The migration and differentiation of hUC-MSCs^CXCR4/GFP encapsulated in BDNF/chitosan scaffolds for brain tissue engineering

hUC-MSCs were purchased from the stem cell bank at the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in 75 cm² flasks in 10 ml Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1, v/v) containing 10% fetal bovine serum (FBS; Gibco/Life Technologies, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The medium was replaced every 3 d. When cultures reached 80% confluence, cells were resuspended with 0.25% Trypsin Express (Gibco/Life Technologies) and reseeded in 75 cm² flasks at a density of 3  ×  10⁵ cells/flask. Third-passage hUC-MSCs were transfected with pLV-null-enhanced green fluorescent protein (eGFP) and pLV-CXCR4-eGFP lentiviral vectors. Blasticidin (5 μg ml⁻¹) was used to screen stable hUC-MSC^CXCR4/GFP and hUC-MSC^GFP lines. Transfection efficiency was confirmed by fluorescence microscopy [21].

CXCR4 and GFP expressions levels in hUC-MSCs, hUC-MSCs^GFP, and hUC-MSCs^CXCR4/GFP were evaluated by qRT-PCR and western blotting. The following forward and reverse primer sequences were used: CXCR4, 5'-AGT CTG GAC CGC TAC CTG G-3' and 5'-ATG GTG AGC AAG GGC GAG GAG-3' and GFP, 5'-GCA AAG ATG AAG TCG GGA ATA-3' and 5'-TCA AAG ATC TAC CAT GTA CAG CTC GT-3'. Total RNA was isolated using RNAqueous-4PCR Total RNA Isolation kit (AM1914; Invitrogen, Carlsbad, CA, USA) and qRT-PCR was carried out in triplicate using FastStart Universal SYBR Green Master (Roche, Indianapolis, IN, USA). Each 20 μl reaction contained 9 μl SYBR Green I mix, 5 μl cDNA, 2 μl each forward and reverse primers, and 2 μl RNase-free H₂O. The mixture was incubated at 95 °C for 10 min, followed by 42 cycles of 95 °C for 15 s and 60 °C for 60 min. The specificity of the PCR product was confirmed by melting curve analysis (obtained between 60 °C and 95 °C) and 2% agarose gel electrophoresis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and western blotting were performed according to standard protocols. Briefly, cells were lysed in lysis buffer containing 15 mM Tris–HCl (pH 7.5), 120 mM NaCl, 25 mM KCl, 1 mM EDTA, 0.5% Triton X-100 (Sigma, St. Louis, MO, USA), and 100  ×  Halt Protease Inhibitor Single-Use cocktail (Thermo Scientific, Waltham, MA, USA). Whole cells lysates (50 μg per group) were separated using 4%–12% Novex Bis-Tris SDS-acrylamide gels (Invitrogen), then electro-transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were washed three times in phosphate-buffered saline with 0.05% Tween-20 (PBST) followed by overnight incubation at 4 °C with rabbit antibodies against HIF-1a (ab2185) and glyceraldehyde 3-phosphate dehydrogenase (ab181602; all from Abcam, Cambridge, MA, USA). After three washes with PBST, membranes were incubated with secondary antibody conjugated with horseradish peroxidase in 0.5% milk for 2 h at room temperature. Immunoreactivity was detected using the ECL Western Blotting Substrate (ID 32109; Pierce, Rockford, IL, USA). Signal intensity was quantified with a Quantity One analysis system (Bio-Rad).

To confirm the identity of transfected hUC-MSCs^CXCR4/GFP, surface antigen expression was evaluated by flow cytometry using phycoerythrin-conjugated antibodies against cluster of differentiation CD34, CD73, and CD105; fluorescein isothiocyanate-conjugated antibodies against CD45, CD90, and CD14; and an antibody against human leukocyte antigen HLA-DR (all from BD Pharmingen. San Jose, CA, USA). After incubation with the antibodies, cells were centrifuged and washed twice with PBS before they were sorted with a BD FACscan instrument (BD Pharmingen).

The viability of hUC-MSCs^CXCR4/GFP was measured with Cell Counting Kit (CCK)-8 (Dojindo, Kumamoto, Japan). Cells transfected with pLV-CXCR4-eGFP or pLV-null-eGFP—i.e. hUC-MSCs^CXCR4/GFP and hUC-MSCs^GFP, respectively—served as positive and negative controls. Untransfected hUC-MSCs were also used as controls. Cells were seeded in 96-well plates at a density of 1  ×  10⁴ cells in 100 μl DMEM/F12 medium (Gibco/Life Technologies) supplemented with 10% FBS and incubated at 37 °C in 5% CO₂/95% air. After 0, 12, 24, 36, 48 and 72 h, 10 μl CCK-8 solution were added to each well and the cells were incubated for 30 min at 37 °C; absorbance was measured at 450 nm on a Synergy 2 multimode microplate reader (BioTek, Winooski, VT, USA).

Scaffolds were constructed as previously described [4]. Crosslinked scaffold solution was generated by adding 9 mg genipin (Yuanyebio, Shanghai, China) and 2 mg recombinant human BDNF (PeproTech, Rocky Hill, NJ, USA) to 10 ml acetic acid (1%) containing 0.2 g chitosan. The solution was stored in a freeze dryer (Thermo Scientific) at  −56 °C for 24 h after vigorous mixing; the scaffolds were then immersed in 0.1 mol/l NaOH to neutralize the remaining acetic acid. After overnight drying at room temperature, scaffolds were repeatedly rinsed with ultra-pure water and sterilized by ultraviolet radiation for 1 h.

hUC-MSCs^CXCR4/GFP (1  ×  10⁴) were cultured in medium containing 1% penicillin, 1% streptomycin (Solarbio, Beijing, China), and 10% FBS and added to CGB scaffolds in 24-well plates. Samples were incubated at 37 °C in 5% CO₂/95% air. Scaffolds with adherent hUC-MSCs^CXCR4/GFP were examined on day 1 by scanning electron microscopy. In briefly, the CGB scaffolds with or without hUC-MSCs^CXCR4/GFP were fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 30 min, washed in ice-cold PBS, blocked in O.C.T. and processed in a cryostat (Leica CM1950, Germany). Standard 25 mm thick sections were cut, then affixed to poly-lysine coated glass slides, then store in drying case before scanning. The images of scanning electron microscope (SEM) were randomly collected of each group (N  =  3). Cell viability in the CGB and control groups (cells incubated with or without CGB scaffolds, respectively) was assessed with the CCK-8 assay. After adding 10 μl CCK-solution to each well for 1 h at 37 °C, absorbance was measured at 450 nm on a microplate reader. Cell viability was calculated using the equation: viability (%)  =  (absorbance of experimental group—absorbance of control group)/absorbance of control group  ×  100%.

The role of the SDF-1α/CXCR4 signaling axis in hUC-MSC migration was assessed with the transwell assay using 8 μm inserts (BD Pharmingen). A total of 1  ×  10⁴ cells were resuspended in 100 μl serum-free DMEM/F12 medium and loaded into the upper wells while the lower chambers were filled with 500 μl complete medium (DMEM/F12 supplemented with 10% FBS). For migration assays, SDF-1α was added to the lower chamber at a concentration of 50, 75, or 100 ng ml⁻¹; another group was also treated with AMD3100 blocking reagent. After incubation in a humidified chamber of 5% CO₂/95% air at 37 °C for 24 h, cells were fixed with 500 μl methanol for 15 min. For the migration assay, the inner surface of the upper chamber was wiped with cotton swabs to remove non-migrating cells; the chambers were then washed with 500 μl PBS and stained with 500 μl haematoxylin for 1 min at room temperature. After another wash with 500 μl PBS, transwell membranes were removed and placed on slides and stained cells in five random microscopic fields at 40  ×  magnification were counted using Image J software (National Institutes of Health, Bethesda, MD, USA).

hUC-MSCs^CXCR4/GFP at 1.5  ×  10⁴ single-cell suspension in 1 ml media with DMEM/F12 (1:1, v/v) was add to 24-well plate. After 6 h, when the cells attached to the plate, the media was replaced by 1 ml neuronal differentiation medium, which contains DMEM/F12 and added with 50 ng ml⁻¹ bFGF (13256029; Gibco, USA), 250 ng ml⁻¹ SHH (PMC8033; Gibco, USA), 100 ng ml⁻¹ FGF-8 (PHG0184; Gibco, USA), 1  ×  N2 supplement (17502048; Gibco, USA), 50 μg ml⁻¹ Vit-C. After cultured for 9 d, CGB scaffolds were appended, placed in apical chambers of transwell inserts (CGB scaffold group); the group without the scaffold served as a control. After culturing for 3 d, cells in the lower chamber were removed and processed for immunocytochemistry. Cells were fixed with 4% paraformaldehyde and permeabilised with 0.1% Triton X-100 (Sigma) for 10 min. After blocking nonspecific binding in 10% goat serum for 30 min at RT, cells were incubated overnight at 4 °C with a mouse antibody against microtubule-associated protein (MAP)-2 (1:300, ab11267; Abcam) and a rabbit antibody against neuronal nuclei (NeuN) (1:500, ab177487; Abcam). Cells were then labelled with iFluor 633 goat anti-mouse IgG (1:300, 16478; AAT Bioquest, Sunnyvale, CA, USA) and Alexa Fluor 594 goat anti-rabbit IgG (1:300, ab150080; Abcam) and counterstained with 4,6-diamidino-2-phenylindole (DAPI; Lonza, Basel, Switzerland).

Animal experiments were approved by the Nantong University Animal Experimentation Committee and were carried out in accordance with their ethical guidelines, which were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. A TBI model was generated as follows. Male Sprague-Dawley rats (12 weeks old; 380–450 g) were anesthetised with zoletil 50 (55 mg kg⁻¹ body weight). A piece of skull over the left frontal cortex was removed by drilling. A dural incision was made to expose the forebrain, and defects with a diameter and thickness of 2 mm were created with a scalpel in the cortex at 2 mm to the right and left of bregma. After hemostasis, CGB scaffolds containing hUC-MSCs^CXCR4/GFP or hUC-MSCs^GFP were implanted into the injury sites. For control animals, saline was instead injected into the site (N  =  6). After 2 weeks and 6 weeks, brains were harvested after transcardial perfusion with 250 ml saline and 250 ml 4% paraformaldehyde in 0.1 M PB and postfixed with 4% paraformaldehyde (Sigma) in 0.1 M PB (pH 7.4) for 2 h. After dehydration in 20% and 30% sucrose solutions, brains were sectioned and embedded in optimum cutting temperature compound (Surgipath, Richmond, IL, USA) and sectioned on a cryostat (CM1950; Leica, Wetzlar, Germany). The 10 μm thick sections were collected on poly-L-lysine-coated glass slides and stained for hematoxylin-eosin (HE) stain and immunocytochemical analysis. In brief, to identify differentiating neurons derived from hUC-MSC, sections of 6 weeks were incubated overnight at 4 °C with a mouse antibody against microtubule-associated protein (MAP)-2 (1:300, ab11267; Abcam), after blocking nonspecific binding in 10% goat serum for 30 min at RT, then labelled with iFluor 594 goat anti-mouse IgG (1:300, 16468; AAT Bioquest, Sunnyvale, CA, USA). Nuclei were counterstained with DAPI. Sections were mounted with mounting medium and analysed by fluorescence microscopy (Axiostar scope A1, Carl Zeiss, Jena, Germany).

All data are presented as mean  ±  standard deviation and were analysed with SPSS 10.0 software (SPSS Inc., Chicago, IL, USA). Differences between groups were evaluated by one-way analysis of variance (ANOVA) and were considered significant at P  <  0.05 (designated as * in figures).

The efficiency of CXCR4 transfection was evaluated by qRT-PCR and western blotting. Protein localisation was determined by confocal microscopy (figure 1(A)), while flow cytometry was used to quantify transfection efficiency (figure 1(B)). CXCR4 expression was higher in hUC-MSCs^CXCR4/GFP than in control cells (hUC-MSCs^GFP), as determined by qRT-PCR (figure 1(C)) and western blotting (figures 1(D) and (E)). These results indicate that transfected hUC-MSCs^CXCR4/GFP stably express CXCR4.

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Flow cytometry was used to identify immunophenotype of hUC-MSCs^CXCR4/GFP. From the results, we can confirm that hUC-MSCs^CXCR4/GFP expressed the stromal/MSC markers CD73, CD105, and CD90 but not the haematopoietic or endothelial markers CD14, CD34, CD45, CD79a, or HLA-DR (figure 2). These results demonstrate that the immunophenotype of hUC-MSCs^CXCR4/GFP was unaffected by transfection.

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There was no difference in viability between transfected cells and negative controls (P  >  0.05) (figure 3(A)), suggesting that the transfection was only mildly cytotoxic to hUC-MSCs^CXCR4/GFP, which were available in large numbers for the repair of lesion cavities after TBI. The CCK-8 assay also showed no differences in viability between hUC-MSCs^CXCR4/GFP grown in the presence or absence of a CGB scaffold (P  >  0.05) (figure 3(B)). The scaffolds were only mildly cytotoxic to hUC-MSCs^CXCR4/GFP, indicating excellent biocompatibility. Scanning electron micrographs revealed a smooth scaffold surface with adherent and proliferating hUC-MSCs^CXCR4/GFP (figure 4), suggesting that the scaffold can serve as a carrier for hUC-MSC^CXCR4/GFP.

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The migration of hUC-MSCs and hUC-MSCs^CXCR4/GFP in response to 50, 75, and 100 ng ml⁻¹ SDF-1α was examined by the transwell assay. The number of migrating cells was 25  ±  2.1, 56  ±  5.7, 85  ±  9.1, (figures 5(A)–(C)) respectively, for hUC-MSCs cells and 37  ±  4.1, 92  ±  8.6, 128  ±  13.1, respectively, for hUC-MSCs^CXCR4/GFP (figures 5(E)–(G)) in a random microscopic field at 40  ×  magnification (0.2 mm²); the number was higher for cells overexpressing CXCR4 than for control cells at each concentration of SDF-1α. In addition, hUC-MSC^CXCR4/GFP migration at 100 ng ml⁻¹ SDF-1α was abrogated by treatment with AMD3100 (figures 5(D)–(H)), a specific inhibitor of the SDF-1α/CXCR4 axis. The effect of CXCR4 on hUC-MSC migration was concentration-dependent (figure 5(I)). These results indicate that CXCR4 overexpression stimulates hUC-MSC migration in the presence of SDF-1α.

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hUC-MSCs^CXCR4/GFP were cultured in neuronal differentiation medium with or without BDNF for 12 d and examined by immunocytochemistry to detect MAP-2 and NeuN double-positive neurons. The percentage of neurons was 20.5%  ±  2.40% in BDNF-induced cells as compared to 4.3%  ±  1.18% in the control group (P  <  0.05; figure 6). These results indicate that BDNF released from CGB scaffolds enhances the differentiation of hUC-MSC into neurons.

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Rat brains were harvested 14 d after transplantation of CGB scaffolds containing hUC-MSCs^CXCR4/GFP, hUC-MSCs^GFP (figures 7(A)–(C)), or saline. Transplanted hUC-MSCs^CXCR4/GFP and hUC-MSCs^GFP were identified by green fluorescence and all cells were stained with DAPI (figures 7(D) and (F)). In the lesion boundary zone, the number of GFP-positive cells was higher in the hUC-MSC^CXCR4/GFP/CGB scaffold as compared to the hUC-MSC^GFP/CGB scaffold group (figures 7(E) and (G)), indicating that overexpression of CXCR4 promotes the migration of transplanted hUC-MSCs from the scaffold to the lesion. Haematoxylin and eosin staining revealed that the scaffold filled the wound cavity 2 weeks after transplantation (figure 8(A1)), in contrast to the severe tissue loss and distortion of the cerebral cortex tissue observed in the saline control group (figure 8(A2)). The wound size was decreased by implantation of the CGB scaffold and newly formed extracellular matrix was present, indicating that the CGB scaffold containing hUC-MSCs^CXCR4/GFP was compatible with brain tissue. Also, the immunofluorescence was done to indentify nerve regeneration 6 weeks after transplantation of CGB scaffolds containing hUC-MSCs ^CXCR4/GFP and containing hUC-MSCs^CXCR4/GFP. The double stains of GFP and MAP-2 indicating neurons differentiated from hUC-MSCs. In the transplantation area, the number of GFP and MAP-2 positive cells in the hUC-MSC^CXCR4/GFP/CGB scaffold was same with the hUC-MSC^GFP/CGB scaffold group. However, in the lesion boundary zone, the number of GFP and MAP-2 positive cells was higher in the hUC-MSC^CXCR4/GFP/CGB scaffold as compared to the hUC-MSC^GFP/CGB scaffold group (figures 8(E) and (I) white arrow). This result indicated transplantation of hUC-MSC^CXCR4/GFP/CGB scaffold not only benefit to the regeneration of the lesion site but also the lesion boundary zone of TBI.

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