The SCI study included about 2–3 months old 15 female, nonpregnant and five male Wistar albino rats with a weight of 200–300 g. In the first step of the study, five rats (male) were sacrificed in order to obtain rPI-SCs. The remaining rats were divided into three groups (five rats per group) : laminectomy+trauma (group 1), laminectomy+trauma+phosphate-buffered saline (PBS) (group 2); laminectomy+trauma+SCs (group 3). Rats were sacrificed 4 weeks after transplantation. The Ethics Committee of Kocaeli University approved the experimental design and all procedures with a IACUC protocol number of KOU/HAYDEK 1/2/2013.

The pancreatic islets were isolated as described previously [26] and cultured in RPMI 1640 (Invitrogen/GIBCO, Grand Island, NY, USA) with glucose 2 g/L supplemented with 10% fetal bovine serum (FBS; Invitrogen/GIBCO), 100 IU/mL penicilin-100 μg/mL streptomycin (Invitrogen/GIBCO) and glutamine (2 mmol/L; Invitrogen/GIBCO) at 37℃ in a humidified air atmosphere containing 5% CO2. Some islets immediately adhered to the surfaces of the flasks. Within several days, a monolayer of cells was observed growing out and away from the islets and after 13 to 15 days of culturing, cells in the monolayer reached to 70% confluency and named as passage zero (P₀) cells. For passaging, the cells were washed with Ca²⁺-Mg²⁺ free phosphate-buffered saline (PBS) (Invitrogen/GIBCO) and detached by incubating with 0.25% trypsin-ethylenediaminetetraacetic acid solution (Invitrogen/GIBCO) for 5–10 minutes at 37℃. After addition of growth medium to inactivate trypsin, the cells were then centrifugated at 200 g for 10 minutes, resuspended in 1 mL complete medium, counted in duplicate using Thoma chamber and then plated in 75 cm² flasks (BD Biosciences, San Diego, CA, USA) at densities of 1×10⁶ cells/flask. The growth medium was replaced every 3 days over a 10–14 day period.

To confirm that rPI-SCs maintain their phenotypic characteristics after growth in culture, undifferentiated SCs were subjected to flow cytometry analysis. The surface markers of rPI-SCs at passages 3 (P₃) were assayed with antibodies against the following rat antigens : CD29 (integrin β1 chain), CD45 (leukocyte common antigen), CD54 (intercellular adhesion molecule-1), CD90 (Thy-1/Thy-1.1), CD106 (vascular cell adhesion protein-1), major histocompatibility complex (MHC) classes I and II and their three isotype controls (IgG2a, κ).

All of the antibodies were supplied by Becton Dickinson (BD Biosciences). Flow cytometry was performed using a FACSCalibur (BD Biosciences). The data were analyzed with Cell Quest software (BD Biosciences).

Green fluorescent protein (GFP) (Clontech, Palo Alto, CA, USA) was transfected by electroporation (Neon Transfection System; Invitrogen, Carlsbad, CA, USA) with respect to the instructions provided by the manufacturer. The transformed cells were cultured in 1 mL minimum essential media-medium with 15% FBS. After 48 hours of incubation, the cells were selected with respect to the resistance against G418 (200 μg/mL).

For skin preparation of T10–T11 spinal cord surgery, lumbar laminectomy of tracer injection, dermal surface of the related regions was cleared by hair razor, and the skin was washed by antibacterial soap followed with betadine and 70% ethanol application [8]. After an overnight fast with unrestricted access to water, all 15 rats were anesthetized with intramuscular ketamine (50 mg/kg) and xylazine (5 mg/kg) prior to surgery. Under dissection stereomicroscope; 3 mm long laminectomy, encompassing the caudal end of T10 vertebra and the rostral end of T11 vertebra, was performed. For SCI groups (total, 15), after the laminectomy, the animals were moved to stabilization platform. The spine was immobilized stabilizing clamps and a severe T10–T11 contusive injury was introduced by dropping the impounder rod (1 g) from a height of 50 mm. The muscle and fascia layers were sutured and the skin was stapled subsequently.

GFP labeled rPI-SCs (3×10⁵ cells/5 μL) were transplanted into the injured spinal cord via Hamilton syringe (Hamilton Company, Reno, NV, USA) connected to a syringe pump (KD Scientific Inc., Holliston, MA, USA) for 5 minutes, respectively. PBS group received 5 μL of PBS at the injured spinal cord with the same technique. The needle was removed 10 minutes after intraspinal transplantation, and muscle & skin layers were closed in layers. The bladders of SCI rats were evacuated twice daily during the entire study.

Functional tests were performed using the BBB locomotor rating scale at pre-surgery, at days 1, 7, 14, 21, and 28 postinjury (p.i.). Two independent, blinded examiners observed each animal for 4 minutes. Hindlimb movements were recorded by video camera and locomotor functions were assessed [5]. The BBB scores were presented as mean±standard error.

At the end of 4 weeks, rats were anesthetized with ketamine (75 mg/kg, i.p.) and xylazine (20 mg/kg, i.p.) and transcardially perfused with saline (150 mL/per animal) and followed with 4% neutral buffered paraformaldehyde in 0.1 mol/L PBS, pH 7.4. One cm spinal cord segment encompassing the injury site was removed, the tissues were post-fixed in 4% paraformaldehyde approximately 24 hours and then tissues were dehydrated through a graded series of ethanol, cleared with xylene and finally embedded in paraffin wax.

To perform cell tracing after injection of the GFP labeled rPI-SCs, an immunof luorescence double staining protocol was performed on sections. Slides were deparaffinized with two changes of xylene for 5 minutes each and rehydrated in a series of graded alcohol solutions. Sections were antigen retrieved using a steamer-citrate buffer antigen retrieval method. Endogenous peroxidases were inhibited by incubation with fresh 3% H₂O₂ in PBS buffer. Nonspecific staining was blocked with the mixture of two different serums at 1.5% in PBS for 30 minutes at room temperature. The sections were incubated in a mixture of two primary antibodies (Table 1) in a pairwise fashion with the mouse monoclonal anti-GFP antibody (sc-9996) for 1 hour at room temperature and appropriate secondary antibodies 30 minutes at room temperature. The mounted cells with mounting medium containing DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, USA), were examined under fluorescence microscope. Immunoflourescence stainings on the P₃ cells were performed as previously described [26]. The evaluation of the immunoflourescence stainings were accomplished by ImageJ program (National Institutes of Health, Bethesda, MD, USA) as described previously [49]. Briefly, corrected total cell fluorescence was determined through the integrated density, area and the mean gray value of the cells.

All experiments were repeated a minimum of three times. All data presented as mean±standard error. All statistical analyses were performed using SPSS version 10.0 (SPSS Inc., Chicago, IL, USA). The data were analyzed using one-way analysis of variance. Differences between groups were regarded as statistically significant when p<0.05.

Examination of cultured islets under an inverted microscope showed that they had regular cellular structures (Fig. 1A). rPI-SCs spread on culture flask from islets and attached to the culture f lasks sparsely, and the majority of cells displayed a fibroblast-like, spindle-shaped morphology during the early days 20 of explant culture incubation. rPI-SCs reached a monolayer conf luence in the primary culture on 12–15 days 1 of being plated in their first passages. Most of the rPI-SCs exhibited large, flattened or fibroblast-like morphology in the later passages (Fig. 1B and C). We confirmed that rPI-SCs maintained their phenotypic characteristics after growth in culture and undifferentiated SCs were subjected to flow cytometry. Flow cytometry analysis revealed that, specific markers for MSCs were expressed by rPI-SCs such as CD29 (99.57%), CD90 (91.79%), CD54 (98.29%), MHC class I (73.77%). On the other hand, rPI-SCs did not express some of hematopoietic stem cell markers including CD45 (0.12%) and MHC class II (0.01%) and also CD106 (6.90%) (Fig. 1D).

Immunoflourescence analysis revealed that, in addition to MSC markers such as vimentin (Fig. 2D), isolated rPI-SCs also expressed neural and glial cell markers including brain derived neurotrophic factor (BDNF) (Fig. 2A), GFAP (Fig. 2B), fibronectin (Fig. 2C), microtubule associated protein-2a,b (MAP2a,b) (Fig. 2E), β3-tubulin (Fig. 2F), and nestin (Fig. 2G). These cells were also expressing interleukin-1 receptor antagonis (IL-1ra) (Fig. 2H) and EP3 (Fig. 2I), inhibitors of pro-inflammatory cytokines (Fig. 2).

Four weeks after rPI-SC transplantation, the immunofluorescence microscopic analysis of transversal sections of rat spinal cords from experimental groups were performed with double staining of GFP together with vimentin, β3-tubulin, 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNPase), S100β, nestin, vascular endothelial growth factor (VEGF) or BDNF. In all sections of laminectomy+trauma groups, they showed negative staining for GFP (Figs. 3-7), as well as laminectomy & trauma & PBS group (data not shown). However, GFP+ cells were observed near the damage site of laminectomy+trauma+ SC group at the end of 4 weeks (Figs. 3-7). GFP+ SCs migrated into cavitated area from the injection sites and the majority of these still survived and expressed some stem cell and neural cell markers such as vimentin (Fig. 3), CNPase (Fig. 4), S-100β (Fig. 5), BDNF (Fig. 6), and also VEGF (Fig. 7A). Immunostaining intensity level of these factors were higher in laminectomy+trauma +SC group compared to laminectomy+trauma group (Fig. 7B) and laminectomy+trauma+PBS group (data not shown).

Immunoflouresence analysis was applied to rat spinal cord sections of experimental groups. It was revealed that IL-1ra, a suppressor of proinflammatory cytokine IL-1 expression was higher in the spinal cords of rPI-SC injected group compared to the spinal cords of the rats that did not receive rPI-SCs. Conversely, IL-6, transforming growth factor (TGF)-β1, macrophage inflammatory protein (MIP)-2 and myeloperoxidase (MPO), indicators of inflammation, were expressed in a higher level in spinal cords of rPI-SC cell injected rats in compared to the ones without rPI-SC injection (Fig. 8).

To confirm the traumatic impact of the standardized severe weight-drop contusion injury to the T10–T11 spinal cord, the hind limb locomotion of the SCI rats were evaluated. At each assessing time point, consistent functional deficits were noted among SCI rats with the BBB locomotion scores showing profound loss initially, which was then gradually improved and approaching a plateau level of spontaneous recovery typical for this type of injury by 4 weeks p.i. (Fig. 9).

According to BBB locomotor activity test, the performances of the laminectomy+trauma+SC group were statistically different from the laminectomy+trauma group (p<0.05). The injured rats showed marked lower activity score than MSC injected group in the BBB locomotor rating score. All experimental groups showed BBB locomotion score increment to some extent but the highest scores were observed in the stem cell injected group (Fig. 9). Hindlimb movements recorded by video camera at each assessing time points can be seen in Supplementary Video 1.