Plasmid Construction
An EGFP expression vector, pEGFP-N1, was purchased from Clontech BD Bioscience (Palo Alto, CA, USA). BDNF cDNA was obtained using the Marathon cDNA Amplification system (Clontech) and cloned in the TOPO TA Cloning vector (Invitrogen, Carlsbad, CA, USA) and the sequence was verified. Plasmid encoding human BDNF was generated by polymerase chain reaction (PCR) cloning into pEGFP-N1 using KpnI (5" primers) and NotI (3" primers) restriction sites, which was designated pEGFP-BDNF.
hUCB-MSCs and Transfection
Human UCB samples were obtained with consent from the mothers and were separated and maintained in accordance with techniques as previously described [36]. Cells were transfected with pEGFP-N1 or pEGFP-BDNF by liposome-based reagent, Gene Porter2 (Gene Therapy System, San Diego, CA, USA), established electroporators; Nucleofector® (Amaxa, Cologne, Germany), ECM 830 (BTX Harvard Apparatus, Holliston, MA, USA), MicroPulser™ (Bio-Rad, Huston, TX, USA) and a novel electroporator (MicroPorator™, Digital Bio, Seoul, Korea).
For electroporation, cells were trypsinized and resuspended in PBS for electroporation with the ECM 830 or in resuspension buffer for electroporation with the MicroPulser™ and Nucleofector®. Then 2 × 105 cells were used for each electroporation. Briefly, 2 μg DNA was mixed in PBS and electroporated as described previously [23, 24]. For microporation, 2 μg DNA was mixed with 10 μl resuspension buffer. Then, 2 × 105 cells were electroporated with a pre-optimized square pulse condition (1600 V, 20 ms, 1 pulse). Electroporated cells were incubated in 500 μl α-MEM (Invitrogen) supplemented with 10% FBS (Invitrogen) without antibiotics for 24 hr, and then the medium was replaced with a medium containing antibiotics. To assay transient and sustained expression, the hUCB-MSCs were isolated by incubation in 0.25% trypsin and 1 mM EDTA (Invitrogen) and assayed by a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) 24 hr after transfection for EGFP-expressing cells. The results were analyzed by CellQuest software (Becton Dickinson).
Assessment of Cell Viability
hUCB-MSCs were transfected with pEGFP-N1 by various transfection. Medium was removed 24 hr after transfection, and replaced with medium containing antibiotics. Cells were analyzed for viability by CellTiter 96® aqueous non-radioactive cell proliferation assay (Promega, Madison, WI, USA) at different time points after transfection. The assay tests cellular viability and mitochondrial function. Briefly, after transfection, MTS solution was added to each culture plate and plates were incubated at 37°C for 3 hr. The absorbance of the formazan product, which is considered to be directly proportional to the number of living cells in the culture, was measured at 490 nm using an ELISA plate reader.
Animals and Cell Transplantation
Adult male SD rats (6-8 weeks old; Charles River Laboratories, Wilmington, MA, USA) were used in accordance with institutional guidelines under the approved protocols. Cells were transfected with pEGFP-BDNF and maintained further for 48 hr in α-MEM containing 10% FBS. For the intracranial xenografts of hUCB-MSCs, animals were anesthetized with an intraperitoneal injection of ketamine/xylazine and 3 × 105 cells were stereotactically transplanted into the right frontal lobe (2.6 mm lateral and 1.2 mm anterior to bregma, at 5.0 mm depth from the skull base) via a Hamilton syringe (Hamilton Company, Reno, NV, USA) using a microinfusion pump (Harvard Apparatus, Holliston, MA, USA).
ELISA for Expressed BDNF
To investigate the persistence of transgene expression in vitro, hUCB-MSCs were transfected with pEGFP-BDNF by MicroPorator™. Culture supernatants were harvested, fresh medium (α-MEM containing 10% FBS) was added, and secreted BDNF was assessed at various time intervals. To investigate the persistence of BDNF expression in vivo, brain tissues (2 mm segment centered on the injection site) were harvested and lysed in a RIPA buffer at 1, 3, 7, 10, and 14 days after treatment of cells. BDNF protein secreted into the culture supernatants or brain tissues was analyzed by ELISA assay kits (R&D Systems, Minneapolis, MN, USA).
Immunophenotyping of hUCB-MSCs
To analyze the cell surface expression of typical marker proteins in hUCB-MSCs, cells were labeled with the following anti-human antibodies: CD29-PE, CD34-PE, CD44-PE, CD73-PE, CD90-PE, and HLA-DR-PE (BD Biosciences). Ten thousand cells were measured using a FACSCalibur flow cytometer (Becton Dickinson) and the results were analyzed with CellQuest software (Becton Dickinson).
Multidifferentiation of Cultured hUCB-MSCs
Differentiation to adipogenic and osteogenic lineages was induced according to previously described procedures [2]. After 2-3 weeks of culture, differentiated cells were fixed with 3% formaldehyde. Adipocytes were detected by staining the lipid droplets in the cell using 0.3% Oil red-O staining for 10 min. Osteocytes were detected by calcium phosphate deposits after von Kossa staining. In brief, cells were fixed with ethanol and stained with 5% silver nitrate for 1 hr. After rinsing with distilled water, the cells were incubated in 5% sodium thiosulfate for 2 min to allow precipitation of insoluble black silver granules around calcium phosphate.
Immunocytochemistry
After transfection, cells were fixed with 4% paraformaldehyde for 10 min and processed for immunocytochemistry to identify neural marker-positive cells. Nonspecific antibody reactions were blocked with 5% horse serum for 1 hr at room temperature (RT). Next, the fixed cells were incubated overnight at 4°C with primary antibodies directed against BDNF (Chemicon, Temecula, CA, USA). After three washes, cells were incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 1 hr at RT, followed by 1 hr of incubation in avidin-biotinylated peroxidase complex (Vector Elite Kit, Vector Laboratories) at RT. Diaminobenzidine (0.05%) with nickel chloride (0.04%) was used as the chromagen, and reactions were sustained for 1-6 min at RT. The fixed cells were then coverslipped with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL, UK).
Immunohistochemistry
Rat brains were perfused with PBS followed by 4% paraformaldehyde under deep anesthesia at 3 days after transplantation of hUCB-MSCs or transfected hUCB-MSCs. The excised brains were postfixed overnight and then equilibrated in PBS containing 30% sucrose for 2 days. Fixed brains were embedded, snap frozen in liquid nitrogen, and stored at -70°C until use. Tissues were cryosectioned (16 μm) and then stained with primary antibodies for anti-BDNF (Chemicon). After three washes, tissues were incubated with biotinylated secondary antibodies (Vector Laboratories) for 1 hr at RT, followed by 1 hr of incubation in avidin-biotinylated peroxidase complex (Vector Elite Kit, Vector Laboratories) at RT. Vector NovaRED (Vector Laboratories) was used for visualizing the immunoreaction products, and reactions were sustained for 1-6 min at RT. Nuclei were counterstained with Hematoxylin.
In vitro Migration Assay
The migratory ability of hUCB-MSCs was determined using Transwell plates (Corning Costar, Cambridge, MA, USA) that were 6.5 mm in diameter with 8 μm pore filters. 1 × 106 of cells were incubated in 5 ml serum free MEM for U-87MG or DMEM for astrocytes for 48 hr, and the resulting conditioned media were used as chemoattractants. hUCB-MSCs or hUCB-MSCs transfected with pEGFP-N1 (2 × 104) were suspended in 100 μl serum free medium (SFM) containing 0.1% bovine serum albumin (Sigma) and seeded into the upper well, and 600 μl of conditioned medium (CM) was placed in the lower well of the Transwell plate. Following incubation for 5 hr at 37°C, cells that had not migrated from the upper side of the filters were scraped off with a cotton swab, and filters were stained with the three-step stain set (Diff-Quik; Sysmex, Kobe, Japan). The number of cells that had migrated to the lower side of the filter was counted under a light microscope (×200). Experiments were performed in triplicate.
Neural Induction Procedure
Neural induction was performed in accordance with the procedure described by Lim et al. [27]. In brief, cells were plated in α-MEM containing 10% FBS at 4000 cells/cm2. Twenty-four hours before induction, the medium was replaced with a preinduction medium composed of α-MEM, 10% FBS and 10 ng/ml of basic fibroblast growth factor (bFGF, Invitrogen). The cells were induced by replacing the pretreatment medium with neural induction medium (NIM), consisting of 100 μM butylated hydroxyanisole (Sigma), 2% dimethylsulfoxide (Sigma), 25 mM KCl, 5 U/ml heparin (Sigma), 20 ng/ml bFGF, 5 μg/ml insulin (Sigma), 100 μg/ml transferring (Sigma), 20 nM progesterone (Sigma), 100 μM putrescine (Sigma), 30 nM sodium selenite (Sigma), and 0.5 μM all-trans-retinoic acid (Sigma). After induction, the cells were maintained in NIM for up to 7 days.
Western Blot Analysis
Antibodies were obtained from commercial sources: BDNF, Nestin, βIII tubulin, NeuN, and MBP antibodies from Chemicon; GFAP antibody from Dako; p-ERKs, and p-Raf-1 antibodies from New England Biolabs (Ipswich, MA). For the Western blot analysis, the cells were rinsed with PBS and subsequently lysed for 30 min on ice in RIPA-B buffer (0.5% Nonidet P-40, 20 mM Tris, pH 8.0, 50 mM NaCl, 50 mM NaF, 100 μM Na3 VO4, 1 mM DTT, and 50 μg/ml PMSF). The insoluble material was removed by centrifugation at 12,000 rpm for 20 min at 48 C. Next, the supernatant was subjected to SDS-PAGE, and the Western blot analysis was then performed. The blots were blocked in PBS with 5% skim milk and 0.05% Tween 20, incubated with the appropriate antibodies and subsequently incubated with the secondary antibodies conjugated with horseradish peroxidase. Next, the blots were assayed using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway NJ, USA).