Ethical issue and study design
In the current study, 54 adult male Wister rats (weight: 270-300g) were purchased from the animal laboratory and maintained according to the guide line of ethics committee of Tabriz University of Medical Sciences (registered number 95/5-10/7). All animals were housed in a standard condition under a 12h light/dark schedule with enough food and water. The rats were randomly divided in to 9 groups (six rats per group), these nine groups included:Control, SCI, (SCI + DMEM, IP), (SCI + CM, IP), (SCI + MSCs, Focally), (SCI + Astrocyte), (SCI + Astrocyte+ DMEM, IP), (SCI + Astrocyte+ CM, IP) and SCI + Astrocyte+ MSCs). In all groups, laminectomy was performed at the T9–T10 vertebral level in the dorsal surface of the spinal cord using the Infinite Horizons Impactor with an impact force of 150 (moderate SCI) kdyn (1 dyn=1g⋅cm/s 2=10-5kg⋅m/s 2=10-5 N) by impactor device.
hAF-MSCs isolation, cultivation and characterization
Briefly, isolation of hAF-MSCs was done undergoing amniocentesis for the routine karyotype screening of about 5 ml of amniotic fluid samples from eight mothers in Al-Zahra hospital (Tabriz, Iran). Prior to the amniocentesis, patients written informed consent for donating amniotic fluid samples voluntarily for this research. Amniotic fluid extraction carried out under supervision the gynecologist with using a 22G Needle. After sending the samples to the desired laboratory, samples were centrifuged at 450 g for 10 minutes, next the settled pellet was washed twice by PBS (Gibco; Thermo Fisher Scientific, Darmstadt, Germany). Then, the cells transferred in 6 well plates with AmnioMAX II Complete Medium (Gibco, cat# 11269) for 1-2 weeks in condition 37°C and 5% CO2. In the cultivation period, the medium was changed twice a week, and in the 90% confluence, the cells trypsinized with trypsin-EDTA [0.25%] (Gibco) and centrifuged, and finally, cells pellet re-seeded in DMEM-low glucose media with 15% FBS, 1% penicillin/streptomycin and10 ng/mL bFGF in the optimized condition. The phenotype profile of the hAF-MSCs was examined by flow cytometry analysis. For this purpose, the cells at passage 3-5 were trypsinzed and two times washed with PBS and centrifuge at 1500 RPM for 3 min, then, the cell pellets were stained with antibodies including CD 105 (Catalog No. 1p-298-To25 Exbio), CD 73 (Catalog No. 561260 BD Biosciences) antibodies as mesenchymal stem cell markers and CD 45 (Catalog No. 1F-222-T025 Exbio), CD 14 (Catalog No. 12-0149-42 eBioscience) as hematopoietic stem cell markers) with dilution 1/30 in the PBS for 20 min on ice.
Preparation of CM
The MSCs at the 3rd-5rd passage and 90% confluent, were trypsinized and washed with PBS three times, and re‐fed with DMEM-low glucose culture medium in serum‐free condition for 48 h. Then, CM was harvested and centrifuged at 450g for 10 min up to eliminate free-floating cells. Finally, CM was sterilized through 0.22 µm filters and concentrated by freeze-drying processes and was stored at -80 ℃ until use.
CM was collected from MSCs culture and was sterilized by 0.22 mm filters, then CM was concentrated 2, 4, 8, 16 and 32 folds by freeze dryer device. Sox2 (Sex determining region Y-box 2) secreted by HAF-MSCs into MSCs-CM, was measured using western blot analysis.
Human astrocyte culture
Human astrocytes (line 1321N1) (Ghasemi-Kasman et al. 2015) were purchased from Pasteur Institute of Iran and cultured in DMEM low glucose medium with 10% FBS and 1% penicillin/streptomycin. This medium was changed twice in a week.
Spinal Cord Surgery
The rats were anesthetized by inhalation of 5% isoflurane and oxygen (1 L/min) in a closed space. After deep anesthesia, an adequate level of anesthesia was determined by checking withdrawal to painful stimuli applied to the hind limb. The rats were shifted to the surgical location and via the mask received an isoflurane vapor inhalation (3-5%) and oxygen (0.8-1 L/min) to the end of surgical procedure. Briefly, animals under anesthesia conditions, their back was shaved and in the midline, the skin ՚s incision was performed around 2cm, and in order to laminectomy, Paravertebral muscles were cut up on the T9-T11 spinal processes, and with the dental drill, a hole of 1.5 mm diameter was made in the vertebral arch of T10 as far as dura mater could be seen. Then, using the Horizons Impactor, animals received a force of 150-kilodyne (moderate SCI) on the targeted spinal cord segments, subsequently, the muscles and skin were closed. Also for bilateral injury, transverse process of the rats throughout the surgery and injury fixed by a clamp. To prevent infection following SCI, ciprofloxacin (350 ml units/days) was injected via IP for one week. After SCI, the bladder sac was discharged manually twice a day for one week.
Infusion of MSCs- CM
In order to explore the effect of the CM on the rate expression of the endogenous neuroblasts and astrocytes, 500 µl of CM, following SCI was infused through intraperitoneal (IP) per day for 7 days.
Transplantation of hAF-MSCs
The next, to examine the effect of the hAF-MSCs on endogenous expression of neuroblasts and astrocytes, the MSCs were detached and harvested using 0.25% Trypsin-EDTA. Prior to the transplantation, the number of cells was estimated by counting in a neobar lam and washed by PBS three times. Following SCI, 5×105 cells per 5µl PBS were transplanted to the proximal, central, and distal parts of the lesion site using a capillary glass needle through a Hamilton syringe. For immunosuppression, the rats received cyclosporine (1 mg/100 g body weight) two days before cell transplantation until the end of the experiment(Springer et al. 2018).
Injection of the exogenous human astrocytes
In the next series of experiments, we decided to investigate the effect of the MSCs and their CM on astrocyte reprogramming and converting the human astrocytes to neuroblasts. To this end, the human astrocytes (Cell line 1321N1) were injected focally into the lesion site of the spinal cord concomitantly with transplantation of MSCs and infusion of CM.
Tissue preparation and immunofluorescence staining
For immunofluorescence examinations, animals were sacrificed two weeks after SCI by ketamine (100mg/kg) and xylazine (5mg/kg) overdose. The rats were transcardially perfused with normal saline (NaCl 9%) and 4% paraformaldehyde, respectively. The animal’s spinal cord was carefully removed and post-fixed overnight with 4% paraformaldehyde solution. Then, samples of the spinal cord were processed and embedded in paraffin and 5-µm thick histological sections prepared by microtome and mounted on poly-lysine-coated slides. After overnight incubation at 4 °C temperature, the sections were deparaffinized and rehydrated in decreasing ethanol and washed in tap water, and finally stored at 4 ◦C until use. Following the washing in PBS (0.1M, pH 7.4, and 0.9% NaCl), antigen retrieval was done by incubating the sections in preheated 10mM sodium citrate buffer for 15 min at 100 °C and blocking endogenous peroxidase step was performed using incubation the sections in 0.6% H2O2 in PBS for 30 min. Then the Sections were exposed to the primary antibodies, Anti- Doublecortin antibody (ab18723), Anti-GFAP antibody [2A5] (ab 4648), Human Anti- Doublecortin antibody (A83146) and Human GFAP antibody MAB2594, for overnight at 4 °C. After three times PBS wash, the sections were incubated with secondary antibodies including Goat Anti-Mouse IgG H&L (Phycoerythrin) (ab 97024), Goat Anti-Rabbit IgG H&L (FITC) (ab 6717), Donkey Anti-Goat IgG H&L (FITC) (ab6881) and Goat Anti-Mouse IgG H&L (Texas Red ®) (ab6787) at room temperature for 1 hour. Also, nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (ab 104139). Images were taken and observed by an Olympus fluorescence microscope and data was analyzed using the Image J program software plugin.
Locomotor performance on 1, 7 and 21 days after SCI was assessed with the use of the 21 point (a score from 0 (complete paralysis) to 21 (normal gait)) BBB (Basso, Beattie and Bresnahan) score by two examiners blinded.
The results were presented as mean±SEM. One-way analysis of variance (ANOVA) and post hoc Tukey tests were performed to detect the statistically significant difference between groups. P<0.05 was considered as statistically significant. All the statistics presented in the article were analyzed and drawn using Graph Pad Prism (version 6.01; Graph Pad Software, CA, USA).