Experimental Neurology 253 (2014) 138–145
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Neuroprotective effects of human spinal cord-derived neural precursor cells after transplantation to the injured spinal cord Mia Emgård a,1, Jinghua Piao a,1,2, Helena Aineskog a,b, Jia Liu a, Cinzia Calzarossa a, Jenny Odeberg a, Lena Holmberg a, Eva-Britt Samuelsson a, Bartosz Bezubik c, Per Henrik Vincent a, Scott P. Falci d, Åke Seiger a,b, Elisabet Åkesson a,b,1, Erik Sundström a,b,⁎,1 a
Division of Neurodegeneration, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Geriatric Clinic Res Lab., Novum, S-14186 Stockholm, Sweden Stockholms Sjukhem Foundation, Mariebergsgatan 22, S-11235 Stockholm, Sweden c Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, S-14186 Stockholm, Sweden d Department of Neurosurgery, Craig Hospital, 3425 S. Clarkson St., Englewood, CO 80110, USA b
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Article history: Received 12 July 2013 Revised 1 November 2013 Accepted 27 December 2013 Available online 8 January 2014 Keywords: Spinal cord injury Neural precursor cells Transplantation Neuroprotection Motor function
a b s t r a c t To validate human neural precursor cells (NPCs) as potential donor cells for transplantation therapy after spinal cord injury (SCI), we investigated the effect of NPCs, transplanted as neurospheres, in two different rat SCI models. Human spinal cord-derived NPCs (SC-NPCs) transplanted 9 days after spinal contusion injury enhanced hindlimb recovery, assessed by the BBB locomotor test. In spinal compression injuries, SC-NPCs transplanted immediately or after 1 week, but not 7 weeks after injury, significantly improved hindlimb recovery compared to controls. We could not detect signs of mechanical allodynia in transplanted rats. Four months after transplantation, we found more human cells in the host spinal cord than were transplanted, irrespective of the time of transplantation. There was no focal tumor growth. In all groups the vast majority of NPCs differentiated into astrocytes. Importantly, the number of surviving rat spinal cord neurons was highest in groups transplanted acutely and subacutely, which also showed the best hindlimb function. This suggests that transplanted SC-NPCs improve the functional outcome by a neuroprotective effect. We conclude that SC-NPCs reliably enhance the functional outcome after SCI if transplanted acutely or subacutely, without causing allodynia. This therapeutic effect is mainly the consequence of a neuroprotective effect of the SC-NPCs. © 2014 Elsevier Inc. All rights reserved.
Introduction The last decade has seen an impressive number of articles on experimental cell therapies for spinal cord injury (SCI), involving embryonic stem cells, adult (Parr et al., 2008) and fetal (Ogawa et al., 2002) somatic neural stem cells, oligodendrocyte progenitor cells (Keirstead et al., 2005), umbilical cord blood cells (Cho et al., 2008), olfactory mucosa (Iwatsuki et al., 2008), different Schwann cell preparations (Paino Abbreviations: SCI, spinal cord injury; NPCs, neural precursor cells; SC-NPCs, spinal cord-derived NPCs; FBr-NPCs, forebrain-derived NPCs; GRPs, glial restricted progenitor cells; CNTF, ciliary neurotrophic factor; PFA, paraformaldehyde; PBS, phosphate-buffered saline; Hsp-27, heat shock protein 27; Hnp, human nuclear antigen; PCNA, proliferating cell nuclear antigen; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; IR, immunoreactive. ⁎ Corresponding author at: Division of Neurodegeneration, Geriatric Clinic Res Lab., Novum 5th floor, S-14186 Stockholm, Sweden. E-mail address: [email protected] (E. Sundström). 1 These authors contributed equally to the study. 2 Present address: Memorial Sloan-Kettering Cancer Center, RRL 469, 411 East 67th Street, New York, NY 10065, USA. 0014-4886/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.12.022
et al., 1994) and olfactory ensheathing cells (Li et al., 1997). Several of these cell types are now used in on-going clinical trials, or have been approved for clinical trial by regulatory authorities. However, there are a depressing number of treatments that have shown effects in animal disease models but failed in clinical trials. We therefore set out to evaluate a promising human cell type in two very different animal models of SCI. Neurospheres are free-floating heterogeneous cultures of immature neural precursor cells (NPCs) (Reynolds and Weiss, 1996), composed of neural stem cells, more committed progenitor cells, as well as occasional differentiated astrocytes and post-mitotic neurons (Piao et al., 2006). A number of studies have shown that there are differences between in vitro-expanded neural stem and progenitor cells due to their regional origins (Armando et al., 2007; Kelly et al., 2009; Onorati et al., 2011). Transplantation of NPCs, murine or human, has been shown to improve functional parameters in SCI animals (Iwanami et al., 2005; Ogawa et al., 2002; Watanabe et al., 2004). NPCs are therefore possible candidates for clinical application in SCI patients. It is however not clear if the regiondependent differences seen in neural stem and progenitor cells translate into different therapeutic effects in SCI.
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There are several possible mechanisms through which cell therapy may restore functions after SCI. Neuroprotection is one, the transplant acting by reducing endogenous toxic substances, inhibiting inflammatory reactions and/or releasing growth factors and other supportive substances (Madhavan et al., 2008; Yasuhara et al., 2006). Since the reversible degenerative processes after experimental SCI most likely proceed for a few weeks in rats, neuroprotection may be a mechanism through which acutely and subacutely transplanted cells act. In later stages, functional effects of transplanted cells may be due to the support of host axon re-growth through release of trophic factors, reduction of neurite growth-inhibitory substances, enhancement of re-myelination of axons, or by supplying the injured tissue with an extracellular matrix that is more permissive to regeneration. In rodent SCI models it is also possible that functional effects of a graft is due to modulation of the central pattern generators, without any improved supraspinal control of movements. In vitro studies have shown that NPCs have neuroprotective effects, which have also been suggested by in vivo studies (Mothe et al., 2013). Others have reported that these cells act by supporting regenerating host axons (Gamm et al., 2007; Pfeifer et al., 2004). The timing of transplantation also has other implications. After measuring the levels of potentially cytotoxic cytokines, Nakamura and coworkers argued that the spinal cord is a hostile environment to transplanted cells the first week after injury (Nakamura et al., 2003). As a consequence, experimental transplantation studies are with few exceptions carried out 9 days after injury. However, there are to our knowledge no systematic comparisons between transplantations to the spinal cord at different time-points, a very important issue with regard to clinical applicability. In the present study we have analyzed the functional effects of human spinal cord-derived NPCs (SC-NPCs) transplanted to SCI animals. To evaluate the potential of these NPCs as cell therapy for human SCI, we initially applied the NPC transplantation to dorsal contusion injuries with very rapid onset using the force-feedback IH impactor (Scheff et al., 2003). This was followed by a more extensive study using another type of SCI model, lateral compression injury, with a slow onset as a result of application of an aneurysm clip (von Euler et al., 1997), comparing NPC transplantation at different time points after injury. We found that human SC-NPCs improve functional outcome in both SCI models, but only after acute and subacute transplantations, and the functional improvement correlated with a neuroprotective effect of the transplanted NPCs, which reduced the loss of host spinal cord neurons. Materials and methods Animals Adult immunodeficient athymic female rats (Hsd: RH-rnu/rnu, Harlan) were housed in an isolated environment, with autoclaved water and food pellets ad lib. The use of research animals was done in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Swedish Animal Protection Act, and the experimental procedures were approved by the Regional Ethics Committee on Animal Research, Stockholm, Sweden. Human neurosphere culture NPCs transplanted as neurospheres were derived from two types of donor tissue; SC-NPCs from the spinal cord, and FBr-NPCs from the subcortical forebrain, 5.5–9 weeks of gestation. The embryonic–fetal tissue was retrieved from elective routine abortions, with written consent from the pregnant women. All procedures for the use of human cells for experimental studies on SCI were approved by the Regional Ethical Committee, Stockholm. Tissue was dissected and cultured as earlier described by us (Piao et al., 2006). Briefly, dissection was made under sterile conditions in DMEM with F12 (DMEM/F12, 1:1, Life Technologies,
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Gaithersburg, MD, USA), and mechanical dissociation of the tissue was performed using a glass-Teflon homogenizer, using the entire spinal cord or sub-cortical forebrain of one fetus to establish NPC cultures. The cells were cultured as free-floating neurospheres in DMEM/F12 complemented with glucose (0.6%, Sigma, St. Louis, MO, USA), Hepes (5 mM, Life Technologies), heparin (2 μg/ml, Sigma), N2 supplement (1%, Life Technologies), basic fibroblast growth factor (20 ng/ml, R&D Systems, Minneapolis, MN, USA), epidermal growth factor (20 ng/ml, R&D Systems) and ciliary neurotrophic factor (CNTF, 10 ng/ml, R&D Systems) at an initial density of 40,000–50,000 cells/cm2 in 20 ml of medium. The NPCs were maintained at 37 °C in 5% CO2, with addition of fresh medium twice a week. They were passaged every 7 to 10 days with enzymatic dissociation using TrypLE Express (Life Technologies) for 4–5 min at 37 °C followed by gentle mechanical dissociation. Selection of neurospheres for transplantation Using quantitative cell counts in immersion-fixed, sectioned mediumsized neurospheres, we established the equation n = exp (k × s + m), k = 0.009 and m = 6.26 describing the relation between the diameter (s) and the number of cells in the neurosphere (n) for neurospheres with diameters between 100 and 500 μm. Neurospheres at passage 3–8 were used for all experiments. In the neurosphere cultures, spheres with a diameter between 150 and 300 μm were identified, the number of cells per neurospheres calculated, and 10–12 neurospheres were chosen to give a total of 100,000 cells ± 10% to be transplanted to each rat. The neurospheres were kept in cell culture medium without growth factors pending transplantation. Animal surgery The weight of the rats was 170–200 g at the time of surgery. They were injected with Atropin (0.05 mg/kg i.p., NM Pharma AB) 30 min before surgery, and anesthetized using Hypnorm (fentanyl citrate, 0.22 mg/kg, and fluanisone, 6.8 mg/kg, Janssen Pharmaceutical) and Dormicum (midazolam, 3.4 mg/kg, Hoffman-La Roche). Body temperature was kept at 38 °C throughout surgery. Lumbar spinal cords were surgically exposed by partial laminectomy of vertebra Th13, the dura was cut open, and a few drops of Xylocaine (lidocaine hydrochloride 20 mg/ml, AstraZeneca) were placed on the exposed spinal cord. Spinal cord contusion injury was achieved with an IH spinal cord impactor (Precision Systems and Instrumentation, LLC), the force set to 175 kdyn with no dwell time. For lateral compression injuries, a modification of the method of Rivlin and Tator (1978) was used. A bulldog clamp was applied to compress the spinal cord for 30 s at the lower half of the spinal cord segment Th13. In acutely transplanted animals, NPCs were injected in the same session as the injury (see below). For the other animals, a layer of Lyoplant (B/Braun Aesculap AG) was placed on the spinal cord as dura substitution before the wound was sutured. The rats were subcutaneously injected twice with 3 ml Ringer/glucose (2.5%) before and after surgery. After surgery, the rats were given intramuscular injections of Temgesic (buprenorphine, 7 μg/kg, Reckitt & Colman) twice a day for four days to avoid allodynia, and Borgal (trimetoprim sulfa, 15 mg/kg s.c., Intervet International B.V.) to prevent from urinary infection. The urinary bladders were emptied manually twice daily until spontaneous evacuation was present. Transplantation procedures Transplantation to dorsal spinal cord contusion injuries was performed 9 days after lesion. Rats were randomized for transplantation, and either received 100,000 SC-NPCs or a 3 μl injection of growth factor-free cell culture medium as sham transplantation. At the time of transplantation, animals were anesthetized, the wound re-opened and the spinal cord exposed. A glass capillary (0.3 mm end
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inner diameter) was attached to a 10 μl Hamilton syringe with Teflontubing, to allow control that all neurospheres entered the spinal cord. The capillary, loaded with 3 μl of neurosphere suspension containing 10–12 neurospheres (=100,000 ± 5000 cells), was placed at the site of the injury, immediately lateral to the midline and inserted approximately 1 mm. The neurospheres suspended in 3 μl were injected during 2–3 min, the glass capillary then kept in place for another minute, before being slowly retracted, carefully making sure no neurospheres leaked out of the cord. For transplantation of NPCs after lateral compression injuries, four groups of rats received 100,000 human NPCs in neurospheres as follows: 1) transplantation of SC-NPCs 10 min after compression injury (SC-Acute), 2) transplantation of SC-NPCs one week after compression injury (SC-Subacute), 3) transplantation of FBr-NPCs one week after compression injury (FBr-Subacute), and 4) transplantation of SC-NPCs 7 weeks after compression injury (SC-Chronic).
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Time post-lesion (weeks) Fig. 2. BBB-locomotor score in rats injured by a lateral compression SCI, and transplanted acutely (0 days) (n = 8), subacutely (7 days) (n = 10) and chronically (7 weeks) (n = 12) after injury with SC-NPCs, or subacutely (n = 10) after injury with FBr-NPCs, and the sham graft group (n = 10). Each data point represents the mean ± SD, **p b 0.01.
Behavioral assessments To study hindlimb sensory motor function, rats were scored according to the Basso, Beattie, Bresnahan (BBB) locomotor rating scale (Basso, 2004; Basso et al., 1995) during 4 min ambulation on an elevated 65 × 150 cm surface. The tests were video recorded, and two independent observers blinded to the treatment evaluated each rat. The rats were evaluated pre-operatively and then on regular intervals for 18 weeks (see Figs. 1 & 2). Hindlimb motor function was also assessed using a grid-walk test. The equipment consisted of a 150 × 20 cm grid with 3 × 3 cm holes and a platform at the end with a small wooden shelter and sweet rat chow pellets. During three consecutive trials, the number of times hind-paws slipped or was misplaced was recorded, and the mean number of mistakes calculated. To be able also to include rats that lacked weight-bearing ability, their score was set to 47 for each hindlimb which equals a stepping mistake at every hole in the grid, giving a maximum score of 94. To assess increased pain sensitivity, pain threshold to pressure to the torso and hindlimbs was tested before the injury, immediately after injury and 1, 2, 6, 10 and 18 weeks later. The test was carried out using von Frey filaments (Stoelting) ranging from 0.4 to 447 g (Yu et al., 1999). Starting with the thinnest filament, rats were tested with increasing pressure until an avoidance response occurred three times for the same pressure.
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Immunohistochemistry After 20 weeks, the rats were transcardially perfused with Ca2+-free Tyrode's solution, followed by phosphate-buffered 4% paraformaldehyde (PFA, Merck Millipore). The tissue was post-fixed for 2 h, rinsed and cryo-preserved in 20% sucrose at 4 °C for at least 24 h. The spinal cords were sectioned at 10 μm in a cryostat and mounted on gelatincoated slides. Primary antibodies used were human-specific rabbit anti-heat shock protein 27 (hsp-27, 1:1500, Medical & Biological Laboratories), mouse anti-human nuclear protein (hnp, 1:150, Merck Millipore), mouse anti-proliferating cell nuclear antigen (PCNA, 1:100, Cell Signaling Technology), rabbit anti-nestin (1:250, Millipore Merck), rabbit antiβ-tubulin type III (1:1200, Nordic Biosite), mouse monoclonal humanspecific anti-glial fibrillary acidic protein (GFAP, 1:200,000, Sternberger Monoclonals), and rabbit anti-microtubule-associated protein 2 (MAP2, 1:50, Merck Millipore). Primary antibodies were diluted in 0.1 M phosphate buffer with 0.3% Triton X-100 (TPBS). The secondary antibodies used were conjugated to Cy3 (Jackson ImmunoResearch Laboratories Lab. Inc.) or Alexa Fluor 488 (Life Technologies). Sections were treated with 1.5% normal goat serum (Sigma) at room temperature for 30 min, incubated with primary antibodies at 4 °C overnight, rinsed before a two hour incubation with secondary antibodies at room temperature. All sections were counterstained with nuclear marker Hoechst 33342 (30 μg/ml, Life Technologies). The immunolabeled tissue sections were captured using the Openlab software for MacOS (Improvision) and a CCD camera (Hamamatsu ORCAER) connected to the fluorescence microscope (Zeiss). Quantification was performed by one person blinded to the identity of the tissue sections. Quantitative histological analysis
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Time post-lesion (weeks) Fig. 1. BBB-locomotor score in rats subjected to a contusion SCI, transplanted with SCNPCs (n = 10) or sham-treated (n = 8) 9 days after injury. Animals were functionally evaluated for 18 weeks post-lesion. Data points represent mean ± SD, **p b 0.01 vs controls.
Human cells were defined as hsp-27 immunoreactive (IR) cell somata surrounding Hoechst 33342-stained nuclei. After an initial screening, a starting section outside the human graft was randomly chosen. From this section, every 30th section was used for analysis. In each tissue section, a random starting point was chosen in the upper left quadrant, from which every 4th field of view moving to the lower right quadrant was used. This resulted in 4–6 fields of view being evaluated at 40× magnification. In each field of view, all human cells were counted, and the total sum of human cells was calculated using the Abercrombie correction. To quantify the proportion of human donor cells expressing phenotype markers, tissue sections were double-stained for hsp-27 or hnp in
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Time post-lesion (weeks) Fig. 3. Total number of paw misplacing in the grid-walk test, performed in rats injured by a lateral compression SCI, transplanted immediately (acute, 0 days), 7 days (subacute) and 7 weeks (chronic) after injury with SC-NPCs, or 7 days after injury with FBr-NPCs, and the sham graft group. Each data point represents the median of 8–12 rats with the 0.25 and 0.75 percentiles indicated at day 2–18, but for the sake of clarity only for the acute SCNPC group (error bar caps the left side), the subacute SC-NPC group (error bar caps on the right side) and the control group (bilateral error bar caps). *p b 0.05.
combination with nestin, GFAP, β-tubulin type III or MAP2. For each antibody, one tissue section 1.2 mm rostral and one section 1.2 mm caudal from the epicenter were used. At least 100 hsp-27-IR cells, and 20 cells labeled by the other antibody were sampled, if necessary additional adjacent tissue sections were added to the analysis. In each tissue section, all labeled cells were counted, and corrected using the Abercrombie formula. For quantification of rat spinal cord neurons, every 60th section of a 4.2 mm spinal cord segment centered around the injury epicenter was used. Rat host neurons – β-tubulin type III-IR cytoplasm surrounding a Hoechst 33342-stained nucleus negative human hsp-27 – were counted, and the total number of rat neurons in the 4.2 mm segment was calculated with the Abercrombie correction. For all quantifications of immunohistochemically labeled tissue, sections from 6 rats per treatment group were used. Statistics and exclusion criteria In the in vivo experiments, occasional rats in each group were much more severely injured. Therefore, a blinded person excluded all rats that did not reach a BBB score of at least 7 within 5 weeks after injury. The number of excluded rats was similar in all treatment groups (contusion injury experiment: transplanted n = 1, control n = 1; compression injury experiment: SC-Acute n = 2, SC-Subacute n = 2, FBr-Subacute n = 4, SC-Chronic n = 2, control n = 2). In two of the groups of the compression injury experiment (SC-Subacute and SC-Chronic), two animals died before the end of the evaluation period. For statistical analyses we used ANOVA for repeated measures with Fisher's post-hoc test for inter-group comparisons (BBB scores and pain thresholds), Kruskal–Wallis test (grid-walk due to skewed data), onefactor ANOVA and Fisher's post-hoc test (morphological evaluations), p b 0.05 was considered statistically significant. Statistical analysis was performed using StatView 4.5 and Instat 3.0. Group data are expressed as mean ± standard deviation (SD). Results Functional analyses We first studied the effects of subacute (9 days) intraspinal transplantation of neurospheres of SC-NPCs on functional outcome after
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Time post-lesion (weeks) Fig. 4. Assessment of pressure-induced pain thresholds in rats injured by a lateral compression SCI and transplanted as described. Each data point represents the mean of 8–12 rats ± SD. There were no significant differences among the treatment groups.
contusion SCI. All animals improved rapidly during the first weeks after injury. Thereafter improvement progressed slower, and with different courses for the two groups. Six weeks after injury, the control group did not improve further, and remained at an average score of approximately 13.5, while animals that received human SC-NPCs 9 days after injury continued to improve during the 18 weeks of study, reaching a mean BBB score of 16.6, significantly better than the control group (Fig. 1). We then investigated the therapeutic effects of human NPCs in a different SCI animal model, and the duration of the therapeutic window. In this experiment we used a SCI model with a sustained compression during 30 s, in contrast to the brief impact in the contusion injury experiment. SC-NPCs were transplanted in the acute (0 day), subacute (7 days) and chronic (7 weeks) stages of injury while FBr-NPCs were only transplanted subacutely (7 days). Acutely transplanted rats reached the highest BBB score, 16.7, of all the groups, compared to 12.6 for the control group. Subacutely transplanted rats also performed significantly better than control rats throughout the 18 weeks of study, and significantly better than rats transplanted with FBr-NPCs (Fig. 2). We did not observe any systematic difference between the right and left hindlimb performance. In the grid-walk test to assess hindlimb function the acutely transplanted rats made significantly fewer mistakes than the control group, while the other transplanted groups did not perform significantly better than the control group (Fig. 3). Pain sensitivity to pressure was analyzed using von Frey filaments to apply localized pressure to the skin. The mean threshold for an avoidance response was between 160 and 270 g for the different experimental groups. This decreased to 100–140 g two weeks after injury, and increased slightly up to 18 weeks post-lesion. We found no significant difference between the groups, or indication of increased sensitivity (i.e. lowered response threshold) in the transplanted groups (Fig. 4).
Morphological analyses At post-mortem analysis, cells positive for the two human-specific markers hsp-27 and hnp could be seen throughout more than 6 mm of the spinal cord in contusion and compression injuries. The transplanted cells were distributed from the remaining ventral white matter to the dorsal horns in contusion (Fig. 5A) and compression SCI. Farther from the lesion, the human cells were mainly located in the white matter. Although the NPCs were injected in the lesion center, there were still large cystic cavities in many animals, with human cells in the tissue surrounding the cavity.
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To determine if survival of transplanted NPCs could explain the differences in therapeutic effect, we compared the number of human cells 20 weeks after injury. Surprisingly, in almost every rat there were many more human cells than the 100,000 NPCs that were transplanted. Due to the large differences within the groups, the groups were not significantly different. However, it is noteworthy that the mean number of human cells was highest in rats transplanted acutely with SC-NPCs (517,000 ± 304,000). There were almost as many cells (426,000 ± 276,000) in rats transplanted subacutely with FBr-NPCs, while the mean numbers were slightly lower in those who received SC-NPCs subacutely (325,000 ± 165,000) and chronically (239,000 ± 180,000) rats. The number of human cells in individual rats did not correlate with functional outcome (data not shown). We also compared the proportion of human cells expressing nestin, GFAP, β-tubulin type III and MAP2 in the treatment groups (Fig. 5B). Cells positive for the early neuronal marker β-tubulin type III (Fig. 5D) and the more mature neuronal marker MAP2 (Figs. 5E–F) comprised less than 10%. In all groups, 80–90% of human cells were positive for the astrocyte marker GFAP, and 4–10% of these cells co-expressed nestin, indicative of a multipotent phenotype (Figs. 5G–H). The proportion of phenotypes was similar in all groups, indicating that differentiation of transplanted NPCs does not determine functional outcome. To evaluate possible neuroprotective effects of the grafted NPCs, we quantified the number of remaining host rat neurons in a 4.2 mm long segment of the injured spinal cord, centered around the lesion epicenter. In non-transplanted lesioned animals, an average of 27,000 neurons in the 4.2 mm spinal cord segment survived the contusion injury (Fig. 6). As a reference, the mean number of neurons in 5 uninjured rats of the same age was 289,200 ± 10,200 in a similar spinal cord segment (Fig. 6, hatched area). Thus, N90% of the neurons in these segments were lost after injury. In acutely transplanted rats more than 3 times as many host neurons could be found in the corresponding spinal cord segment (≈ 77% loss of host neurons). In animals acutely transplanted with SC-NPCs, almost twice as many neurons as in control animals were found (≈82% loss of host neurons). The survival of host neurons in these groups was significantly higher than in the control group, demonstrating a neuroprotective effect of the transplanted NPCs. We found no protective effect in rats transplanted in the chronic stage, or acutely with FBr-NPCs (Fig. 6). Discussion Before proceeding to clinical trial of cell therapy in SCI patients can take place, robust functional treatment effects of the particular cell type should be demonstrated in relevant animal models. Since every disease model has significant differences from the clinical condition it is intended to mimic, we believe a minimum requirement is to show functional effects in more than one injury model. In addition, issues such as the timing of transplantation and risks of side effects should be addressed. In the present study, we have examined the functional and morphological outcomes of human NPCs expanded in vitro, transplanted to two different animal models of SCI. The two rat SCI models used differ with respect to the biomechanics of injury. The contusion injury has an immediate onset of injury and duration of tissue displacement less than a second, while the compression develops during seconds and is sustained for 30 s.
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In both animal SCI experiments we found that hindlimb motor function was significantly improved by SC-NPCs transplanted in the subacute stage. The effect of acutely transplanted SC-NPCs studied in the compression injury model was even more pronounced, reaching a difference between transplanted and non-transplanted groups of more than 4 points of the BBB score, while transplantation at the chronic stage was without beneficial effect. Animals transplanted subacutely with FBr-NPCs showed no significant improvements better than sham. This contrasts to several previous studies, such as Watanabe et al. (2004), who showed similar functional improvement with rodent SCNPCs and FBr-NPCs in a SCI contusion model. We want to emphasize that the lack of effect in a single study does not prove that forebrainderived NPCs are without therapeutic effect. Second, the FBr-NPCs may have resulted in significant functional improvement and neuroprotection if transplanted acutely, or at other time-points not included in our study. Thus, from the present data we can only conclude that SCNPCs have therapeutic effects when transplanted acutely or subacutely. With regard to timing, our data indicate that SC-NPCs should be transplanted as soon after injury as possible to increase the chances of a positive effect, probably also in a clinical situation. Several studies have shown that the levels of potentially toxic cytokines in the spinal cord increase rapidly after a SCI (Nakamura et al., 2003; Pineau and Lacroix, 2007), which may harm the transplanted cells. This has however not been investigated in transplantation experiments. We addressed this issue by analyzing the number of human cells in the host spinal cord. Although there were no significant differences between the groups, the mean number of human cells was highest in the group transplanted acutely. Hence, the acutely injured cord does not seem to be a hostile milieu for transplanted human NPCs. The reason may be that NPCs are less sensitive to apoptotic stimuli, as has been found for other neural stem cells (Tamm et al., 2004). Transplanted NPCs can also suppress several inflammatory mechanisms (Fainstein et al., 2008) and reduce the local synthesis of proinflammatory cytokines, thereby protecting the transplant, and possibly also improving conditions for recovery by axonal regeneration. The number of human cells 20 weeks after transplantation greatly exceeded the number of transplanted NPCs, in some animals as much as 9-fold. This contrasts to most of earlier transplantation studies (Hofstetter et al., 2005; Karimi-Abdolrezaee et al., 2006; Tarasenko et al., 2007), but similar proliferation was reported after transplantation of human neural stem cells to chronic SCI in mice (Salazar et al., 2010). We have previously reported that cell death in transplanted neurospheres is surprisingly low, and some proliferation of the grafted SC-NPCs continues, but peaks already at 1 day and then declines slowly (Emgard et al., 2009). This results in a net increase of human cells. Importantly, in spite of this proliferation, we did not find any signs of tumor-like cell aggregates, which are in alignment with a previous study comparing NPCs derived from human fetal tissue and embryonic stem cells (Sundberg et al., 2011). The lack of tumor growth was also corroborated by immunostaining with the proliferation marker PCNA at 20 weeks post-grafting, showing very few mitotic cells. The therapeutic effect of SC-NPCs declined when the delay between injury and transplantation increased. This suggested to us that neuroprotection may be the major mechanism of the grafted NPCs, rescuing spinal cord neurons endangered by the injury. In previously published studies, neuroprotective effects of transplanted cells have been assessed
Fig. 5. In (A), an overview of a transplant of SC-NPCs to contusion SCI, with cells immunolabeled with the human-specific antibodies hsp-27 (red) and hnp (green) in the neural parenchyma surrounding several cystic cavities (c). (C) shows the same staining in higher magnification. The graph in (B) shows the proportion of cells IR to GFAP, nestin, β-tubulin type III and MAP2 of human NPCs transplanted to lateral compression SCI. Each bar represents the mean of 6 rats ± SD. There were no significant differences among the treatment groups with respect to any of the markers. In the following images examples of a human β-tubulin type III (red) expressing cell with a hnp-IR nucleus (green) are shown, indicated by an asterisk (D); hsp-27IR human cells (E) of which one (arrow) is also identified as MAP2-IR (F) ventral to a central lesion cavity (cav); nestin-IR human cells (G) with partial overlap (arrow) with GFAP immunoreactivity (H) and some structures only expressing GFAP (arrowhead). In (I), PCNA-IR proliferating cells (green) can be identified as human cells by the co-staining with hnp (red) resulting in yellow color of the nuclei. The intensely stained green spot on the left side of the picture is an artifact. Scale bars correspond to 10 μm (D–H) and 20 μm (C, I).
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Fig. 6. The number of rat host β-tubulin type III-IR neurons in a 4.2 mm long segment of the spinal cord, centered around the lesion epicenter, in the different transplantation groups of the lateral compression SCI study. Each bar represents the mean of 6–7 rats ± SD, *p b 0.05.
by calculating total tissue volume, in some studies using histological staining to delineate the graft and exclude it from the analysis of the host tissue. However, since grafted cells integrate in the host tissue such delineation is not accurate, and the volume of host spinal tissue will inevitably include human cells. Consequently, volume measurements will not reliably reflect the extent of tissue sparing. We therefore counted surviving rat neurons in a 4.2 mm long segment of the injured spinal cord, including the epicenter of the injury and the site of NPC transplantation. All rats suffered a major loss of neurons in the injured part of the cord, but the number of surviving neurons in the group with the best functional outcome – the acutely transplanted rats – was almost 3 times as large as in sham-transplanted rats. In the rats transplanted subacutely, the second best functional outcome was paralleled by twice as many surviving rat neurons as in the control group. The other groups, which did not achieve better functionality than the control group, also did not contain more surviving host neurons than the control group. This is a strong indication that neuroprotection is the main therapeutic effect of the grafted NPCs. The cell type(s) responsible for the protective effect are most likely the immature NPCs, which is the cell type present during the 7–9 days therapeutic window, while differentiation takes place weeks–months after grafting. In accordance with this, the ratios of differentiated cell types were not correlated to functional improvement. Whether the neuroprotective effect of the SC-NPCs is mediated by release of growth factors (Bottai et al., 2008; Madhavan et al., 2008), by gap junctions between transplanted NPCs and host cells (Jäderstad et al., 2010) or by other mechanisms remains to be established. Transplanted NPCs have however also been shown to support regeneration of severed or sprouting axons (Boido et al., 2009), and although our results suggest that neuroprotection is the most important mechanism of transplanted SC-NPCs, we can not exclude that improved regeneration of host spinal cord axons contributed to the functional effect. The functional recovery data show that there was no enhanced functional recovery in transplanted rats until three weeks after injury, while neuroprotective treatments often result in functional improvement much earlier (see e.g. von Euler et al., 1994). This may point to regenerative processes contributing to the effects of NPCs, but an alternative explanation is that the neuroprotection is delayed because NPCs protect neurons by interfering with late steps in the degeneration process, while many drugs such as glutamate receptor antagonists inhibit the initial trigger of cell death.
Furthermore, the fact that SC-NPCs transplanted to chronic SCI did not improve function may be taken as an argument against regeneration as a treatment mechanism. However, a recent study showed that antisera to the neurite growth-inhibitory Nogo-A had to be administered within a week after injury (Gonzenbach et al., 2012). Their data show that regeneration-enhancing treatments also can have a short therapeutic window. The lack of effect of our chronic transplantation does therefore not rule out regeneration as a contributing mechanism. Concerning differentiation of grafted NPCs, we found that the vast majority of NPCs derived from the spinal cord and forebrain differentiated into astrocytes. It has been claimed that regeneration in the injured spinal cord can be enhanced by human glial restricted progenitors (GRPs), but only if these progenitors were first differentiated to astrocytes by exposure to bone morphogenetic protein (BMP) in vitro, while differentiation with CNTF did not have the same effect (Davies et al., 2011). However, Haas and Fischer (2013) found that GRP as well as astrocytes produced by differentiating GRP with BMP or CNTF all enhanced regeneration equally well. In our study CNTF was used during culture of NPCs – which we have found to be necessary for long-term culture of SC-NPCs (Akesson et al., 2007) – but we did not apply any pre-differentiation step. It has previously been suggested that astrocyte differentiation of grafted stem or progenitor cells is associated with allodynia (Hofstetter et al., 2005; Macias et al., 2006). The lack of allodynia in our rats shows that astrocyte differentiation is not per se responsible for allodynia after transplantation of neural stem and progenitor cells. Regarding possible mechanisms of the grafted NPCs, Hawryluk and colleagues recently showed that grafts of mouse adult NPCs from the subventricular zone remyelinate host spinal cord axons to improve function (Hawryluk et al., 2013). However, differentiation of human SC-NPCs to oligodendrocytes in our study was negligible, ruling out this mechanism. To summarize these various view points, our data are compatible with transplanted SC-NPCs having a primary effect which is neuroprotective, rescuing compromised neurons at the periphery of the lesion. The NPC grafts may in addition also enhance regeneration of severed axons, re-connecting neurons below the injury with supraspinal nuclei, or re-establishing local neuronal circuitry, explaining the delayed effect of the graft, thereby contributing to the functional effects of the transplanted SC-NPCs. However, we have no direct evidence to support this. From a clinical perspective, the neuroprotective effect of transplanted SC-NPCs has an important advantage. Neuroprotective drugs typically have to be administered minutes to hours after injury. SC-NPC transplantation can reduce the tissue loss even if treatment is delayed for a week. It should be emphasized that in the present study we investigated SCI of moderate severity. The efficacy of transplanted NPCs may be different in more severe SCI. In light of the finding that the therapeutic window for neuroprotective treatments probably depends on the severity of injury (Andrade et al., 2008), the window for SC-NPC transplantation may also be different in severe SCI, an issue that should be clarified for any cell type that is a candidate for clinical application. Conclusion We have demonstrated a robust therapeutic effect of human SCNPCs, improving the functional outcome in two biomechanically different animal SCI models when transplanted up to 9 days after SCI. Twenty weeks after transplantation the number of human cells was several folds higher than transplanted, with no signs of tumor-like growth and only occasional human cells still proliferating. The functional improvement correlated with rescue of host spinal cord neurons, providing evidence for SC-NPCs being neuroprotective. Importantly, we found no signs of mechanical allodynia in transplanted animals. These data show that human SC-NPCs are suitable candidates for clinical trials in acute–subacute SCI.
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Neuroprotective effects of human spinal cord-derived neural precursor cells after transplantation to the injured spinal cord Mia Emgård a,1, Jinghua Piao a,1,2, Helena Aineskog a,b, Jia Liu a, Cinzia Calzarossa a, Jenny Odeberg a, Lena Holmberg a, Eva-Britt Samuelsson a, Bartosz Bezubik c, Per Henrik Vincent a, Scott P. Falci d, Åke Seiger a,b, Elisabet Åkesson a,b,1, Erik Sundström a,b,⁎,1 a
Division of Neurodegeneration, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Geriatric Clinic Res Lab., Novum, S-14186 Stockholm, Sweden Stockholms Sjukhem Foundation, Mariebergsgatan 22, S-11235 Stockholm, Sweden c Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, S-14186 Stockholm, Sweden d Department of Neurosurgery, Craig Hospital, 3425 S. Clarkson St., Englewood, CO 80110, USA b
a r t i c l e
i n f o
Article history: Received 12 July 2013 Revised 1 November 2013 Accepted 27 December 2013 Available online 8 January 2014 Keywords: Spinal cord injury Neural precursor cells Transplantation Neuroprotection Motor function
a b s t r a c t To validate human neural precursor cells (NPCs) as potential donor cells for transplantation therapy after spinal cord injury (SCI), we investigated the effect of NPCs, transplanted as neurospheres, in two different rat SCI models. Human spinal cord-derived NPCs (SC-NPCs) transplanted 9 days after spinal contusion injury enhanced hindlimb recovery, assessed by the BBB locomotor test. In spinal compression injuries, SC-NPCs transplanted immediately or after 1 week, but not 7 weeks after injury, significantly improved hindlimb recovery compared to controls. We could not detect signs of mechanical allodynia in transplanted rats. Four months after transplantation, we found more human cells in the host spinal cord than were transplanted, irrespective of the time of transplantation. There was no focal tumor growth. In all groups the vast majority of NPCs differentiated into astrocytes. Importantly, the number of surviving rat spinal cord neurons was highest in groups transplanted acutely and subacutely, which also showed the best hindlimb function. This suggests that transplanted SC-NPCs improve the functional outcome by a neuroprotective effect. We conclude that SC-NPCs reliably enhance the functional outcome after SCI if transplanted acutely or subacutely, without causing allodynia. This therapeutic effect is mainly the consequence of a neuroprotective effect of the SC-NPCs. © 2014 Elsevier Inc. All rights reserved.
Introduction The last decade has seen an impressive number of articles on experimental cell therapies for spinal cord injury (SCI), involving embryonic stem cells, adult (Parr et al., 2008) and fetal (Ogawa et al., 2002) somatic neural stem cells, oligodendrocyte progenitor cells (Keirstead et al., 2005), umbilical cord blood cells (Cho et al., 2008), olfactory mucosa (Iwatsuki et al., 2008), different Schwann cell preparations (Paino Abbreviations: SCI, spinal cord injury; NPCs, neural precursor cells; SC-NPCs, spinal cord-derived NPCs; FBr-NPCs, forebrain-derived NPCs; GRPs, glial restricted progenitor cells; CNTF, ciliary neurotrophic factor; PFA, paraformaldehyde; PBS, phosphate-buffered saline; Hsp-27, heat shock protein 27; Hnp, human nuclear antigen; PCNA, proliferating cell nuclear antigen; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; IR, immunoreactive. ⁎ Corresponding author at: Division of Neurodegeneration, Geriatric Clinic Res Lab., Novum 5th floor, S-14186 Stockholm, Sweden. E-mail address: [email protected] (E. Sundström). 1 These authors contributed equally to the study. 2 Present address: Memorial Sloan-Kettering Cancer Center, RRL 469, 411 East 67th Street, New York, NY 10065, USA. 0014-4886/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.12.022
et al., 1994) and olfactory ensheathing cells (Li et al., 1997). Several of these cell types are now used in on-going clinical trials, or have been approved for clinical trial by regulatory authorities. However, there are a depressing number of treatments that have shown effects in animal disease models but failed in clinical trials. We therefore set out to evaluate a promising human cell type in two very different animal models of SCI. Neurospheres are free-floating heterogeneous cultures of immature neural precursor cells (NPCs) (Reynolds and Weiss, 1996), composed of neural stem cells, more committed progenitor cells, as well as occasional differentiated astrocytes and post-mitotic neurons (Piao et al., 2006). A number of studies have shown that there are differences between in vitro-expanded neural stem and progenitor cells due to their regional origins (Armando et al., 2007; Kelly et al., 2009; Onorati et al., 2011). Transplantation of NPCs, murine or human, has been shown to improve functional parameters in SCI animals (Iwanami et al., 2005; Ogawa et al., 2002; Watanabe et al., 2004). NPCs are therefore possible candidates for clinical application in SCI patients. It is however not clear if the regiondependent differences seen in neural stem and progenitor cells translate into different therapeutic effects in SCI.
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There are several possible mechanisms through which cell therapy may restore functions after SCI. Neuroprotection is one, the transplant acting by reducing endogenous toxic substances, inhibiting inflammatory reactions and/or releasing growth factors and other supportive substances (Madhavan et al., 2008; Yasuhara et al., 2006). Since the reversible degenerative processes after experimental SCI most likely proceed for a few weeks in rats, neuroprotection may be a mechanism through which acutely and subacutely transplanted cells act. In later stages, functional effects of transplanted cells may be due to the support of host axon re-growth through release of trophic factors, reduction of neurite growth-inhibitory substances, enhancement of re-myelination of axons, or by supplying the injured tissue with an extracellular matrix that is more permissive to regeneration. In rodent SCI models it is also possible that functional effects of a graft is due to modulation of the central pattern generators, without any improved supraspinal control of movements. In vitro studies have shown that NPCs have neuroprotective effects, which have also been suggested by in vivo studies (Mothe et al., 2013). Others have reported that these cells act by supporting regenerating host axons (Gamm et al., 2007; Pfeifer et al., 2004). The timing of transplantation also has other implications. After measuring the levels of potentially cytotoxic cytokines, Nakamura and coworkers argued that the spinal cord is a hostile environment to transplanted cells the first week after injury (Nakamura et al., 2003). As a consequence, experimental transplantation studies are with few exceptions carried out 9 days after injury. However, there are to our knowledge no systematic comparisons between transplantations to the spinal cord at different time-points, a very important issue with regard to clinical applicability. In the present study we have analyzed the functional effects of human spinal cord-derived NPCs (SC-NPCs) transplanted to SCI animals. To evaluate the potential of these NPCs as cell therapy for human SCI, we initially applied the NPC transplantation to dorsal contusion injuries with very rapid onset using the force-feedback IH impactor (Scheff et al., 2003). This was followed by a more extensive study using another type of SCI model, lateral compression injury, with a slow onset as a result of application of an aneurysm clip (von Euler et al., 1997), comparing NPC transplantation at different time points after injury. We found that human SC-NPCs improve functional outcome in both SCI models, but only after acute and subacute transplantations, and the functional improvement correlated with a neuroprotective effect of the transplanted NPCs, which reduced the loss of host spinal cord neurons. Materials and methods Animals Adult immunodeficient athymic female rats (Hsd: RH-rnu/rnu, Harlan) were housed in an isolated environment, with autoclaved water and food pellets ad lib. The use of research animals was done in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Swedish Animal Protection Act, and the experimental procedures were approved by the Regional Ethics Committee on Animal Research, Stockholm, Sweden. Human neurosphere culture NPCs transplanted as neurospheres were derived from two types of donor tissue; SC-NPCs from the spinal cord, and FBr-NPCs from the subcortical forebrain, 5.5–9 weeks of gestation. The embryonic–fetal tissue was retrieved from elective routine abortions, with written consent from the pregnant women. All procedures for the use of human cells for experimental studies on SCI were approved by the Regional Ethical Committee, Stockholm. Tissue was dissected and cultured as earlier described by us (Piao et al., 2006). Briefly, dissection was made under sterile conditions in DMEM with F12 (DMEM/F12, 1:1, Life Technologies,
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Gaithersburg, MD, USA), and mechanical dissociation of the tissue was performed using a glass-Teflon homogenizer, using the entire spinal cord or sub-cortical forebrain of one fetus to establish NPC cultures. The cells were cultured as free-floating neurospheres in DMEM/F12 complemented with glucose (0.6%, Sigma, St. Louis, MO, USA), Hepes (5 mM, Life Technologies), heparin (2 μg/ml, Sigma), N2 supplement (1%, Life Technologies), basic fibroblast growth factor (20 ng/ml, R&D Systems, Minneapolis, MN, USA), epidermal growth factor (20 ng/ml, R&D Systems) and ciliary neurotrophic factor (CNTF, 10 ng/ml, R&D Systems) at an initial density of 40,000–50,000 cells/cm2 in 20 ml of medium. The NPCs were maintained at 37 °C in 5% CO2, with addition of fresh medium twice a week. They were passaged every 7 to 10 days with enzymatic dissociation using TrypLE Express (Life Technologies) for 4–5 min at 37 °C followed by gentle mechanical dissociation. Selection of neurospheres for transplantation Using quantitative cell counts in immersion-fixed, sectioned mediumsized neurospheres, we established the equation n = exp (k × s + m), k = 0.009 and m = 6.26 describing the relation between the diameter (s) and the number of cells in the neurosphere (n) for neurospheres with diameters between 100 and 500 μm. Neurospheres at passage 3–8 were used for all experiments. In the neurosphere cultures, spheres with a diameter between 150 and 300 μm were identified, the number of cells per neurospheres calculated, and 10–12 neurospheres were chosen to give a total of 100,000 cells ± 10% to be transplanted to each rat. The neurospheres were kept in cell culture medium without growth factors pending transplantation. Animal surgery The weight of the rats was 170–200 g at the time of surgery. They were injected with Atropin (0.05 mg/kg i.p., NM Pharma AB) 30 min before surgery, and anesthetized using Hypnorm (fentanyl citrate, 0.22 mg/kg, and fluanisone, 6.8 mg/kg, Janssen Pharmaceutical) and Dormicum (midazolam, 3.4 mg/kg, Hoffman-La Roche). Body temperature was kept at 38 °C throughout surgery. Lumbar spinal cords were surgically exposed by partial laminectomy of vertebra Th13, the dura was cut open, and a few drops of Xylocaine (lidocaine hydrochloride 20 mg/ml, AstraZeneca) were placed on the exposed spinal cord. Spinal cord contusion injury was achieved with an IH spinal cord impactor (Precision Systems and Instrumentation, LLC), the force set to 175 kdyn with no dwell time. For lateral compression injuries, a modification of the method of Rivlin and Tator (1978) was used. A bulldog clamp was applied to compress the spinal cord for 30 s at the lower half of the spinal cord segment Th13. In acutely transplanted animals, NPCs were injected in the same session as the injury (see below). For the other animals, a layer of Lyoplant (B/Braun Aesculap AG) was placed on the spinal cord as dura substitution before the wound was sutured. The rats were subcutaneously injected twice with 3 ml Ringer/glucose (2.5%) before and after surgery. After surgery, the rats were given intramuscular injections of Temgesic (buprenorphine, 7 μg/kg, Reckitt & Colman) twice a day for four days to avoid allodynia, and Borgal (trimetoprim sulfa, 15 mg/kg s.c., Intervet International B.V.) to prevent from urinary infection. The urinary bladders were emptied manually twice daily until spontaneous evacuation was present. Transplantation procedures Transplantation to dorsal spinal cord contusion injuries was performed 9 days after lesion. Rats were randomized for transplantation, and either received 100,000 SC-NPCs or a 3 μl injection of growth factor-free cell culture medium as sham transplantation. At the time of transplantation, animals were anesthetized, the wound re-opened and the spinal cord exposed. A glass capillary (0.3 mm end
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inner diameter) was attached to a 10 μl Hamilton syringe with Teflontubing, to allow control that all neurospheres entered the spinal cord. The capillary, loaded with 3 μl of neurosphere suspension containing 10–12 neurospheres (=100,000 ± 5000 cells), was placed at the site of the injury, immediately lateral to the midline and inserted approximately 1 mm. The neurospheres suspended in 3 μl were injected during 2–3 min, the glass capillary then kept in place for another minute, before being slowly retracted, carefully making sure no neurospheres leaked out of the cord. For transplantation of NPCs after lateral compression injuries, four groups of rats received 100,000 human NPCs in neurospheres as follows: 1) transplantation of SC-NPCs 10 min after compression injury (SC-Acute), 2) transplantation of SC-NPCs one week after compression injury (SC-Subacute), 3) transplantation of FBr-NPCs one week after compression injury (FBr-Subacute), and 4) transplantation of SC-NPCs 7 weeks after compression injury (SC-Chronic).
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Time post-lesion (weeks) Fig. 2. BBB-locomotor score in rats injured by a lateral compression SCI, and transplanted acutely (0 days) (n = 8), subacutely (7 days) (n = 10) and chronically (7 weeks) (n = 12) after injury with SC-NPCs, or subacutely (n = 10) after injury with FBr-NPCs, and the sham graft group (n = 10). Each data point represents the mean ± SD, **p b 0.01.
Behavioral assessments To study hindlimb sensory motor function, rats were scored according to the Basso, Beattie, Bresnahan (BBB) locomotor rating scale (Basso, 2004; Basso et al., 1995) during 4 min ambulation on an elevated 65 × 150 cm surface. The tests were video recorded, and two independent observers blinded to the treatment evaluated each rat. The rats were evaluated pre-operatively and then on regular intervals for 18 weeks (see Figs. 1 & 2). Hindlimb motor function was also assessed using a grid-walk test. The equipment consisted of a 150 × 20 cm grid with 3 × 3 cm holes and a platform at the end with a small wooden shelter and sweet rat chow pellets. During three consecutive trials, the number of times hind-paws slipped or was misplaced was recorded, and the mean number of mistakes calculated. To be able also to include rats that lacked weight-bearing ability, their score was set to 47 for each hindlimb which equals a stepping mistake at every hole in the grid, giving a maximum score of 94. To assess increased pain sensitivity, pain threshold to pressure to the torso and hindlimbs was tested before the injury, immediately after injury and 1, 2, 6, 10 and 18 weeks later. The test was carried out using von Frey filaments (Stoelting) ranging from 0.4 to 447 g (Yu et al., 1999). Starting with the thinnest filament, rats were tested with increasing pressure until an avoidance response occurred three times for the same pressure.
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Immunohistochemistry After 20 weeks, the rats were transcardially perfused with Ca2+-free Tyrode's solution, followed by phosphate-buffered 4% paraformaldehyde (PFA, Merck Millipore). The tissue was post-fixed for 2 h, rinsed and cryo-preserved in 20% sucrose at 4 °C for at least 24 h. The spinal cords were sectioned at 10 μm in a cryostat and mounted on gelatincoated slides. Primary antibodies used were human-specific rabbit anti-heat shock protein 27 (hsp-27, 1:1500, Medical & Biological Laboratories), mouse anti-human nuclear protein (hnp, 1:150, Merck Millipore), mouse anti-proliferating cell nuclear antigen (PCNA, 1:100, Cell Signaling Technology), rabbit anti-nestin (1:250, Millipore Merck), rabbit antiβ-tubulin type III (1:1200, Nordic Biosite), mouse monoclonal humanspecific anti-glial fibrillary acidic protein (GFAP, 1:200,000, Sternberger Monoclonals), and rabbit anti-microtubule-associated protein 2 (MAP2, 1:50, Merck Millipore). Primary antibodies were diluted in 0.1 M phosphate buffer with 0.3% Triton X-100 (TPBS). The secondary antibodies used were conjugated to Cy3 (Jackson ImmunoResearch Laboratories Lab. Inc.) or Alexa Fluor 488 (Life Technologies). Sections were treated with 1.5% normal goat serum (Sigma) at room temperature for 30 min, incubated with primary antibodies at 4 °C overnight, rinsed before a two hour incubation with secondary antibodies at room temperature. All sections were counterstained with nuclear marker Hoechst 33342 (30 μg/ml, Life Technologies). The immunolabeled tissue sections were captured using the Openlab software for MacOS (Improvision) and a CCD camera (Hamamatsu ORCAER) connected to the fluorescence microscope (Zeiss). Quantification was performed by one person blinded to the identity of the tissue sections. Quantitative histological analysis
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Time post-lesion (weeks) Fig. 1. BBB-locomotor score in rats subjected to a contusion SCI, transplanted with SCNPCs (n = 10) or sham-treated (n = 8) 9 days after injury. Animals were functionally evaluated for 18 weeks post-lesion. Data points represent mean ± SD, **p b 0.01 vs controls.
Human cells were defined as hsp-27 immunoreactive (IR) cell somata surrounding Hoechst 33342-stained nuclei. After an initial screening, a starting section outside the human graft was randomly chosen. From this section, every 30th section was used for analysis. In each tissue section, a random starting point was chosen in the upper left quadrant, from which every 4th field of view moving to the lower right quadrant was used. This resulted in 4–6 fields of view being evaluated at 40× magnification. In each field of view, all human cells were counted, and the total sum of human cells was calculated using the Abercrombie correction. To quantify the proportion of human donor cells expressing phenotype markers, tissue sections were double-stained for hsp-27 or hnp in
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Time post-lesion (weeks) Fig. 3. Total number of paw misplacing in the grid-walk test, performed in rats injured by a lateral compression SCI, transplanted immediately (acute, 0 days), 7 days (subacute) and 7 weeks (chronic) after injury with SC-NPCs, or 7 days after injury with FBr-NPCs, and the sham graft group. Each data point represents the median of 8–12 rats with the 0.25 and 0.75 percentiles indicated at day 2–18, but for the sake of clarity only for the acute SCNPC group (error bar caps the left side), the subacute SC-NPC group (error bar caps on the right side) and the control group (bilateral error bar caps). *p b 0.05.
combination with nestin, GFAP, β-tubulin type III or MAP2. For each antibody, one tissue section 1.2 mm rostral and one section 1.2 mm caudal from the epicenter were used. At least 100 hsp-27-IR cells, and 20 cells labeled by the other antibody were sampled, if necessary additional adjacent tissue sections were added to the analysis. In each tissue section, all labeled cells were counted, and corrected using the Abercrombie formula. For quantification of rat spinal cord neurons, every 60th section of a 4.2 mm spinal cord segment centered around the injury epicenter was used. Rat host neurons – β-tubulin type III-IR cytoplasm surrounding a Hoechst 33342-stained nucleus negative human hsp-27 – were counted, and the total number of rat neurons in the 4.2 mm segment was calculated with the Abercrombie correction. For all quantifications of immunohistochemically labeled tissue, sections from 6 rats per treatment group were used. Statistics and exclusion criteria In the in vivo experiments, occasional rats in each group were much more severely injured. Therefore, a blinded person excluded all rats that did not reach a BBB score of at least 7 within 5 weeks after injury. The number of excluded rats was similar in all treatment groups (contusion injury experiment: transplanted n = 1, control n = 1; compression injury experiment: SC-Acute n = 2, SC-Subacute n = 2, FBr-Subacute n = 4, SC-Chronic n = 2, control n = 2). In two of the groups of the compression injury experiment (SC-Subacute and SC-Chronic), two animals died before the end of the evaluation period. For statistical analyses we used ANOVA for repeated measures with Fisher's post-hoc test for inter-group comparisons (BBB scores and pain thresholds), Kruskal–Wallis test (grid-walk due to skewed data), onefactor ANOVA and Fisher's post-hoc test (morphological evaluations), p b 0.05 was considered statistically significant. Statistical analysis was performed using StatView 4.5 and Instat 3.0. Group data are expressed as mean ± standard deviation (SD). Results Functional analyses We first studied the effects of subacute (9 days) intraspinal transplantation of neurospheres of SC-NPCs on functional outcome after
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Time post-lesion (weeks) Fig. 4. Assessment of pressure-induced pain thresholds in rats injured by a lateral compression SCI and transplanted as described. Each data point represents the mean of 8–12 rats ± SD. There were no significant differences among the treatment groups.
contusion SCI. All animals improved rapidly during the first weeks after injury. Thereafter improvement progressed slower, and with different courses for the two groups. Six weeks after injury, the control group did not improve further, and remained at an average score of approximately 13.5, while animals that received human SC-NPCs 9 days after injury continued to improve during the 18 weeks of study, reaching a mean BBB score of 16.6, significantly better than the control group (Fig. 1). We then investigated the therapeutic effects of human NPCs in a different SCI animal model, and the duration of the therapeutic window. In this experiment we used a SCI model with a sustained compression during 30 s, in contrast to the brief impact in the contusion injury experiment. SC-NPCs were transplanted in the acute (0 day), subacute (7 days) and chronic (7 weeks) stages of injury while FBr-NPCs were only transplanted subacutely (7 days). Acutely transplanted rats reached the highest BBB score, 16.7, of all the groups, compared to 12.6 for the control group. Subacutely transplanted rats also performed significantly better than control rats throughout the 18 weeks of study, and significantly better than rats transplanted with FBr-NPCs (Fig. 2). We did not observe any systematic difference between the right and left hindlimb performance. In the grid-walk test to assess hindlimb function the acutely transplanted rats made significantly fewer mistakes than the control group, while the other transplanted groups did not perform significantly better than the control group (Fig. 3). Pain sensitivity to pressure was analyzed using von Frey filaments to apply localized pressure to the skin. The mean threshold for an avoidance response was between 160 and 270 g for the different experimental groups. This decreased to 100–140 g two weeks after injury, and increased slightly up to 18 weeks post-lesion. We found no significant difference between the groups, or indication of increased sensitivity (i.e. lowered response threshold) in the transplanted groups (Fig. 4).
Morphological analyses At post-mortem analysis, cells positive for the two human-specific markers hsp-27 and hnp could be seen throughout more than 6 mm of the spinal cord in contusion and compression injuries. The transplanted cells were distributed from the remaining ventral white matter to the dorsal horns in contusion (Fig. 5A) and compression SCI. Farther from the lesion, the human cells were mainly located in the white matter. Although the NPCs were injected in the lesion center, there were still large cystic cavities in many animals, with human cells in the tissue surrounding the cavity.
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To determine if survival of transplanted NPCs could explain the differences in therapeutic effect, we compared the number of human cells 20 weeks after injury. Surprisingly, in almost every rat there were many more human cells than the 100,000 NPCs that were transplanted. Due to the large differences within the groups, the groups were not significantly different. However, it is noteworthy that the mean number of human cells was highest in rats transplanted acutely with SC-NPCs (517,000 ± 304,000). There were almost as many cells (426,000 ± 276,000) in rats transplanted subacutely with FBr-NPCs, while the mean numbers were slightly lower in those who received SC-NPCs subacutely (325,000 ± 165,000) and chronically (239,000 ± 180,000) rats. The number of human cells in individual rats did not correlate with functional outcome (data not shown). We also compared the proportion of human cells expressing nestin, GFAP, β-tubulin type III and MAP2 in the treatment groups (Fig. 5B). Cells positive for the early neuronal marker β-tubulin type III (Fig. 5D) and the more mature neuronal marker MAP2 (Figs. 5E–F) comprised less than 10%. In all groups, 80–90% of human cells were positive for the astrocyte marker GFAP, and 4–10% of these cells co-expressed nestin, indicative of a multipotent phenotype (Figs. 5G–H). The proportion of phenotypes was similar in all groups, indicating that differentiation of transplanted NPCs does not determine functional outcome. To evaluate possible neuroprotective effects of the grafted NPCs, we quantified the number of remaining host rat neurons in a 4.2 mm long segment of the injured spinal cord, centered around the lesion epicenter. In non-transplanted lesioned animals, an average of 27,000 neurons in the 4.2 mm spinal cord segment survived the contusion injury (Fig. 6). As a reference, the mean number of neurons in 5 uninjured rats of the same age was 289,200 ± 10,200 in a similar spinal cord segment (Fig. 6, hatched area). Thus, N90% of the neurons in these segments were lost after injury. In acutely transplanted rats more than 3 times as many host neurons could be found in the corresponding spinal cord segment (≈ 77% loss of host neurons). In animals acutely transplanted with SC-NPCs, almost twice as many neurons as in control animals were found (≈82% loss of host neurons). The survival of host neurons in these groups was significantly higher than in the control group, demonstrating a neuroprotective effect of the transplanted NPCs. We found no protective effect in rats transplanted in the chronic stage, or acutely with FBr-NPCs (Fig. 6). Discussion Before proceeding to clinical trial of cell therapy in SCI patients can take place, robust functional treatment effects of the particular cell type should be demonstrated in relevant animal models. Since every disease model has significant differences from the clinical condition it is intended to mimic, we believe a minimum requirement is to show functional effects in more than one injury model. In addition, issues such as the timing of transplantation and risks of side effects should be addressed. In the present study, we have examined the functional and morphological outcomes of human NPCs expanded in vitro, transplanted to two different animal models of SCI. The two rat SCI models used differ with respect to the biomechanics of injury. The contusion injury has an immediate onset of injury and duration of tissue displacement less than a second, while the compression develops during seconds and is sustained for 30 s.
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In both animal SCI experiments we found that hindlimb motor function was significantly improved by SC-NPCs transplanted in the subacute stage. The effect of acutely transplanted SC-NPCs studied in the compression injury model was even more pronounced, reaching a difference between transplanted and non-transplanted groups of more than 4 points of the BBB score, while transplantation at the chronic stage was without beneficial effect. Animals transplanted subacutely with FBr-NPCs showed no significant improvements better than sham. This contrasts to several previous studies, such as Watanabe et al. (2004), who showed similar functional improvement with rodent SCNPCs and FBr-NPCs in a SCI contusion model. We want to emphasize that the lack of effect in a single study does not prove that forebrainderived NPCs are without therapeutic effect. Second, the FBr-NPCs may have resulted in significant functional improvement and neuroprotection if transplanted acutely, or at other time-points not included in our study. Thus, from the present data we can only conclude that SCNPCs have therapeutic effects when transplanted acutely or subacutely. With regard to timing, our data indicate that SC-NPCs should be transplanted as soon after injury as possible to increase the chances of a positive effect, probably also in a clinical situation. Several studies have shown that the levels of potentially toxic cytokines in the spinal cord increase rapidly after a SCI (Nakamura et al., 2003; Pineau and Lacroix, 2007), which may harm the transplanted cells. This has however not been investigated in transplantation experiments. We addressed this issue by analyzing the number of human cells in the host spinal cord. Although there were no significant differences between the groups, the mean number of human cells was highest in the group transplanted acutely. Hence, the acutely injured cord does not seem to be a hostile milieu for transplanted human NPCs. The reason may be that NPCs are less sensitive to apoptotic stimuli, as has been found for other neural stem cells (Tamm et al., 2004). Transplanted NPCs can also suppress several inflammatory mechanisms (Fainstein et al., 2008) and reduce the local synthesis of proinflammatory cytokines, thereby protecting the transplant, and possibly also improving conditions for recovery by axonal regeneration. The number of human cells 20 weeks after transplantation greatly exceeded the number of transplanted NPCs, in some animals as much as 9-fold. This contrasts to most of earlier transplantation studies (Hofstetter et al., 2005; Karimi-Abdolrezaee et al., 2006; Tarasenko et al., 2007), but similar proliferation was reported after transplantation of human neural stem cells to chronic SCI in mice (Salazar et al., 2010). We have previously reported that cell death in transplanted neurospheres is surprisingly low, and some proliferation of the grafted SC-NPCs continues, but peaks already at 1 day and then declines slowly (Emgard et al., 2009). This results in a net increase of human cells. Importantly, in spite of this proliferation, we did not find any signs of tumor-like cell aggregates, which are in alignment with a previous study comparing NPCs derived from human fetal tissue and embryonic stem cells (Sundberg et al., 2011). The lack of tumor growth was also corroborated by immunostaining with the proliferation marker PCNA at 20 weeks post-grafting, showing very few mitotic cells. The therapeutic effect of SC-NPCs declined when the delay between injury and transplantation increased. This suggested to us that neuroprotection may be the major mechanism of the grafted NPCs, rescuing spinal cord neurons endangered by the injury. In previously published studies, neuroprotective effects of transplanted cells have been assessed
Fig. 5. In (A), an overview of a transplant of SC-NPCs to contusion SCI, with cells immunolabeled with the human-specific antibodies hsp-27 (red) and hnp (green) in the neural parenchyma surrounding several cystic cavities (c). (C) shows the same staining in higher magnification. The graph in (B) shows the proportion of cells IR to GFAP, nestin, β-tubulin type III and MAP2 of human NPCs transplanted to lateral compression SCI. Each bar represents the mean of 6 rats ± SD. There were no significant differences among the treatment groups with respect to any of the markers. In the following images examples of a human β-tubulin type III (red) expressing cell with a hnp-IR nucleus (green) are shown, indicated by an asterisk (D); hsp-27IR human cells (E) of which one (arrow) is also identified as MAP2-IR (F) ventral to a central lesion cavity (cav); nestin-IR human cells (G) with partial overlap (arrow) with GFAP immunoreactivity (H) and some structures only expressing GFAP (arrowhead). In (I), PCNA-IR proliferating cells (green) can be identified as human cells by the co-staining with hnp (red) resulting in yellow color of the nuclei. The intensely stained green spot on the left side of the picture is an artifact. Scale bars correspond to 10 μm (D–H) and 20 μm (C, I).
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Fig. 6. The number of rat host β-tubulin type III-IR neurons in a 4.2 mm long segment of the spinal cord, centered around the lesion epicenter, in the different transplantation groups of the lateral compression SCI study. Each bar represents the mean of 6–7 rats ± SD, *p b 0.05.
by calculating total tissue volume, in some studies using histological staining to delineate the graft and exclude it from the analysis of the host tissue. However, since grafted cells integrate in the host tissue such delineation is not accurate, and the volume of host spinal tissue will inevitably include human cells. Consequently, volume measurements will not reliably reflect the extent of tissue sparing. We therefore counted surviving rat neurons in a 4.2 mm long segment of the injured spinal cord, including the epicenter of the injury and the site of NPC transplantation. All rats suffered a major loss of neurons in the injured part of the cord, but the number of surviving neurons in the group with the best functional outcome – the acutely transplanted rats – was almost 3 times as large as in sham-transplanted rats. In the rats transplanted subacutely, the second best functional outcome was paralleled by twice as many surviving rat neurons as in the control group. The other groups, which did not achieve better functionality than the control group, also did not contain more surviving host neurons than the control group. This is a strong indication that neuroprotection is the main therapeutic effect of the grafted NPCs. The cell type(s) responsible for the protective effect are most likely the immature NPCs, which is the cell type present during the 7–9 days therapeutic window, while differentiation takes place weeks–months after grafting. In accordance with this, the ratios of differentiated cell types were not correlated to functional improvement. Whether the neuroprotective effect of the SC-NPCs is mediated by release of growth factors (Bottai et al., 2008; Madhavan et al., 2008), by gap junctions between transplanted NPCs and host cells (Jäderstad et al., 2010) or by other mechanisms remains to be established. Transplanted NPCs have however also been shown to support regeneration of severed or sprouting axons (Boido et al., 2009), and although our results suggest that neuroprotection is the most important mechanism of transplanted SC-NPCs, we can not exclude that improved regeneration of host spinal cord axons contributed to the functional effect. The functional recovery data show that there was no enhanced functional recovery in transplanted rats until three weeks after injury, while neuroprotective treatments often result in functional improvement much earlier (see e.g. von Euler et al., 1994). This may point to regenerative processes contributing to the effects of NPCs, but an alternative explanation is that the neuroprotection is delayed because NPCs protect neurons by interfering with late steps in the degeneration process, while many drugs such as glutamate receptor antagonists inhibit the initial trigger of cell death.
Furthermore, the fact that SC-NPCs transplanted to chronic SCI did not improve function may be taken as an argument against regeneration as a treatment mechanism. However, a recent study showed that antisera to the neurite growth-inhibitory Nogo-A had to be administered within a week after injury (Gonzenbach et al., 2012). Their data show that regeneration-enhancing treatments also can have a short therapeutic window. The lack of effect of our chronic transplantation does therefore not rule out regeneration as a contributing mechanism. Concerning differentiation of grafted NPCs, we found that the vast majority of NPCs derived from the spinal cord and forebrain differentiated into astrocytes. It has been claimed that regeneration in the injured spinal cord can be enhanced by human glial restricted progenitors (GRPs), but only if these progenitors were first differentiated to astrocytes by exposure to bone morphogenetic protein (BMP) in vitro, while differentiation with CNTF did not have the same effect (Davies et al., 2011). However, Haas and Fischer (2013) found that GRP as well as astrocytes produced by differentiating GRP with BMP or CNTF all enhanced regeneration equally well. In our study CNTF was used during culture of NPCs – which we have found to be necessary for long-term culture of SC-NPCs (Akesson et al., 2007) – but we did not apply any pre-differentiation step. It has previously been suggested that astrocyte differentiation of grafted stem or progenitor cells is associated with allodynia (Hofstetter et al., 2005; Macias et al., 2006). The lack of allodynia in our rats shows that astrocyte differentiation is not per se responsible for allodynia after transplantation of neural stem and progenitor cells. Regarding possible mechanisms of the grafted NPCs, Hawryluk and colleagues recently showed that grafts of mouse adult NPCs from the subventricular zone remyelinate host spinal cord axons to improve function (Hawryluk et al., 2013). However, differentiation of human SC-NPCs to oligodendrocytes in our study was negligible, ruling out this mechanism. To summarize these various view points, our data are compatible with transplanted SC-NPCs having a primary effect which is neuroprotective, rescuing compromised neurons at the periphery of the lesion. The NPC grafts may in addition also enhance regeneration of severed axons, re-connecting neurons below the injury with supraspinal nuclei, or re-establishing local neuronal circuitry, explaining the delayed effect of the graft, thereby contributing to the functional effects of the transplanted SC-NPCs. However, we have no direct evidence to support this. From a clinical perspective, the neuroprotective effect of transplanted SC-NPCs has an important advantage. Neuroprotective drugs typically have to be administered minutes to hours after injury. SC-NPC transplantation can reduce the tissue loss even if treatment is delayed for a week. It should be emphasized that in the present study we investigated SCI of moderate severity. The efficacy of transplanted NPCs may be different in more severe SCI. In light of the finding that the therapeutic window for neuroprotective treatments probably depends on the severity of injury (Andrade et al., 2008), the window for SC-NPC transplantation may also be different in severe SCI, an issue that should be clarified for any cell type that is a candidate for clinical application. Conclusion We have demonstrated a robust therapeutic effect of human SCNPCs, improving the functional outcome in two biomechanically different animal SCI models when transplanted up to 9 days after SCI. Twenty weeks after transplantation the number of human cells was several folds higher than transplanted, with no signs of tumor-like growth and only occasional human cells still proliferating. The functional improvement correlated with rescue of host spinal cord neurons, providing evidence for SC-NPCs being neuroprotective. Importantly, we found no signs of mechanical allodynia in transplanted animals. These data show that human SC-NPCs are suitable candidates for clinical trials in acute–subacute SCI.
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