Transplantation of Human Umbilical Cord Blood or Amniotic Epithelial Stem Cells Alleviates Mechanical Allodynia after Spinal Cord Injury in Rats

Animals

Experiments were performed on 30 male Sprague– Dawley rats weighing 180–200 g. All experimental animals were obtained from the Laboratory Animal Center of Seoul National University. They were housed in colony cages with free access to food and water and maintained in temperature- and light-controlled rooms (24 ± 2°C, 12/12 h light/dark cycle with lights on at 07:00) for at least 1 week prior to the study. All of the methods used in the present study were reviewed and approved by the Animal Care and Use Committee at Seoul National University and conformed to US National Institutes of Health guidelines. Every effort was made to minimize animal distress and discomfort and to reduce the number of animals used in the present study.

Spinal Cord Hemisection Surgery

A number of animal models of SCI exist and have been primarily used to determine mechanisms of motor dysfunctions (54,61). Recently, these various SCI animal models have been utilized for pain studies (40), and in particular the partial spinal transection injury model (hemisection) has become popular in neuropathic pain studies (20,32). Thus, this model was used in the present study. A spinal cord hemisection was performed according to the method described by Christensen et al. (9). Briefly, rats were anesthetized with 3% isoflurane (Hana Pharm., Seoul, Korea) in a mixture of N₂/O₂ gas, and the T11–12 vertebrae were identified by counting spinous processes from the sacrum. A laminectomy was performed between the T11–12 vertebral segments, and the lumbar enlargement was identified with the accompanying dorsal blood vessel. The spinal cord was then hemisected just cranial to the L1 dorsal root entry zone with a No. 15 scalpel blade (Feather Safety Razor Co., Osaka, Japan) without damaging the major dorsal vessel or its vascular branches. The musculature and the fascia were then sutured (Won Ind., Siheung, Korea), and the skin was apposed. Sterile procedures were used throughout the surgery. Sham surgery consisted of exposing the spinal cord in the same manner, but without hemisection (n = 6). During recovery, animals were separately housed in a clear plastic cage with a thick layer of sawdust bedding and were monitored for locomotor recovery.

Isolation and Culture of hUCB-MSCs and hAESCs

Isolation and culture of hUCB-MSCs were performed as described previously (46,58). Human umbilical cord blood samples were obtained from the Seoul City Borame Hospital Cord Blood Bank. Samples from term and preterm deliveries were harvested at the time of birth with the mother's informed consent. This work was approved by the Borame Hospital Institutional Review Board and Seoul National University (IRB No. 0603/001-002-07C1). The blood samples were processed within 24 h of collection. The mononuclear cells were separated from the UCB using Ficoll-Paque TM PLUS (Amersham Bioscience, Uppsala, Sweden) and were suspended in a Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA), containing 20% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100 I/ml penicillin (Gibco), 100 mg/ml streptomycin (Gibco), 2 mM l-glutamine (Invitrogen, Carlsbad, CA, USA), and 1 mM sodium pyruvate (Invitrogen).

Isolation and culture of hAESCs were performed as described previously (37,38). Human amniotic tissue was obtained from Kangpuk Samsung Hospital (Seoul, Korea) with informed consent, and isolation and culture were performed with the approval of the Seoul National University Institutional Review Board (IRB No. 0611/001-002). The amniotic tissue was washed several times with phosphate-buffered saline (PBS; EMD Millipore Corp., Billerica, MA, USA) to remove blood and incubated with 0.05% trypsin-EDTA (Invitrogen) for 1 h. The amniotic epithelial cells were collected and suspended in standard culture media, consisting of K-SFM (keratinocyte-serum-free media) supplemented by 0.031 μg/ml human recombinant epidermal growth factor (EGF), 12.4 mg/ml bovine pituitary extract (all from Invitrogen), and 10% FBS.

hUCB-MSC and AESC Transplantation

Two weeks after SCI surgery (Fig. 1A), rats were randomly assigned to groups receiving PBS (n = 8), hUCB-MSCs (n = 8), or hAESCs (n = 8). Following a laminectomy at the same site as the injury, four injections of either PBS, hUCB-MSCs, or hAESCs were made in the following four locations: (1) 1 mm cranial to the lesion site, (2) 1 mm caudal to the lesion site, and (3) two injections were made 1 mm lateral to the lesion site in the contralateral spinal cord (these two contralateral injections were separated by a small distance from each other; see Fig. 1B).

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Figure 1.

The experimental design and timeline, the spinal cord location of stem cell injections, and the morphological appearance of the cultured stem cells. (A) The experimental design and time point of stem cell injection [day 14 after spinal cord injury (SCI) surgery] are depicted in this schematic drawing. (B) This schematic drawing depicts the approximate spinal cord location of the stem cell injection sites with respect to the T13 hemisection area. (C, D) Representative pictures of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) and human amniotic epithelial stem cells (hAESCs), respectively, in culture. Scale bar: 50 μm.

At each site, 2.5 μl of hUCB-MSCs, hAESCs, or PBS was infused for 60 s. Thus, a total of 1×10⁶ hUCB-MSCs or AESCs (10 μl) were delivered to the spinal cord. Following the injection, the syringe was held in place for an additional 10 s before removal to prevent outflow of the injected cells. To allow tracking of the injected hUCB-MSCs and hAESCs, the cells were labeled with a membrane-bound fluorescent marker PKH26 (Sigma-Aldrich, St. Louis, MO, USA) prior to injection.

Behavioral Assessments

Following spinal cord hemisection, rats were examined for motor function in an open-field test space using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale (6). Briefly, the BBB scale ranges from 0 (no hindlimb movement) to 21 (normal movement, including coordinated gait with parallel paw placement). Scores from 0 to 7 indicate the return of isolated movements in the three joints (hip, knee, and ankle). Scores from 8 to 13 indicate the return of paw placement and coordinated movements with the forelimbs. Scores from 14 to 21 show the return of toe clearance during stepping, predominant paw position, trunk stability, and tail position. Only the scores of the ipsilateral hind limb on the hemisected side were recorded, because there were no observable differences in locomotor function of contralateral hind limb throughout the experimentation period.

Pain behavioral tests were performed 1 day before hemisection surgery on all animals to obtain normal baseline values of withdrawal response to mechanical and heat stimuli. Thirteen days after surgery (D+13), rats were again tested and the development of MA and TH were confirmed for each rat prior to randomly assigning each rat to one of the cell treatment groups (PBS, hUCB-MSCs, or hAESCs). All behavioral tests were performed blindly.

MA can be measured in various ways using von Frey filaments. In the present study, the number of paw withdrawal responses to 10 applications of a normally innocuous mechanical stimulus produced by a von Frey filament with a force of 2.0 g (North Coast Medical, Morgan Hill, CA, USA) was measured. Rats were placed on a metal mesh grid under a plastic chamber (Yeong Seong Bio Tech., Seoul, Korea), and the von Frey filament was applied from underneath the metal mesh flooring to each hind paw. The von Frey filament was applied 10 times to each hind paw, and the number of paw withdrawal responses out of 10 was then counted. The results of this mechanical behavioral testing for both the ipsilateral and contralateral hind limbs were expressed as a “percent withdrawal response frequency” (PWF, %), which represented the percentage of paw withdrawals (calculated from the number times the rat withdrew the paw in response to application of the von Frey filament out of the maximum of 10 applications) as previously described (51,52).

To assess nociceptive responses to heat stimuli (i.e., TH), we measured paw withdrawal response latency (PWL) to a heat stimulus using the plantar paw-flick latency test as previously described by Hargreaves et al. (23). Briefly, rats were placed in a plastic chamber with a glass floor (IITC Life Science Inc., Woodland Hills, CA, USA) and were allowed to acclimate for 10 min before testing. A radiant heat source was positioned under the glass floor beneath the hind paw to be tested, and withdrawal latency was measured using a plantar analgesia meter (IITC Life Science Inc.). The test was performed on both the ipsilateral and contralateral hind paws of each animal. Cutoff time in the absence of a response was set at 20 s to prevent any potential skin damage from the radiant heat source.

Immunohistochemistry

Protein phosphorylation occurs on the NMDA receptor subunits, which represents an imperative mechanism for the posttranslational modulation of the properties of the receptor. Overall, NMDA receptor phosphorylation studies suggest that phosphorylation of spinal cord NR1 subunit leads to enhanced NMDA receptor function and central sensitization. Since neuropathic pain is associated with increased phosphorylation of the NR1 subunit in the spinal cord (48,49), we wanted to examine the effect of transplantation of hUCB-MSCs and hAESCs on SCI-induced NR-1 phosphorylation. At the end of the behavioral experiment, rats (n = 3 in each group) were euthanized, and the L4-5 segments of the spinal cord were removed for pNR1 immunohistochemistry. Animals were first deeply anesthetized with 5% isoflurane, perfused transcardially with calcium-free Tyrode's solution (116.4 mM NaCl, 5.4 mM KCl, 1.6 mM MgCl₂•6H₂O, 0.7 mM MgSO₄•H₂O, 1.2 mM NaH₂PO₄•H₂O, 5.6 mM glucose, 26.2 mM NaHCO₃; all reagents were purchased from Sigma-Aldrich), followed by a fixative containing 4% paraformaldehyde and 0.2% picric acid (Sigma-Aldrich) in 0.1 M phosphate buffer (pH 6.9; Santa Cruz Biotech. Inc., Dallas, TX, USA). The spinal cord was then removed immediately, postfixed at 4°C overnight in the same fixative, and then cryoprotected in 30% sucrose (Junsei Chemical Co., Tokyo, Japan) in PBS (pH 7.4) for 48 h. Frozen serial frontal sections (40 μm) were cut through the lumbar L4-5 spinal cord using a cryostat (Microm, Walldorf, Germany). After elimination of endogenous peroxidase activity with 0.3% hydrogen peroxide (Junsei Chemical Co.) and preblocking with 1% normal donkey serum (Vector Laboratories, Burlingame, CA, USA), 0.3% Triton X-100 (Sigma-Aldrich), and 1% bovine serum albumin (Zymed, San Francisco, CA, USA) in PBS, the free-floating sections were incubated with a polyclonal rabbit anti-pNR1 antibody (1:1,000; Upstate, Lake Placid, NY, USA) at 4°C for 48 h. After washing with PBS, the sections were reacted for 2 h at room temperature in a dark chamber with a fluorescent secondary antibody, cyanine 3 (Cy3)-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Some sections were processed in an identical fashion, but without the primary antibody, and these sections served as an immunohistochemical control for nonspecific binding. No positive immunofluorescent staining was detected in tissues processed in this manner. The spinal cord dorsal horn was divided into the following three areas for subsequent immunohistochemical analysis: (1) the superficial dorsal horn (SDH, laminae I and II), (2) the nucleus proprius (NP, laminae III and IV), and (3) the neck of dorsal horn (NECK, laminae V and VI). Five spinal cord sections from the L4-5 lumbar spinal cord segments were randomly selected from each animal. Visualization and localization of pNR1 cells in these spinal cord sections was performed using a confocal laser microscope system (Eclipse TE200, Nikon, Nagano City, Japan). The average number of pNR1-ir cell profiles per section from each animal was obtained, and these values were averaged again across each group and presented as group data (n = 3 in each group). For PKH26 detection, nuclei were counterstained with Hoechst 33238 (1 μg/ml, Sigma-Aldrich). All images per section were also captured and merged by a confocal laser microscope system (Eclipse TE200). To examine the number of surviving/remaining hUCB-MSCs and hAESCs after spinal cord injection, we counted the number of PKH26-labeled cells among the Hoechst-positive cells.

Protein Extraction and Western Blot Assay

The T13 segment and the L4-5 segments of the spinal cord were obtained at the end of the experiment for protein extraction and western blot analysis (n = 3 in each group). The experiments were performed on the following four groups of rats: (1) a sham group, (2) a control PBS injection-SCI group, (3) a hUCB-MSC-treated SCI group, and (4) a hAESC-treated SCI group. Spinal cord tissue from each animal was homogenized independently in lysis buffer containing protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany), followed by vigorous shaking for 2–3 min in the presence of 20 μl of 10% Nonidet-P40 (Sigma-Aldrich). The total protein content of each lysate was quantified using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Foster City, CA, USA). For Western blotting, an equal amount (30 μg) of protein extract was subjected to 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Amersham, Piscataway, NJ, USA) analysis and transferred to a nitrocellulose membrane (Amersham) for incubation with primary antibodies. After immunoblotting with secondary antibodies, proteins were detected with an enhanced chemiluminescence reagent (Intron Biotechnology, Kyungki-Do, Korea). The relative band intensities were measured using NIH ImageJ version 1.34e software (Bethesda, MD, USA). One representative band from three independent experiments was chosen for presentation. The following primary antibodies were used: anti-F4/80 (Abcam, Cambridge, UK), anti-glial fibrillary acidic protein (GFAP, Millipore, Billercia, MA, USA), anti-myelin basic protein (MBP), anti-neuron-specific βIII tubulin (antibody clone TUJ1), anti-inducible nitric oxide synthase (iNOS), anti-cyclooxygenase (COX2, all Abcam), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sigma-Aldrich).

Statistical Analysis

All values are expressed as the mean ± SEM. Statistical analysis was performed using Prism 5.0 (Graph Pad Software, San Diego, CA, USA). A repeated-measures two-way analysis of variance (ANOVA) was performed to determine overall differences in PWL (s) and PWF (%). A one-way ANOVA was also used to determine differences in the number of spinal pNR1-ir cells in spinal cord sections across all experiment groups. Post hoc analysis after repeated-measures two-way or one-way ANOVA was performed using the Bonferroni's multiple comparison test to determine the p value. A value of p < 0.05 was considered statistically significant.

Morphologic Characteristics of hUCB-MSCs and hAESCs

The morphological appearance of the hUCB-MSCs and hAESCs was characteristic of that reported in the literature (27,30). The hUCB-MSCs were spindle shaped and showed a fibroblast-like appearance (Fig. 1C), while the hAESCs were polygonal in shape and exhibited a cobblestone-like morphology (Fig. 1D).

Effect of hUCB-MSCs and hAESCs on SCI-Induced Below-Level Neuropathic Pain

The effect of transplantation of hUCB-MSCs or hAESCs on below-level MA and TH in SCI rats is illustrated in Figure 2. The development of MA (Fig. 2A, B) and TH (Fig. 3A, B) was robust by day 13 postinjury, stem cells were implanted on day 14, and their potential effects on ipsilateral and contralateral MA and TH were evaluated for 28 days posttransplantation. While the transplantation of hUCB-MSCs into the spinal cord showed a trend toward gradually reducing PWF in both hind paws from day 10 to day 28 posttransplantation, this effect did not reach statistical significance. Conversely, the transplantation of hAESCs significantly reduced the SCI-induced increase in PWF from days 13 to 24 in the ipsilateral hind paw and on day 24 in the contralateral hind paw, respectively (*p < 0.05 as compared to PBS-treated SCI rats) (Fig. 2A, B). The initial suppressive effect of hAESCs occurred at approximately 2 weeks after cell transplantation. While transplantation of hAESCs significantly reduced MA, neither hUCB-MSC nor hAESC transplantation had any effect on SCI-induced TH as measured by changes in paw withdrawal latency in the Hargreaves test (Fig. 3A, B).

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Figure 2.

The effect of transplantation of hUCB-MSCs and hAESCs into the spinal cord on SCI-induced mechanical allodynia in the ipsilateral and the contralateral hind paws of rats with a T13 injury. While transplantation of hUCB-MSCs gradually reduced “percent withdrawal response frequency” PWF (%) in both the ipsilateral (A) and contralateral hind paws (B) from day 10 to day 28 following stem cell transplantation, the effect did not attain statistical significance. Conversely transplantation of hAESCs significantly reduced mechanical allodynia as measured by PWF (%) from day 13 to day 24 in the ipsilateral hind paw and at day 24 in contralateral hind paw, respectively (*p < 0.05 as compared to PBS-treated rats) (n = 8 in each group).

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Figure 3.

Graphs summarizing the effect of transplantation of hUCB-MSCs and hAESCs on thermal hyperalgesia in the ipsilateral and contralateral hind paws of SCI rats. Neither hUCB-MSC nor hAESC transplantation altered the SCI-induced decrease in ipsilateral (A) or contralateral (B) paw withdrawal response latency (PWL; s) (n = 8 in each group).

Effect of hUCB-MSCs and hAESCs on pNR1 Expression in Lumbar Dorsal Horn After SCI

SCI caused a significant increase in the number of phosphorylated NMDA receptor NR1 subunit immunoreactive (pNR1-ir) cell profiles in the SDH (laminae I and II), NP (laminae III and IV), and in the NECK region (laminae V and VI) of both the ipsilateral and contralateral spinal cord dorsal horns (*p < 0.05 as compared to that of sham surgery animals) (Fig. 4A–D). An increase in the number of pNR1-ir cells in the spinal cord dorsal horn is thought to be a major mechanism contributing to the induction of the central sensitization phenomenon in chronic pain states, and thus, the SCI-induced increase is consistent with the development of central sensitization. While the transplantation of hUCB-MSCs showed a trend toward suppressing the SCI-induced increase in the number of pNR1-ir cells in the NECK regions of ipsilateral and contralateral spinal dorsal horn (Fig. 4A–E), this did not reach statistical significance. On the other hand, transplantation of hAESCs significantly reduced the SCI-induced increase in the number of pNR1-ir cells in the NP and the NECK regions of ipsilateral and contralateral spinal dorsal horn (#p < 0.05 and ##p < 0.01 as compared with those of PBS-treated animals) (Fig. 4A–D, F).

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Figure 4.

The number of phosphorylated NMDA receptor NR1 subunit immunoreactive (pNR1-ir) cell profiles in different laminae of the spinal cord dorsal horn following transplantation. Graphs illustrating the number of phosphorylated N-methyl-d-aspartate (NMDA) receptor NR1 subunit immunoreactive (pNR1-ir) cell profiles in different laminae of the ipsilateral (A) and contralateral (B) spinal cord dorsal horn and photomicrographs (C–J) of representative pNR1-ir cells in the spinal cord following treatment with hUCB-MSCs and hAESCs. SCI caused a significant increase in the number of pNR1-ir cell profiles in the superficial dorsal horn (SDH, laminae I and II), the nucleus proprius (NP, laminae III and IV), and the neck of the dorsal horn (NECK, laminae V and VI) regions of both the ipsilateral (A) and contralateral spinal cord dorsal horns (B) (*p < 0.05 as compared to that of sham surgery animals). The transplantation of hAESCs significantly reduced the SCI-induced increase in the number of pNR1-ir cells in the NP and the NECK regions of ipsilateral and contralateral spinal dorsal horn (A, B; #p < 0.05 and ##p < 0.01 as compared with those of PBS-treated animals). (C–F) Representative pictures showing the effect of hUCB-MSCs and hAESCs on pNR1 expression. (G–J) The magnified images of rectangular in (C–F), respectively. Scale bar: 100 μm.

hUCB-MSCs and hAESCs Detected in Transplanted Spinal Cord

To determine the position of the injected stem cells in SCI rats, we performed cell tracking using confocal imaging in conjunction with the fluorescent cell dye PKH26, which incorporates a yellow–orange fluorescence into the lipid regions of the stem cell membrane prior to transplantation. PKH26-labeled hUCB-MSCs and hAESCs were present around the site of spinal cord injury (Fig. 5). Hoechst staining of cell nuclei was also conducted for detecting cell nuclei, and the image of the nuclear staining was merged with that of the PKH26-labeled cells (Fig. 5). This allowed confirmation that the injected labeled hUCB-MSCs and hAESCs were present within the injection sites near the site of spinal cord injury. To determine the number of surviving hUCB-MSCs and hAESCs following injection, we performed an analysis of the number of PKH26-labeled cells among the entire cell population (Hoechst-positive cells) at the injection site. PKH26-labeled hUCB-MSCs and hAESCs were counted in each section and measured by percentage. We found that the number of hUCB-MSCs at the injection site represented approximately 12% of the total cell population, and similarly the number of hAESCs at the injection site represented approximately 10% of the total cell population. Thus, the distribution of surviving hUCB-MSCs was comparable to that of surviving hAESCs at the injection site (Fig. 5D).

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Figure 5.

Representative confocal images of PKH26-labeled hUCB-MSCs (A) and hAESCs (B) near the injury site. No labeled cells were present in PBS injected SCI rats. (C) Hoechst staining was conducted for detecting nuclei and the confocal images of cell nuclear staining were then merged with those of the PKH26-labeled cells. These merged images illustrate the nuclei of the transplanted stem cells. Scale bar: 50 μm in low magnification (200×) and 100 μm in high magnification (400× and zoom-merge). (D) Ratio of cell distribution. Detected PKH26-labeled cells are presented as percentage of the total cell population. The percentage of PKH26-labeled surviving cells in hUCB-MSC-treated animals is similar to that in the hAESC-treated group.

Effect of hUCB-MSC and hAESC on iNOS and COX Expression in the Dorsal Horn of At-Level and Below-Level Spinal Cord Segments Following SCI

Since we have shown that the transplantation of hUCB-MSCs and hAESCs reduce the SCI-induced MA component of neuropathic pain, we next examined the spinal cord expression levels of several proteins including F4/80, GFAP, MBP, neuron-specific βIII tubulin (antibody clone TUJ1), iNOS, and COX2 that are known to be involved in spinal cord injury, inflammation, and/or chronic pain. We conducted a Western blot analysis of both the T13 (“at-level”) and L4-5 (“below-level”) spinal cord segments in each experiment group. At the site of spinal cord injury (T13), the expression of F4/80, GFAP, MBP, COX2, and iNOS were increased in PBS-treated SCI animals. While the SCI-induced increase in the expression levels of GFAP, COX2, and iNOS were not changed in the stem cell treatment groups, the SCI-induced increase in F4/80 expression was decreased in the hAESC treatment group compared with that of the PBS-treated SCI group (Fig. 6). Stem cell transplantation produced no changes in the expression of MBP and neuron-specific βIII tubulin (Fig. 6A). In addition, we conducted a Western blot analysis of the expression of these same proteins at the L4-5 (below-level) segments of the spinal cord. SCI increased the expression of both F4/80 and COX2 within the L4-5 spinal cord segments as compared with rats in the sham control group. Importantly, treatment with either hUCB-MSCs or hAESCs dramatically decreased the expression of F4/80 as compared with PBS-SCI rats (Fig. 6B). Conversely, transplantation of either hUCB-MSCs or hAESCs had no effect on the expression of GFAP, MBP, neuron-specific βIII tubulin, iNOS, or COX2 within below-level spinal cord segments (Fig. 6B).

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Figure 6.

Western blots depicting the effect of hUCB-MSCs and hAESCs on neuronal, inflammation, and immune-associated markers. At the site of spinal injury (T13), epidermal growth factor-like module-containing mucin-like, hormone receptor-like 1 (EMR1; F4/80), glial fibrillary acidic protein (GFAP), Myelin basic protein (MBP), cyclooxygenase 2 (COX2), and induced nitric oxide synthase (iNOS) were increased in PBS-treated SCI animals (A). Although the expression levels of GFAP, COX2, and iNOS did not change in the stem cell treatment groups, transplantation of hAESCs caused a decrease in the SCI-induced increase in the expression of F4/80 as compared with that of the PBS-treated group. In addition, within the L4-5 spinal cord segments (below-level site), F4/80 and COX2 also increased in SCI animals as compared to sham animals. This SCI-induced increase in F4/80, but not COX2, was reduced by transplantation of either hUCB-MSCs or hAESCs as compared to PBS-injected SCI rats (B). TUJ1, antibody clone for neuron-specific βIII tubulin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Effect of hUCB-MSC and hAESC on Motor Function After SCI

Prior to hemisection, the BBB scores were 21 in all SCI groups (Fig. 7). Immediately upon emerging from anesthesia, hemisected animals displayed loss of ipsilateral hindlimb function as indicated by the BBB scores of 0 for each group. At day 14 after hemisection, the transplantation of either hUCB-MSCs or AESCs produced no effects on motor function (Fig. 7).

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Figure 7.

A graph illustrating the effect of transplantation of hUCB-MSCs and hAESCs on motor function following SCI. Prior to hemisection, the Basso, Beattie, and Bresnahan (BBB) scores were 21 in all SCI groups. Immediately upon emerging from anesthesia, hemisected animals displayed loss of ipsilateral hindlimb function as indicated by BBB scores of 0 for each group. By day 14 posthemisection, the BBB scores were increased indicating partial recovery and the transplantation of hUCB-MSCs or AESCs had no significant effect on the further recovery of BBB scores indicating of a lack of effect on motor function.