Adult Mouse Post Mortem Neural Precursors Survive, Differentiate, Counteract Cytokine Production and Promote Functional Recovery after Transplantation in Experimental Traumatic Spinal Cord Injury

Research Article

J Stem Cell Res Transplant. 2014;1(2): 1008.

Adult Mouse Post Mortem Neural Precursors Survive, Differentiate, Counteract Cytokine Production and Promote Functional Recovery after Transplantation in Experimental Traumatic Spinal Cord Injury

Carelli Stephana1#, Giallongo Toniella1#, Latorre Elisa1, Caremoli Filippo1, Claudio Gerace1, Basso Michele D2, Di Giulio Anna Maria1 and Gorio Alfredo1*

1Laboratory of Pharmacology, Department of Health Sciences, University of Milan, via A. di Rudinì 8 20142 Milan, Italy

2Department of Neuroscience, College of Medicine, The Ohio State University, 453 W 10th Ave, Columbus, OH 43210-1234 (USA)

#Authors contributed equally

*Corresponding author: Gorio Alfredo, Laboratory of Pharmacology, Department of Health Sciences University of Milan, Polo H. San Paolo, via A di Rudinì 8, 20 142 Milan, Italy

Received: August 08, 2014; Accepted: September 20, 2014; Published: September 22, 2014


Spinal cord injury (SCI) is a debilitating clinical condition, characterized by a complex of neurological dysfunctions. Adult neural stem cells (NSCs) from the subventricular zone of the forebrain have been considered a potential tool for cell replacement therapies. We have recently isolated a subclass of neural progenitors from the cadaver of mouse donors. These cells, named Post Mortem Neural Precursor Cells (PM-NPCs), express both erythropoietin and its receptor and their EPO-dependent differentiation abilities produce a significantly higher percentage of neurons than regular NSCs. The aim of the present study was to compare the reparative properties of PM-NPCs and those expressed by NSCs in a mouse model of traumatic spinal cord injury. PM-NPCs and NSCs were administered intravenously, and then functional recovery and fate of transplanted cells were studied. Animals transplanted with PM-NPCs showed a more remarkably improved recovery of hind limb function than NSCs treated animals. The PM-NPCs effect was accompanied by reduced myelin loss, counteraction of the invasion of lesion site by macrophages, and attenuation of cytokine production. PM-NPCs migrate mostly at the injury site, where they survive at a significantly higher extent than classical NSCs.

Keywords: Spinal cord injury; Neural stem cells; Transplantation; Regenerative medicine; Animal behaviour; Inflammation


Acute spinal cord injury (SCI), with an annual incidence of 22 to 59 cases per million population internationally, is a devastating disease that has a significant impact in society. Moreover, the majority of affected patients are young individuals (10-40 years old) resulting in a substantial burden of impairment and high costs to society [1,2]. There is currently no curative therapy, and the care in the acute phase is often limited to high-dose corticosteroid treatment, surgical stabilization and decompression aiming at the attenuation of further damage [1,3]. The pathophysiology of SCI is biphasic. Primary injury results from the immediate response to the trauma that cause axonal and blood vessels transection, lipid peroxidation and disruption of cell membranes. Secondary injury is due to the activation of degeneration that causes ischemia, inflammation, cytokine production, demyelination and death of multiple cell types [2,4]. A large number of studies have evaluated the effects of transplanting stem cells or stem cell-derived cells in spinal cord injury models, and remarkably, many studies using different strategies have indicated beneficial effects to a certain degree [5]. Transplanted cells can improve the recovery of function either by replacing partially the lost cells, or supplying a favorable environment that attenuates the effects of secondary degeneration thereby enhancing the amount of spared tissue at the injury site, i.e. transplanted stem cells must survive in such an unfavorable environment. We had recently reported the isolation of adult neural stem cells from SVZ several hours after death of the mouse donor which are capable of surviving in such an unfavourable environment [6]. This procedure provides a population of NSCs, named post mortem neural precursors (PM-NPCs), that in vitro differentiate preferentially in neurons. Such a process is dependent on the autocrine EPO release and is prevented by exposure to EPO and EPOR antibodies [6]. In this work we make a comparison between adult mouse PM-NPCs and adult NSCs by evaluating their effects after intravenous transplantation in a mouse model of spinal cord injury. Here we report that after intravenous administration an high number of PM-NPCs migrate to spinal cord lesion site and survive for a long time, differentiate mostly into neuron-like cells and reduced tissue degeneration enhancing preservation of myelin fibres. These events are preceded by the counteraction of cytokine production and associated with the promotion of a stable recovery of hind limb function. Their action is apparently superior to that of regular adult NSCs.

Materials and Methods

Animal care

For this study we used adult CD1 male mice 25–30 g in weight (Charles River, Calco, Italy). All the procedures were taken with the approval of the Review Committee of the University of Milan and met the Italian Guidelines for Laboratory Animals which conform to the European Communities Directive of November 1986 (86/609/ EEC). The animals were kept for at least 3 days before the experiments in standard conditions (22 ± 2°C, 65% humidity, and artificial light between 08:00 a.m. to 08:00 p.m.).

Neural stem cell isolation and culture

Neural stem cells (NSCs) were isolated from sub-ventricular zone of adult mice brain [7,8]. Their maintenance in culture, their differentiation, and their immunostaining were performed as described by Gritti and co-workers [7,8]. Briefly, 8 weeks old CD-1 albino were anesthetized by intraperitoneal (i.p.) injection of 4% chloral hydrate (0.1 mL/10 g body weight) and killed by decapitation. The brains were removed from adult mice and tissues containing the sub-ventricular zone (SVZ), were dissected out and transferred to a phosphate buffer solution containing penicillin and streptomycin 100 U/mL (Life Technologies), glucose (0.6%) at 4°C until the end of the dissection. Enzymatic digestion was performed by transferring the tissue to an Earl’s balanced salt solution (EBSS) (Sigma-Aldrich, Milan, Italy) containing 1 mg/mL papain (27 U/ mg; Sigma-Aldrich), 0.2 mg/mL cysteine (Sigma-Aldrich), and 0.2 mg/mL EDTA (Sigma-Aldrich), and incubated for 45 min at 37°C on a rocking platform. Tissue was then centrifuged at 123g and the supernatant was discarded. The pellet was re-suspended in 1 mL of EBSS and mechanically dissociated with a pipette, than cells were centrifuged at 123g and supernatant was discarded. This step was repeated trice as described [7]. Cells were counted and plated at 3500 cells/cm2 in DMEM-F-12 (Euroclone, Pero, Milan, Italy) containing 2 mm l-glutamine (Euroclone), 0.6% glucose (Sigma-Aldrich), 9.6 gm/ml putrescine (Sigma-Aldrich), 6.3 ng/ml progesterone (Sigma- Aldrich), 5.2 ng/ml sodium selenite (Sigma-Aldrich), 0.025 mg/ml insulin (Sigma-Aldrich), 0.1 mg/ml transferrin (Sigma-Aldrich), and 2 μg/ml heparin (sodium salt, grade II; Sigma-Aldrich), bFGF (human recombinant, 10 ng/mL; Life Technologies) and EGF (human recombinant, 20 ng/mL; Life Technologies). Spheres formed after 5–7d were harvested, collected by centrifugation (10 min at 123g), mechanically dissociated to a single-cell suspension, and re-plated in medium indicated above [8]. The total number of viable cells was assessed at each passage by Trypan blue exclusion. Stem cells used in these experiments were between the fifth to the fifteenth passage in culture.

Post Mortem-Neural precursor’s cells derivation, differentiation, and labelling

PM-NPCs were obtained from 6 weeks old CD-1 albino mice; their isolation, growth and characterization were performed following methods described for NSCs and set up by Gritti et al. [8]. Briefly, cells were isolated from the sub ventricular zone (SVZ) of adult male mice (CD1) six hours after their killing by cervical dislocation. Brains were removed and tissues containing the SVZ region were dissected, transferred to Earl’s Balanced Salt Solution (Life Technologies, Monza, Italy) containing 1 mg/ml papain (27 U/mg; Sigma-Aldrich, Milan, Italy), 0.2 mg/ml cysteine (Sigma- Aldrich), and 0.2 mg/ml EDTA (Sigma-Aldrich) and processed as described above. At the end of isolation procedure cells were collected by centrifugation and resuspended in the same growth medium indicated above [7,8]. Differentiation of both NSCs and PM-NPCs was performed by plating the dissociated stem cells at the density of 40,000 cells/cm2 in presence of adhesion molecules (Matrigel™, BD Biosciences, Buccinasco, MI, Italy) and bFGF (10ng/ml) for 48 hours, then cells were exposed to the same medium without bFGF and the addition of foetal bovine serum (1% vol/vol; Euroclone) for the following 5 days as previously described [6,8]. Then, the extent of differentiation was determined by immunocytochemical staining [6]. For transplantation NSCs and PM-NPCs were labelled with PKH26 (Sigma-Aldrich) just before the injection, following manufacturer’s instructions and previously described [9]. PKH26 is an not toxic cell dye characterized by a long aliphatic tails (PKH26) that allow the dye incorporation in lipid regions of the cell membrane [10,11].

Dead cells administration: PM-NPCs (1x106 cells) were treated with 1 ml of 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature, then 10 mL of PBS were added and cells were spun down at 123g, and suspended in PBS. This washing step was performed twice, and, finally, the ready to use dead PM-NPCs were suspended at the final concentration of 3,3 x 105 cells/50μl in saline solution.

Cell quantification at the lesion site: Cells were considered PKH26 positive, when fluorescence was visible by the confocal microscope (Leica TSC2; Leica Microsystems, Heidelberg, Germany) and if the spots had an emission wave length of 567 nm which correspond to the emission of PKH26. As negative reference the confocal analysis we used sections obtained from animals transplanted with unlabeled PM-NPCs. The counting of the cells was performed assessing the PKH26-positive cells in the transversal sections in a region of 4 mm centered at the lesion site. The lesion epicenter was defined in cross sections as the region with the minimum tissue sparing. The PKH26- positive cells present in a group of three consecutive sections (10 μm thick) were averaged, and such count was repeated every 400 μm. The total number of labeled cells was obtained integrating the curve obtained calculating the average of each sections across a 4 mm span across the epicenter of the lesion [12].

Spinal Cord Injury, experimental groups and cell administration

The traumatic SCI was performed using a commercially available Infinite Horizon (IH; Precision Systems and Instrumentation, LLC, Lexington, KY, USA) spinal cord injury device [13] at the T8 level. Surgery on the animals was performed as described elsewhere [13]. Briefly, animals were anesthetized with 2.5% isoflurane in oxygen (1 L/min; Farmagricola, San Donato, Milan, Italy) for 5 minutes before surgery. A dorsal vertical incision was made through the skin from T7 to T12, superficial fat pad was removed and T7 and T10 bilateral paravertebral muscles were cut. Laminectomy was performed and spinal cord exposed. The impactor tip was then positioned just above the cord following the constructor instructions, the force of 70 Kdyne was applied for 1 s to the cord. After the contusion the animal muscles were sutured and the skin closed by means of clips (2Biological Instruments, Besozzo, Varese, Italy). Experimental animals were divided into four groups: 1) Laminectomies mice (n=18); 2) Lesioned mice treated by i.v. route with phosphate buffer (PBS, n=24); 3) Lesioned mice transplanted by i.v. route with PM-NPCs (n=24); 4) Lesioned mice transplanted by i.v. route with dead PM-NPCs (n=12); 5) Lesioned mice transplanted by i.v. route with NSCs (n= 24). Intravenous administration was performed by injections in the tail vein. PM-NPCs, PBS. NSCs or dead PM-NPCs were administered after spinal cord lesion. The first treatment was a slow i.v. injection of 50 μl in the tail vein performed within 30 min after injury, followed by a second injection 6 h later and a third one 18 h after the lesion. Each cellular administration consisted of 3,3 x 105 cells in PBS for a total of 1x 106 cells. The choice of a time limit of 18 h after SCI for administering PM-NPCs was determined by the optimal permeability of the blood brain barrier at this time [14]. PM-NPCs between the 5th and the 9th passage in culture were used for these experiments, the cultures were tested for proliferation and differentiation ability before being transplanted [6]. Up to now we observed that transplanted animals can survive until 90 days after grafting, then were sacrificed to perform further investigations.

Behavioural tests and hind limb function

All outcome measures were assessed in a blinded fashion. Neurological function was evaluated first 24 h after injury and then twice a week for the first 4 weeks. The methods utilized are well known in the field of behavioural evaluation of recovery of function after SCI. Locomotor function and hind limb recovery after contusion were evaluated with the open field test according to the Basso mouse rating scale [15]. For behavioural experiments we used 5 animals in laminectomies group, and at least 12 animals for other groups. Allodynia-like responses in the unaffected forepaw were assessed by means of standard hotplate test and cold stimulation. For hotplate testing, mice were placed on hotplate and the latency to licking was measured. Non-responders were removed after 60s. The response to cold was tested by the application of ethyl chloride spray (Gebauer Company, Cleveland, OH, USA) to the palm surface. The response was rated 1 (no response), 2 (brief withdrawal with licking), and 3 (vocalization, withdrawal with licking, and aversion) [16].

Tissue collection and processing, histology and immunohistochemistry

At the end of the experimental period, animals were anesthetized by i.p. injection of cloralium hydrate (Sigma-Aldrich) 4% in distilled water, and perfused with 4% paraformaldehyde in phosphate buffer (PB) 0.1 M pH 7.4 by transcardial perfusion. Spinal cords were post-fixed overnight in the same fixative, cryoprotected with 30% sucrose (Sigma-Aldrich), and quickly frozen, stored at -80°C and sectioned by means of a cryostat (Leica). Every twentieth section was stained with thionin (Sigma-Aldrich). Cross-sections containing the lesion epicenter and the complete T8 segment cavitations were analyzed by computer-assisted image analysis. The percent of lesion was calculated as the area of the injured tissue divided by the area of the total cross-section at the level of the injury. Cryostat coronal sections (15 μm) were also collected onto glass slides and processed for immunocytochemistry. Sections were rinsed with PBS (Euroclone), treated with blocking solution (Life-Technologies) and incubated with primary antibodies overnight at 4°C. After treatment with primary antibodies, the sections were washed with PBS and incubated with appropriate secondary antibodies (Alexa Fluor® 488, Molecular Probes®, Life Technologies) for 2 hours at room temperature. Sections were washed in PBS, nuclei were stained with DAPI (1μg/ml final concentration, 10 minutes at room temperature; Sigma-Aldrich) and then mounted using the FluorSave Reagent (Calbiochem, Merck Chemical, Darmstadt, Germany) and analyzed by confocal microscopy. In control determinations, primary antibodies were omitted and replaced with equivalent concentrations of unrelated IgG of the same subclass. The following primary antibodies were used:, β-Tubulin III (1:150; Covance). For immunofluorescence, the following secondary antibodies were used: 488 goat-anti-mouse IgG (1:200; Alexa), 488 donkey-anti-rabbit IgG (1:200; Alexa).

Assessment of myelin preservation

In order to perform a homogeneous analysis, the staining was carried out on sections of non lesioned, Lesion+PBS and Lesion+cells animals placed on the same coverslip. Myelin preservation was evaluated comparing the levels of myelin in the ventral white matter at 0.4 mm (rostral and caudal) laterally from the lesion epicenter in healthy, saline and cells treated animals. The choice of the ventral white matter was based on the knowledge that the reticular spinal pathway descends mostly in the ipsilateral dorso- and ventrolateral funiculi and is directly involved in the regulation of the movement of the mouse foot [17]. We previously reported that the quantification of the spared ventral myelin evaluated in a semi-thin section gave comparable results when fluoromyelin was used (FluoroMyelin Green, Molecular Probes, Life Technologies). The confocal microscope images for the laminectomies animals and saline and cells-treated mice were obtained using the same intensity, pinhole, wavelength and thickness of the acquisition. As reference we used sections close to the ones analyzed and not treated with fluoromyelin. Briefly, the procedure of the staining was carried out by incubating the cryosections with fluoromyelin diluted 1:300 in PBS for 20 minutes; slides were then washed three times for 10 min each with PBS and mounted with FluorSave (Merck, Darmstadt, Germany), and qualitatively and quantitatively analyzed by confocal microscopy (Leica TSC2; Leica Microsystems, Heidelberg, Germany).

Estimate of the macrophages number at the site of lesion

ED1- positive cells were counted in transversal sections made at the lesion epicenter (1 mm extension) and 0.4 mm rostral. As negative reference for the confocal analysis we used a consecutive section that was stained by omitting primary antibody anti rat macrophages/ monocytes and replacing it with equivalent concentrations of unrelated IgG of the same subclass. The zero level was adjusted on this reference and used for all the further analysis (we used a new zero reference for each new staining). The ED-1 positive cells present in a group of three consecutive sections (10 μm thick) were averaged, and we repeated this count each 100 μm. The total number of ED1- positive cells was obtained integrating the average within volume analyzed, i.e. the 1mm around the epicenter of the lesion [12].

RNA Isolation and Real-Time PCR Analysis

Mice (n=6 48h post injury group, n=6 1 week post injury group) were anesthetized [4,7,18] and killed by decapitation. Laminectomy was performed at the T5–T12. The spinal cord region corresponding at the lesion site was removed (we took 4 mm of tissue rostral and 4 mm of tissue caudal to the lesion epicenter). The tissue was put in 1 ml of Trizol® Reagent (Life Technologies), shock frozen, and kept at –80°C until performing the RNA isolation. Total RNA was isolated by using Trizol® Reagent (Life Technologies) in accordance with the manufacturer’s instructions. The genomic DNA was removed by DNase I treatments (2 U/μg of RNA) (Ambion, Austin, Texas, USA). The synthesis of single-strand cDNA was carried out on 1 μg of RNA, using U M-MLV Reverse Transcriptase III (Life Technologies) following the manufacturer’s instructions.

Real-time (RT)–PCR was performed in an MJ Opticon 2 (Biorad, SEgrate, Milan, Italy) using Brilliant SYBR Green qPCR Master Mix (Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions. The housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used for normalization of cytokine expression. The relative expression of cytokine genes, with GAPDH as reference gene, was determined using the 2-ΔΔCt method. We used this method because both target and reference genes were amplified with similar efficiencies near 100%. The primer design was performed using the DNASTAR Lasergene program. Primers used were the following:

GAPDH (F: cgacttcaacagcaactcccactcttcc; R: tgggtggtccagggtttcttactcctt), BDNF (F: cattaccttcctgcatctgttgg; R: cgtggacgtttacttctttcatgg), IL-6 (F: gacaaccacggccttccctac; R: cgttgttcatacaatcagaattgcc), NGF (F: tgggcccaataaaggttttgcc; R: tgggcttcagggacagagtctcc), TNFα (F: tctatggcccagaccctcacac; R: cagccactccagctgctcctc), MIP2 (F: acgcccccaggaccccactg;

R: ggacagcagcccaggctcctcc), LIF (F: aacgtggaaaagctatgtgcg; R: gcgaccatccgatacagctc).

Statistical analysis

Data were expressed as the mean ± SD. Multiple group comparison were made by ANOVA with a post hoc Tukey test. The analyses were performed using Prism 3.0 software (GraphPad Software, Inc.). Statistical significance was accepted for a P < 0.05.


PM-NPCs improve recovery of hind limb function

The 70 Kdyne traumatic impact to the mouse cord caused a transient loss of ability in hind limb function, that was followed by a progressive gradual recovery reaching the maximum extent within 2-3 weeks (3.0 ± 0.220 points of the BMS scale; n = 12) (Table 1). The recovery was far better and reached the extent of 5.0 ± 0.35 at day 30 (n = 12, corresponding to frequent or consistent plantar stepping without coordination, or frequent or consistent plantar stepping with some coordination) when injured mice were treated with adult PM-NPCs (see Materials and Methods). The behavioral improvement was particularly evident during the first 3 weeks after SCI, the recovery was then steadily improving. Monitoring was done up to 90 days (n = 6). The injection of adult neural stem cells (NSCs) determined an early locomotor recovery of hind limb function at day 7 that improved also during the following week. However the locomotor tests failed to show further improvements in the following weeks (Table 1). Differently, the application of killed PM-NPCs used as controls, failed to promote recovery of hind limb function and the rate of recovery was comparable to that of the saline group (Table 1). No signs of allodynia-like forelimb hypersensibility [16] were recorded at any time in any experimental group throughout the observational period.