Isolation and culture of aMSCs, bMSCs and cMSCs
For isolation of MSCs, we collected abdominal adipose tissue, femur, tibia, and cranial bone from adult female Sprague–Dawley (SD) rats (body weight, 250-300g). The adipose tissues collected were digested with Collagenase-A type AFA (560 U/mg; Worthington Biochemical Corporation, Lakewood, NJ, USA) and filtered. The extracts were centrifuged and then seeded into tissue culture dishes (Sumitomo Bakelite Co., Tokyo, Japan) containing culture medium, consisting of low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Corporation, St Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL, both from Sigma-Aldrich). The adhesion of aMSCs was detected the next day after seeding. In the present study, bMSCs and cMSCs were established as described previously [24]. Briefly, bone marrow was collected from the femur and tibia, filtered, and the resulting cells were seeded into culture dishes. Once the cells had adhered to the bottom of the culture dishes at about 5-7 days, non-adherent cells were eliminated by changing the culture medium. The adherent cells were used as bMSCs. After removing muscle, periosteum, and dura matter, cranial bones were seeded into culture dishes. The adhesion of cMSCs was detected at 5-7days after seeding. Cells were maintained at 37°C under an atmosphere of 5% CO2/95% air and the culture medium was changed every 3 days after the detection of cell adhesion. The aMSC, bMSC, and cMSC collected were passaged to more than 80% confluence.
Flow cytometry analysis
aMSCs, bMSCs, and cMSCs at passage 3 were collected with TrypLE™ Select Cell Dissociation Solution (Thermo Fisher Scientific), centrifuged at 1500 × g for 5 min, and then resuspended in phosphate-buffered saline (PBS) (n = 3). Aliquots containing 1 × 105 cells were incubated with fluorochrome-conjugated fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibodies (Abs) against rat CD29 (562154; BD Biosciences, San Jose, CA, USA), CD34 (sc-7324; Santa Cruz Biotechnology Inc., Dallas, TX, USA), CD44 (203906; Biolegend, San Diego, CA, USA), CD45 (554877; BD Biosciences), and CD90 (554898; BD Biosciences). PE-conjugated mouse immunoglobulin (Ig)G1 and FITC-conjugated mouse IgG1 (400108 and 400112; both from Biolegend) were used as an isotype control. FITC-conjugated antibody (Ab) against CD44 and PE-conjugated Abs against CD29, CD90, were used as MSC markers. FITC-conjugated Ab against CD45 and PE-conjugated Ab against CD34 were used as hematopoietic markers. Flow cytometry was conducted using the BD FACSVerse™ Flow Cytometer and data acquisition and analyses were performed using BD FACSuite™ software (both, BD Biosciences).
Multi-lineage cell differentiation
aMSCs, bMSCs, and cMSCs at passage 3 or 4 were used for differentiation into osteoblasts, adipocytes, or neurons. To induce osteogenic differentiation, cells were cultured in Mesenchymal Stem Cell Osteogenic Differentiation Medium (PromoCell GmbH, Heidelberg, Germany) for 21 days, which changes to the medium every 3 or 4 days. To confirm calcium deposition, the cells were stained with alizarin red S solution (Sigma-Aldrich). To induce adipogenic differentiation, cells were cultured in Mesenchymal Stem Cell Adipogenic Differentiation Medium (PromoCell) for 14 days, which change to the medium was changed every 3 or 4 days. Afterward, the cells were finally stained with oil red O solution (Wako Pure Chemical Industries, Osaka, Japan) to confirm the formation of lipid droplets. To induce neural differentiation, cells were cultured under modified neural differentiation conditions with the use of neural conditioning medium and neural differentiation medium, as described in previous reports [23, 24]. MSCs were cultured in neural conditioning medium, which consisted of DMEM: Nutrient Mixture F-12 (Invitrogen Corporation, Carlsbad, CA, USA), 1% FBS, basic fibroblast growth factor (100 ng/mL; PeproTech Inc., Rocky Hill NJ, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL). After culturing maintained in neural conditioning medium for 3 days, the medium was changed to the neural differentiation medium composed of neural conditioning medium with forskolin (10 µM; Sigma-Aldrich) for 7 days. The differentiation medium was changed every 3 or 4 days. After the neural differentiation process, the cultured cells were fixed in 4% (w/v) paraformaldehyde and incubated with anti-β-tubulin III mouse monoclonal Ab (T8578; 1:200, Sigma-Aldrich) as the primary Ab overnight at 4°C, followed by Alexa Fluor 488-conjugated anti-mouse IgG Ab (H+L) (A11029; dilution, 1:500, Molecular Probes Europe BV Co., Leiden, Netherlands) as the fluorescent secondary Ab at room temperature. The nuclei were stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (71-03-00; dilution, 1:800, Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). The stained cells were examined under a multifunctional microscope (BZ-9000, KEYENCE Co., Osaka, Japan).
Reverse transcription and real-time polymerase chain reaction (PCR) of MSCs
At confluence, the cultured cells after reached confluency were collected in PBS. Total RNA was extracted using the NucleoSpin® RNA kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany), in accordance with the manufacturer’s protocol. The purified RNA was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using the ReverTra Ace-α-™ reverse transcription kit (Toyobo Co., Ltd., Osaka, Japan). Using cDNA as a template, real-time PCR was performed with the 7900 HT Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions. Real-time PCR amplification and analyses of rat brain-derived neurotrophic factor (Bdnf), glial cell-derived neurotrophic factor (Gdnf), fibroblast growth factor 2 (Fgf2), nerve growth factor (Ngf), and sortilin 1, which is also known as neurotrophin 3 (Sort1/Nt-3), were performed using the FastStart™ Universal Probe Master Mix (Roche, Basel, Switzerland) and TaqMan™ Gene Expression Assays (Applied Biosystems). Actin beta (Actb) was used as the loading control to normalize the relative quantity of the specific messenger ribonucleic acid (mRNA) in each sample. The TaqMan Gene Expression Assays used in this study are listed in Table 1.
Table 1. TaqMan Assay primers used for real-time PCR of mRNA expression of each MSC type.
Gene symbol
|
Gene name
|
Assay ID
|
Actb
|
Actin beta
|
Rn00667869_m1
|
Bdnf
|
Brain-derived neurotrophic factor
|
Rn02531967_s1
|
Fgf2
|
Fibroblast growth factor 2
|
Rn00570809_m1
|
Gdnf
|
Glial cell-derived neurotrophic factor
|
Rn00569510_m1
|
Ngf
|
Nerve growth factor
|
Rn01533872_m1
|
Sort1 (Nt-3)
|
Sortilin 1 (Neurotrophin 3)
|
Rn01521847_m1
|
Surgical procedure
Adult female SD rats (body weight, 250-300 g) were used to construct a SCI model with the weight-dropping method [26, 28]. The rats were anesthetized and a midline linear incision was made over the thoracic (Th) 9-11 spinous processes. After exposing the laminae of Th9-11, laminectomy was carried out at Th10. An impactor rod was set on the surface of the SC at Th10 and a cylindrical brass weight (10 g) was dropped onto the impactor to create a SC contusion was made with a force of 50 g/cm. Following the contusion, the skin was sutured to close the lesion. After the surgical procedure, the rats were administered antibiotics for 5 days postoperatively to prevent infection. The bladders of the SCI rats were compressed manually twice daily until autonomic bladder function had recovered sufficiently. After the SCI, the rats also received passive joint motion exercises daily to prevent contracture of after the hind-limb joints.
Experimental groups and cell transplantation
SCI rats were assigned to one of the following four treatment groups (n = 10 each): only PBS group; aMSCs group; bMSCs group; cMSCs group. Rats in the aMSC, bMSC, and cMSC groups were injected intravenously with 1.0 × 106 MSCs in 300 µL of PBS at 4week post the SCI. To identify the transplanted MSCs in the recipient SCI rats, the MSCs were labeled with PKH26 (Sigma-Aldrich) just before transplantation.
Motor functional assessment of SCI model rats
The inclined plane test and the Basso-Beattie-Bresnahan locomotor rating scale (BBB scale) were used to evaluate hind limb function. The BBB scale is a 22-point scale that systematically follows the recovery of hind-limb function, and ranges from a score of 0, indicative of no observed movement of the hind-limb, to a score of 21, normal ambulation [29]. The inclined plane test assesses the maximum angle at which the rats can hold a position for 5 s on an inclined plane [30]. Motor function was assessed just before the SCI, on day 1 post the SCI, and weekly until sacrifice at week 8 post the SCI.
Detection of transplanted cells in SC
To detect the PKH26-labeled transplanted cells, SCs were collected and cryoprotected. Rats were euthanized by deep anesthesia and transcardial perfusion of 4% paraformaldehyde. The fixed SCs that were removed from the vertebral columns, soaked in 30% sucrose solution, embedded in Tissue-Tek O.C.T compound (Sakura Finetechnical Co., Tokyo, Japan), and frozen in liquid nitrogen. Afterward, the spinal columns were sliced at a thickness of 10 μm with a cryostat and the sections were encapsulated by VECTASHIELD Mounting Medium with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA) to stain the cell nuclei. The encapsulated sections were examined under a multifunctional microscope (KEYENCE Co.).
real-time PCR analysis of SC tissue
Five rats from each group were sacrificed 4 weeks after MSC transplantation. RNA from the SCs was isolated with the ISOGEN RNA extraction reagent (Nippon Gene, Tokyo, Japan) by centrifugation and reverse transcribed into cDNA, as described above. Real-time PCR analyses of rat Bdnf, tropomyosin receptor kinase B (TrkB), growth-associated protein 43 (Gap-43), and synaptophysin (Syn) were performed using TaqMan Gene Expression Assays. Actb was used as the loading control to normalize the relative quantity of the specific mRNA in each sample. The TaqMan Gene Expression Assays used in this study are listed in Table 2.
Table 2. TaqMan Assay primers used for real-time PCR of mRNA expression in SC tissue.
Gene symbol
|
Gene name
|
Assay ID
|
Actb
|
Actin beta
|
Rn00667869_m1
|
Bdnf
|
Brain-derived neurotrophic factor
|
Rn02531967_s1
|
Gap-43
|
Growth associated protein 43
|
Rn01474579_m1
|
Syn
|
Synaptophysin
|
Rn00561986_m1
|
TrkB
|
Tropomyosin receptor kinase B
|
Rn01441749_m1
|
Western blotting analysis
After RNA extraction, total protein was extracted from the remaining lysate and concentrations were measured with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins (20 μg per lane) were loaded into the lanes of a 15% polyacrylamide gel, separated by electrophoresis, and transferred to nitrocellulose membranes (HybondTM-ECL; GE Healthcare, Little Chalfont, UK). Membranes were blocked with a blocking buffer (20 mM Tris–HCl [pH 7.4], 137 mM NaCl, 0.1% Tween-20, 1% bovine serum albumin) for 60 min at room temperature and then incubated overnight at 4°C with primary Abs against BDNF (GTX132621; dilution 1:2000; GeneTex Inc., Irvine, CA, USA), GAP-43 (GTX127937; dilution 1:10000; GeneTex), and SYN (ab32127; dilution 1:5000; Abcam, Cambridge, UK). After washing with Tris-buffered saline with Tween 20, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (PI-1000; dilution 1:2000; Vector Laboratories) as the secondary Ab. The immunoreaction was visualized using either the SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). Images were acquired with the VersaDoc™ imaging system (Bio-Rad). The membranes were then stripped with a solution of 62.5 mM Tris-HCl, 2% sodium dodecyl sulfate, and 100 mM 2-mercaptoethanol (pH 6.8), and labeled with HRP-conjugated anti-Actb Ab (A3854; Sigma-Aldrich). The expression levels of the target proteins were quantified by measuring the band densities using ImageJ software (version 1.53; National Institutes of Health, Bethesda, MD, USA). Actb was used as the loading control for data normalization.
Immunohistochemical analysis
For immunohistochemical analyses, the fixed SCs were embedded in paraffin using standard methods. Paraffin blocks were sliced into 10-μm-thick sections, which were rehydrated using xylene and ethanol solutions. After treatment with antigen retrieval solution at 98°C for 15 min, nonspecific binding was blocked with 1% bovine serum albumin in PBS for 60 min. Afterward, the sections were incubated overnight with rabbit anti-SYN Ab (dilution, 1:500; Abcam) at 4°C, followed by FITC-conjugated goat anti-rabbit IgG (F9887; dilution, 1:100; Sigma-Aldrich) for 60 min at room temperature. Then, the stained sections were incubated overnight with mouse anti-Tuj1 Ab (GTX631836, 1:250, GeneTex) at 4°C, followed by Rhodamine Red-X-AffiniPure Fab Fragment Donkey Anti-Mouse IgG (715-297-003; dilution, 1:100; Jackson ImmunoResearch Europe Ltd., Suffolk, UK) for 60 min at room temperature. The stained sections were encapsulated by VECTASHIELD Mounting Medium with DAPI (Vector Laboratories) to stain the cell nuclei. The encapsulated sections were examined under a multifunctional microscope (KEYENCE Co.).
NG108-15 neural cell culture
NG108-15 neuroblastoma /glioma hybrid cells (ECACC, Porton Down, UK), which were used as a model of motor neurons, were seeded into culture dishes and cultured in high-glucose DMEM (Sigma-Aldrich), supplemented with 10% FBS (Thermo Fisher Scientific), penicillin (100 U/mL), streptomycin (100 µg/mL, both from Sigma-Aldrich), and HAT supplement (Thermo Fisher Scientific) at 37°C under an atmosphere of 5% CO2/95% air.
Co-culture of MSCs and NG108-15 neural cells
aMSCs, bMSCs, and cMSCs at passage 3 were seeded at 20,000 cells per cm2 in the wells of six-well plates (Sumitomo Bakelite). As a negative control (no co-culture), only the MSC growth medium was added to the well. After 24 h, the NG108-15 cells formed a monolayer at a density of 2,000 cells and the co-culture was continued in DMEM/HAM’s F12 (Wako Pure Chemical Industries), supplemented with 1% FBS (Thermo Fisher Scientific), penicillin (100 U/mL), streptomycin (100 µg/mL, both from Sigma-Aldrich) for an additional 48 h. Afterward, the cells were fixed in 4% (w/v) paraformaldehyde. NG108-15 neurite outgrowth was assessed by fluorescent immunocytochemical analysis. After blocking with 1% bovine serum albumin for 60 min at room temperature, the cells were incubated overnight with mouse anti-Tuj1 Ab (dilution, 1:1000; GeneTex) incubated at 4 °C overnight followed by Alexa Fluor 488-conjugated anti-mouse IgG Ab (H+L) (dilution, 1:500; Molecular Probes Europe BV) as the fluorescent secondary Ab for 60 min at room temperature. DAPI (dilution, 1:800; Kirkegaard & Perry Laboratories) was used to stain the cell nuclei. The stained cells were examined under a multifunctional microscope (KEYENCE Co.) and neurite analysis was performed using ImageJ software with the NeuronJ plugin [31]. The following neurite parameters were assessed: (1) the percentage of neurite-like processes; (2) the number of neurites expressed per cell; (3) the average neurite length (μm); and (4) the longest neurite length in each group (μm). At least 100 neuronal cells from each co-culture were examined. The results are reported as the mean of six independent experiments for each co-culture.
Statistical analysis
Statistical analyses were performed using JMP software from SAS (version 14; SAS Institute, Cary, NC, USA). Comparisons among groups were performed by one-way analysis of variance (ANOVA) with the post-hoc Tukey’s honestly significant difference (HSD) test. Motor functional analysis was performed by two-way ANOVA with the post-hoc Tukey’s HSD test. A probability (p-) value of < 0.05 was considered statistically significant.