effects. As shown in Figure 1, although CD34− cells or saline therapy was ineffective on all tests at these time points, the SCI-induced motor deficits, measured by maximal angle an animal could hold to the inclined plane, were completely abated at days 4 to 7 after SCI by an i.v. dose of CD34+ cells administered immediately after SCI (Fig. 1).
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Fig. 1: Maximal angle animals could cling to an inclined plane. *Maximum grip angle 1 to 7 days after SCI was significantly (P < 0.05) decreased for spinal cord-injured animals treated with i.v. saline (▤) or CD34− cells (5 × 105 per 0.3 mL) (◩) compared with sham controls (□). †Maximum grip angle 4 to 7 days after SCI was significantly (P < 0.05) increased for spinal cord-injured animals treated with i.v. CD34+ cells (5 × 105 per 0.3 mL) (▩) compared with CD34− cells-treated spinal cord-injured animals (◩) (ANOVA followed by Duncan test). The bars represented means ±SEM of eight rats per group.
CD34+ cells grafts reduce spinal cord infarcts and apoptosis
Here we conducted TTC and TUNEL stainings at days 1 to 7 after SCI. Data revealed that i.v. CD34+ cells, but not CD34− cells or saline, significantly limited the spinal cord infarct (Fig. 2) 4 to 7 days after SCI. Again, apoptosis, evidenced by increasing numbers of TUNEL-positive cells, was significantly reduced by CD34+ cells, but not CD34− cells or saline therapy, when evaluated 7 days after SCI (Figs. 3 and 4).
Fig. 2: Spinal cord infarct zone. *Percentage of area at risk 7 days after SCI was significantly (P < 0.05) increased for spinal cord-injured animals treated with i.v. saline (0.3 mL) (▤) or CD34− cells (5 × 105 per 0.3 mL) (◩) compared with sham controls (□). †Percentage of area at risk 7 days after SCI was significantly (P < 0.05) decreased for spinal cord-injured animals treated with i.v. CD34+ cells (5 × 105 per 0.3 mL) (▩) compared with CD34− cells-treated spinal cord-injured animals (◩). The bars represented means ± SEM of eight rats per group. The volume of infarction, as revealed by negative TTC stains indicating dehydrogenase-deficient tissue, of a spinal cord section from a spinal cord-injured rat treated with CD34− cells is depicted in the top panel.
Fig. 3: The TUNEL staining of spinal cord 7 days after SCI was greatly increased for spinal cord-injured animals treated with i.v. saline (SCI + saline) or CD34− cells (SCI + CD34− cells), which would be reversed by treating with i.v. CD34+ cells (SCI + CD34+ cells).
Fig. 4: *Numbers of the TUNEL-positive cells in spinal cord section 7 days after SCI was significantly ( P < 0.05) increased for spinal cord-injured animals treated with i.v. saline (0.3 mL) (▤) or CD34− cells (5 × 105 per 0.3 mL) (◩) compared with sham controls (□). †Numbers of the TUNEL-positive cells in spinal cord section 7 days after SCI was significantly (P < 0.05) decreased for spinal cord-injured animals treated with i.v. CD34+ cells (5 × 105 per mL) (▩) compared with CD34− cell-treated spinal cord-injured animals (◩). The bars represented means ± SEM of eight rats per group.
CD34+ cells graft secrete both GDNF and VEGF
To elucidate whether GDNF or VEGF can be secreted in spinal cord-injured area by the CD34+ cells, analysis of spinal cord homogenate supernatants by specific ELISA for GDNF or immunofluorescence for VEGF was done. These revealed that GDNF (863-1,115 pg/mL) could be detected in the spinal cord homogenate medium 4 to 7 days after transplantation of CD34+ cells, but not CD34− cells or isotonic sodium chloride solution (as shown in Table 1). The concentration of GDNF in the supernatants obtained approximately 1 h after incubation of CD34+ cells (5 × 105) with PBS by centrifugation was found to be in a negligible amount. Immunofluorescence staining also revealed that the spinal cord-injured rat 7 days after CD34+, but not CD34− cells or saline therapy, displayed more VEGF−positive cells (∼80) in their injured spinal cord section (Figs. 5 and 6) as compared with those of saline-treated or CD34− cell-treated groups.
Fig. 5: The immunofluorescence staining. Numbers of the VEGF-positive cells in spinal cord section 7 days after SCI was increased for spinal cord-injured animals treated with i.v. CD34+ cells (SCI + CD34+ cells) compared with sham controls, SCI + saline, or SCI + CD34− cells.
Fig. 6: *Numbers of the VEGF-positive cells in spinal cord sections 7 days after SCI was significantly ( P < 0.05) increased for spinal cord-injured animals treated with i.v. CD34+ cells (5 × 105 per 0.3 mL) (▩) compared with sham controls or spinal cord-injured rats treated with isotonic sodium chloride solution or CD34− cells. The bars represent means ± SEM of eight rats per group.
Table 1: The concentration of GDNF in the tissue homogenates of spinal cord at T8 or T9 of rats measured at 1 to 7 days after SCI
Delivered CD34+ cells were localized to spinal cord-injured area as determined by immunohistochemistry
Human nuclei immunoreactive cells were detected in the spinal cord-injured area by DAPI staining (Fig. 7) of animals injected with 5 × 105 CD34+, but not CD34−, cells immediately after SCI. This revealed that CD34+ cells could be detected in the spinal cord-injured section 7 days after transplantation of CD34+ cells.
Fig. 7: CD34+ cells identification by human nuclei antibody immunoreactivity in the spinal cord of a spinal cord-injured rat that received CD34+ cells (5 × 105 per 0.3 mL) (SCI + CD34+), CD34− cells (5 × 105 per 0.3 mL) (SCI + CD34−) or saline (0.3 mL) (SCI + saline) and a sham control. FITC - fluorescein isothiocyanate.
DISCUSSION
In the present study, HUCBCs that were CD34+ improved functional recovery and reduced both infarction and apoptosis in the injured spinal cord. Immunohistochemical examination revealed that systemically transplanted CD34+ cells survived in the host spinal cord for at least 1 week after transplantation. CD34+ cells were transplanted via the tail vein immediately after SCI in the present study, whereas CD34+ cells transplanted directly into the spinal cord 1 week after SCI were still able to improve functional recovery in rats (7). These investigators reported that transplanted CD34+ cells survived in the host spinal cord for at least 3 weeks after transplantation but had disappeared by 5 weeks (7). In addition, it was found that i.v. HUCBC alone showed therapeutic effects when administered 24 h to 7 days after stroke (23, 24) or traumatic brain injury (25). The results reported here are consistent with several previous findings. For example, intraspinal transplantation of CD34+ cells after spinal cord hemisection (6) or contusion (7) injury improves functional recovery in adult rats. It should be stressed that the present acute neuroprotection was induced without immunosuppression, which is generally requisite for long-term graft survival and often accompanied by deleterious side effects.
According to the findings of Nishio et al. (7), the transplanted CD34+ cells did not express neural lineage markers, but they did express CD45 (a marker for hemopoietic lineage cells) 1 and 3 weeks after transplantation (7). Although the transplanted CD34+ cells did not differentiate into neural lineage cells and survived only transiently in the injured spinal cord, they reduced hind limb dysfunction. It has also been suggested that HUCBC grafts, instead of the host tissues per se, were likely the source of neuroprotective trophic factors (15).
The expression of several neurotrophic factors in the brain are influenced by cerebral ischemia (26, 27). The GDNF is a patent neurotrophic factor that promotes the survival and morphological differentiation of dopaminergic neurons (28) and motoneurons (29). Tropical application of GDNF and adenovirus-mediated GDNF gene transfer significantly reduced infarct size in a rat middle cerebral artery occlusion model (30, 31). Mesenchymal stem cells that produced GDNF reduced ischemic damage in the rat middle cerebral artery occlusion model (32). In the present results, although the effluent of CD34+ cells did not increase the concentration of GDNF in the injured spinal cord, systemic delivery of CD34+ cells, but not CD34− cells or saline, significantly increased the production of GDNF in the injured spinal cord 4 to 7 days after injury. Thus, it is likely that CD34+ cells may reduce ischemic damage in the rat SCI model by producing GDNF and/or other neurotrophic factors. To further confirm the potential involvement of GDNF in the observed neuroprotection, one set of animals shall receive SCI + i.v. CD34+ cells, whereas a second set of spinal cord-injured animals shall be treated identically, except that CD34+ cells shall be exposed to antibodies against GDNF before transplantation in future studies.
A recent report has demonstrated that systemic administration of human cord blood-derived CD34+ cells to immunocompromised mice subjected to stroke 48 h earlier accelerates neovascularization of the ischemic zone (17, 33). Such a rich vascular environment, along with generation of other nurturing neuronal mediators by CD34+ cells, such as VEGF, enhances subsequent neuronal regeneration. On the basis of these data, accelerated neovessel formation seems to be essential for enhancing endogenous neurogenesis and improving functional recovery. Indeed, as shown in the present results, systemic transplantation of CD34+ cells stimulated production of VEGF in the injured spinal cord area. These findings support the hypothesis that the transplanted CD34+ cells promote an environment conducive to neovascularization of ischemic spinal cord so that neuronal regeneration can proceed.
Cell death has been examined by studying the spinal cords of rats subjected to traumatic insults of mild to moderate severity (18). Within minutes after mild weight drop, impact neurons in the immediate impact area showed a loss of cytoplasmic Nissl substances. Over the next 7 days, this lesion area expanded and cavitated. The TUNEL-positive neurons were noted primarily restricted to the gross lesion area 4 to 24 h after injury, with a maximum presence at 8 h after injury. TUNEL-positive glia were present at all stages studied between 4 h and 14 days, with a maximum within the lesion area 24 h after injury. However, 7 days after injury, a second wave of TUNEL-positive glial cells was noted in the white matter peripheral to the lesion and extending at least several millimeters away from the lesion. It is believed that apoptosis contributes to the neuronal and glial cell death, as well as to the neurological dysfunction, induced by SCI (18). To our knowledge, the data reported here are the first to provide direct support for the idea that CD34+ cell therapy may improve hind limb dysfunction after SCI by reducing apoptosis in the injured spinal cord. The CD34+ cells may promote regeneration of the injured spinal cord rather than interacting with injured axons by secreting trophic factors such as GDNF that acts as a neuroprotectant or promotes axon regeneration. It should be mentioned that the motor function was evaluated by maximal angle an animal could hold to the inclined plane. It would be better to show specificity to resolution of the SCI for motor deficits by comparing performance for upper and lower extremity strength. That is, one might expect that no difference in upper extremity strength would be seen at any time, whereas lower extremity strength might return as shown in the existing figure (Fig. 1). However, inclusion of upper body function would assure that no other artifacts or effects of CD34+ cells were responsible.
A more recent result has demonstrated that CD34+ cells cause attenuation of hypotension, hepatic and renal failure, hypercoagulable state, activated inflammation, and cerebral ischemia and injury during experimental heatstroke (34). Therefore, it is likely that CD34+ cells may suppress a deleterious aspect of injury or systemic response as opposed to providing a signal for nerve repair.
In summary, the current findings demonstrate that systemic administration of CD34+ cells are beneficial in restoring the hind limb function by stimulating production of both GDNF and VEGF in an SCI model. Collection of HUCBCs can take place at any hospital or birthing center. The procedures take about 5 min and pose no risk to mother or baby. Human umbilical cord blood banking provides enough number of CD34+ cells needed for clinical benefit in man. In addition, CD34+ cells are safe to use and are associated with few ethical issues. Thus, it seems that CD34+ cell therapy is one of the potentially useful strategies for SCI.
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