Isolation of pMSCs
The pMSCs were isolated as described previously 4. Bone marrow (BM) was aspirated from juvenile pig iliac crest using a syringe containing 10ml PBS with 200µl heparin (Polfa, Warsaw, Poland). The BM was diluted with PBS (Gibco, Gaithersburg, USA)) in a ratio of 1:2. Next, the mixture was layered on a Ficoll-Paque Plus (Sigma Aldrich, LGC Standards, UK) and centrifuged at 1300rpm at room temperature (RT) for 25 min. A ring of mononuclear cells was collected into a new tube with 20ml of PBS and centrifuged at 1500 rpm for 10 min in RT. Next, the pellet of cells was washed twice with PBS. Cells were suspended in the BMMSC medium (Gibco) and plated on 25ml flasks at 37°C in a humified incubator with 5% CO2. Cultured cells were maintained for 15-20 days (2-3 passages), harvested with Accutase (Gibco), cryopreserved in a freezing medium (Sigma Aldrich), and stored in vapor-phase liquid nitrogen.
Isolation of cGRPs and cMSCs
cGRPs were isolated from canine fetuses between E32-37, as described previously4. For cMSC isolation, the lower abdomen adipose tissue was isolated during the sterilization of the bitch, with the agreement of the owner. The adipose tissue was washed with PBS (Gibco) 3 times and treated with Collagenase II (Gibco) for 3 hours at 37°C. Next, the collagenase was neutralized with serum from the donor and centrifuged at 1550 rpm for 10 min in RT. The pellet was then passed through cell strainers, washed with PBS, and centrifuged at 1350 rpm for 10 min twice. Then, the cells were counted and cryopreserved in a freezing medium (Sigma Aldrich,), and stored in vapor-phase liquid nitrogen until used.
Animals
All animal experiments were approved by the University of Warmia and Mazury’s ethics committee. The ARRIVE guidelines was followed for all animal studies. Experiments were performed according to the EU Directive 2010/63/EU as well as Poland Act 2015/01/15 “Act on the protection of animals used for scientific or educational purposes”. Additionally, for transplantation procedures on dogs, written permission of dog owners was obtained.
Experimental Animals (Pigs)
Three juvenile, Large White domestic pigs (40kg, both sexes) were used. At least two weeks before transplantation, animals were acclimated to the new environment and human presence to minimize the stress associated with the experiment. Pigs had access to water and food ad libitum.
Experimental Animals (Dogs)
Eight dogs were recruited for the study based on neurological exam identifying symptoms of DM and genetic test confirming mutations in the SOD-1 gene, for immunosuppression dogs received cyclosporin (Equoral, Teva Pharmaceuticals, Poland), as described previously4.
Cell preparation for the transplantation procedure
All types of cells (cGRP, cMSC, pMSC) were thawed three days before transplantation and plated on cell culture bottles in dedicated media. One day before transplantation, the medium was supplemented with 25 µg/ml SPION particles (Molday, BioPal, Worcester, MA, USA) for labeling and detection in MRI. The next day cells were washed with phosphate buffered saline (PBS), harvested, and centrifuged at 1100 rpm for 5 min. For transplantation, the cells pellet was suspended in PBS (4-5x106/ml).
MRI-guided intra-arterial transplantation of pMSCs
During the transplantation procedure, animals were anesthetized with a combination of sevoflurane (2%) and propofol (3 mg/kg/h). Anesthetized pigs were placed under C-arm, where 5F sheath (TerumoMedical) was introduced percutaneously to the femoral artery. Next, an angiographic catheter (5F, Balton) was navigated under X-ray guidance to the common carotid artery over hydrophilic 0.35 guidewire (Merit Laureate, Merit Medical). Microcatheter (UltraFlow™ HPC Flow Directed Micro Catheter, ev3™) was introduced over a micro guidewire (Hybrid 0,008", Balt) to the ascending pharyngeal artery with the tip placed proximally to the rete mirabile. Continuous flow flushing of heparinized (5000 U/l) saline was maintained to avoid occlusion of the microcatheter. Cells were transplanted under real-time MRI with the acquisition of dynamic T2*-weighted images (GE-EPI TR/TE:1750/30ms). The MRI protocol also included T2 (TR/TE=5851/83ms) and T1 (TR/TE=1111.4/10 ms) scans prior and post cell delivery, SWI (TE/TR= 20/28) and diffusion-weighted with ADC mapping (TE/TR=104/4800) after transplantation to assess if the procedure and the cells caused brain damage.
MRI-guided intra-arterial transplantation of cGRPs and cMSCs
Eight dogs (5-12 years) of various breeds (1 German Shepherd, 3 Hovawart, 3 Bernese mountain, one crossbred dog) were divided into two groups: 1. with GRP transplantation (three animals) and 2. with cMSC transplantation (five animals). During the procedure, animals were anesthetized as described above. The 5-French introducer (ProCardia TerumoMedical) was inserted into the femoral artery and navigated to the carotid artery using fluoroscopy. Next, a 1.2-French microcatheter was advanced into the middle cerebral artery under roadmap guidance as described previously 32. Then, dogs were transferred to a 3T MRI scanner (Magnetom Trio, Siemens) and positioned supine. MRI protocol was the same as that for pig studies with T2*-weighted dynamic imaging of cells biodistribution in real-time and T2, T1 scans prior and post cell delivery. Just after the procedure during recovery from anesthesia, dogs were hydrated with Ringer's fluid and were returned to their owners for recovery. The follow up monthly visits included blood analysis and neurological examination. MRI was done six months post-transplantation. For calculations of a brain covered by cells, Horos software was used.
Tissue samples
Brain tissues of pigs were collected immediately after slaughter, 24hours after pMSC transplantation, fixed in 4% paraformaldehyde, for 48 h at 4ºC, cryo-protected in 30% sucrose until sunk, frozen on dry ice for 5 min and kept at -80ºC for histological analysis. Brains from dogs that succumbed to the disease were harvested immediately after euthanasia (2 and 7 months post-transplantation), and each hemisphere was divided into two parts from which one was protected in liquid nitrogen and stored at -80 ºC for gene expression analysis and the other was protected for histology as described above. The rest of the transplanted dogs are still alive.
Histopathological analysis
For the detection of SPION labeled cells, Prussian Blue staining was performed. Porcine brain tissues were dried in RT for 10 min and washed in distilled water. The next sections were flooded in a mixture of equal parts of aqueous potassium ferrocyanide (5%) and aqueous hydrochloric acid (5%) for 30 min. Then sections were washed in distilled water three times for 5 min and counterstained with an aqueous solution of eosin for 1 min. After rinsing in distilled water, sections were dehydrated, cleared, and mounted in DPX (Sigma Aldrich). Sections were analyzed on a scanner (3D Histech).
Total RNA extraction and reverse transcription
Canine brain tissues form dogs that succumbed to disease were used for total RNA extraction using a commercial kit (A&A Biotechnology, Poland). The quality of RNA and its concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., DE, USA). Subsequently, reverse transcription reactions were done using the Reverse Transcription System Kit (Applied Biosystems, CA, USA). Two types of RT controls were used, one without RNA and another in the absence of the reverse transcriptase.
Real-time polymerase chain reaction (RT-PCR)
The cDNA obtained was used for real-time quantitative PCR analysis using the Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Each sample contained 3 µL (36 ng) cDNA, 1.5 µL RNAse-free water (Promega, USA), 5 µL TaqMan Universal MasterMix II (Life Technologies, USA) and 0.5 µL TaqMan assays (MBP -Cf02641118_m1, GFAP-Cf02655693_g1, IBA-Cf02653363_m1, Olig1-Cf02685151_s1, MCT4-Cf02702665_g1, ChaT-Cf02724445_m1, RbFox3-Cf02658562_m1, GAPDH-Cf04419463_gH, β-actin-Cf04931159_m1, cyclophilin (Cf02665149_m1. Olig2 and MCT2 were made to order; Life Technologies, USA). All PCR runs were performed as described previously.4 Data obtained from the RT-PCR were normalized using the ratio of mRNA examined to the GAPDH mRNA. Quantification of gene expression was performed using the comparative CT method.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc, San Diego, CA). The distribution of normality was evaluated with Kolmogorov-Smirnov test. The student's t-test was used to determine the difference in mRNA expression. The two-way ANOVA followed by Bonferroni's post hoc test was used to determine the size of hemispheres and ventricles and to assess differences between brain areas covered by MSC and GRP. All numerical data are presented as mean with standard deviation (SD) and differences were considered as statistically significant at the 95% confidence level (P < 0.05).