Localized SDF-1alpha gene release mediated by collagen substrate induces CD117+ stem cells homing

Abstract Stromal cell-derived factor-1α (SDF-1α) mediated mobilization and homing of stem cells showed promising potential in stem cell based tissue engineering and regenerative medicine. However local and sustained release of SDF-1α is indispensable for stem cell mediated regenerative process due to its short half-life under inflammatory conditions. In this study, a gene activated collagen substrate (GAC) was formed via assembly of plasmid encoding SDF-1α into a collagen substrate to create a microenvironment favoring stem cell homing. Local release of SDF-1α from the transfected cells on GAC and its effect on CD117+ stem cell homing were investigated. Non-viral poly-ethyleneimine (25kDa PEI)/DNA complexes were mixed with rat tail collagen solution to form the GAC. Optimization of GAC was carried out based on collagen effects on the PEI/DNA complexes, viability and luciferase expression of COS7 cells on GAC. CD117+ stem cells homing in response to SDF-1α local expression from transfected cells on GAC were investigated in a flow chamber in vitro and in a mouse hind limb model in vivo. The gene expression, migration of CD117+ stem cells and the induced inflammation were investigated with immunostaining, reverse transcription polymerase chain reaction (RT-PCR) and H&E staining. The optimized parameters for GAC were DNA dosage 10 μg/cm2, molar ratio of PEI nitrogen in primary amine to DNA phosphate (N/P ratio) 4 and mass ratio of collagen to DNA (C/D ratio) 1.0. It kept cell viability above 75% and transfection efficiency around 5.8 × 105 RLU/mg protein. GAC allowed the sustained gene release up to 60 days. GAC mediated SDF-1α gene release induced migration and homing of CD117+ stem cells in vitro and in vivo significantly, and the inflammation of GAC reduced significantly two weeks after transplantation. GAC is a promising stem cell based therapeutic strategy for regenerative medicine.

CD26/dipeptidyl peptidase IV, which are two abundant proteases under inflammatory conditions [18][19][20]. Thus, precisely controlled and sustained delivery of SDF-1␣ may be crucial for driving stem cells homing to the injured site and enhancing tissue regeneration.
Gene activated matrix allows local and sustained release of plasmid DNA to the desired site. It has been used effectively in a variety of applications, including regeneration of injured bone, blood vessel and heart tissue [21][22][23][24]. It offers an effective platform of controlled and prolonged chemotactic factors release to enhance the beneficial efficacy of homing circulating stem cell population to the injured site for tissue regeneration.
In the present study, we formed a gene-activated collagen substrate (GAC) via assembly of plasmid encoding SDF-1␣ into a biocompatible and biodegradable collagen substrate to create a microenvironment favouring stem cell homing. The SDF-1␣ was sustainably expressed in situ by the adhesive cells on the GAC. The sustained SDF-1␣ release from the transfected cells promoted CD117 ϩ stem cells tethering and homing both in a flow chamber and after implantation into mouse hindlimb.

Materials and methods
All used animals received humane care and animal experiments conformed to the 'guide for the care and use of laboratory animals' published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).
All plasmids were transformed in Escherichia coli DH5␣ strain and amplified in LB medium at 37ЊC for overnight at 200 rpm. The amplified plasmid DNA was purified by plasmid DNA purification kit (Macherey-Nagel, Diiren, Germany). The purified plasmid DNA was dissolved in Tris-EDTA (TE) buffer and condensed in a lyophilizer (BETA 1-16, Christ, Osterode, Germany).
The concentration and purity of plasmids were determined by ultraviolet (UV) absorbance at 260 and 280 nm with a spectrophotometer (Thermo Electron, Waltham, MA, USA). Finally, the purified plasmid DNA was resuspended in TE buffer and stored in aliquots at a concentration of 2 or 4 mg/ml and stored at Ϫ20ЊC prior to use.

Preparation and characterization of GAC
The PEI/DNA complex was prepared according to Li et al. [26] [27]. Briefly, GAC (PEI/pcDNA3.1-LacZ/collagen, N/P ratio ϭ 4, C/D ratio ϭ 1) complexes were added into 24-well cell culture plate at the final DNA dose of 100 g/well. The aqueous solvent was evaporated by airdrying at room temperature under a sterile condition. Then, 600 l 1 ϫ PBS was added into each well and the plate was incubated at 37ЊC. At the specific time points, 300 l supernatant of each sample was removed and replaced with 300 l fresh 1 ϫ PBS was added. The complex concentration in the supernatant was measured by mixing with ethidium bromide. In brief, 100 l released PEI/DNA complexes were mixed with 50 l 1 ϫ PBS and 100 l ethidium bromide (0.02 mg/ml). The mixtures were visualized under the UV illuminator and the intensities of the emitted fluorescence were analyzed with Quantity One 1-D Analysis Software (Bio-Rad). A standard curve was made by a series of PEI/DNA complexes (N/P ratio ϭ 4) with known concentration. The amounts of released PEI/DNA complexes were quantified via the standard curve.

MTT cytotoxicity
The kidney fibroblast (African green monkey) cell line, COS7 (ATCC) was cultured in Dulbecco's minimum essential medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and the antibiotics streptomycin (100 g/ml) and penicillin G (100 IU/ml). The cells were grown at 37ЊC in a humidified atmosphere of air and 5% CO2. GAC (PEI/pREP4-Luc/ collagen) complex solution was coated into a 96-well plate by air-drying at room temperature under sterile condition for 96 hrs. Then, COS7 cells were seeded into the coated plates with the density of 1.2 ϫ 10 4 cells/well. Collagen free group was used as control. The cells were seeded at 1.2 ϫ 10 4 cells/well and the PEI/DNA complex solution was added immediately after seeding to make the cells have the same complexes-treated time as that in GAC group. After 40 hrs of incubation at 37ЊC, 15 l MTT (5 mg/ml in 1ϫPBS) was added into each well. And after another 4 hrs of incubation at 37ЊC, the medium was removed and the purple crystals were dissolved in 100 l dimethylsulfoxide (DMSO). Absorbance was measured at a wavelength of 550 nm and a reference wavelength of 655 nm using a microplate reader (Model 680, Bio-Rad). The results were expressed as the percentage of viability with respect to the control cells, which were cultured without complexes treatment. Cell viability was calculated using the equation below:

In vitro transfection
The in vitro transfection with GAC was optimized using COS7 cell line. GAC (PEI/pREP4-Luc/collagen) complex solution was coated into 48-well plates by air-drying at room temperature under sterile condition for 96 hrs. The cells were seeded at the density of 0.4 ϫ 10 5 cells/well. In addition, collagen substrate free group was also investigated as comparison. COS7 cells were pre-seeded into 48-well plate at the density of 0.2 ϫ 10 5 cells/well. After 24 hrs of culture, the PEI/DNA complexes were added into the cell culture medium directly. After 60 hrs of transfection the culture medium was removed. The cells were washed twice by 1 ϫ PBS and permeabilized by 100 l cell lysis buffer (Promega, Madison, WI, USA). The luciferase activity in cell extracts was measured using a luciferase assay kit (Bright-Glo, Promega) on a 96-well microplate luminometer (MicroLumatPlus LB 96V, EG&G Berthold, Bad Wildbad, Germany) for 5 sec. The relative light units (RLU) were normalized against protein concentration in the cell extracts, which was measured using a protein assay kit with microplate reader (Model 680, Bio-Rad).
Based on the optimized parameters the EGFP expression was investigated for confirmation. GAC (PEI/pEGFP-N3-SDF-1␣/collagen) was prepared with the optimized N/P ratio and C/D ratio. The GAC was coated into cell culture dish. In brief, one drop of solution was dropped onto the predesigned coated area in dish bottom and aspirated with pipette after 5 min. of incubation at room temperature. After another 5 min., the procedure was repeated for additional four times with 'dropping-5 min. of incubationaspirating-5 min. of drying'. After coating COS7 cells were seeded with the density of 2.0 ϫ 10 4

Statistical analysis
Statistical analysis was performed with one-way analyses of variance (ANOVA). Results were expressed as average Ϯ standard deviation. A P-value Ͻ 0.05 was considered to be statistically significant.

Characterization of GAC
Besides plasmid DNA, GAC is composed of PEI and collagen. To investigate whether collagen affects the DNA condensing abilities of PEI, DNA condensing abilities of PEI and collagen were studied by gel retardation assay. Electrophoretic mobility of DNA after being condensed by PEI and collagen are shown in Fig. 1A and B. Both of PEI and collagen can retard DNA motility. And the retardation ability increased with the PEI or collagen amount. At PEI/DNA ratio (N/P ratio) 1 and collagen/DNA mass ratio (C/D ratio) 6, the DNA was retarded completely. Hence we select C/D ratio 1 as the optimal ratio for GAC assembly to reduce the effect of collagen on condensing ability of PEI.
The effect of collagen on the complexes size of PEI/DNA is shown in Fig. 1C. There were no apparent effects of collagen on the size of PEI/DNA complexes under N/P ratios ranging from 2 to 6.
PEI/DNA complex release speed from the GAC, one crucial property of the substrate, was investigated and the result is shown in Fig. 1D. On the first day, about 37% of complexes were released from GAC. After that, the release speed slowed down and sustained release up to 2 months was observed. The cumulative amount of released complexes after 67 days was approximately 41%.

Cytotoxicity and in vitro transfection
The optimization of GAC was carried out by measuring the cytotoxicity and transfeciton efficiency at different N/P ratios ranging from 1 to 12 and DNA dosages ranging from 2.5 to 15.0 g/cm 2 . The collagen free group was used to evaluate the effect of collagen substrate. For GAC group, both of the cell viability and transfection efficiency were statistically analyzed.
MTT analysis results (Fig. 2) showed that the cell viability cultured on the GAC was significantly improved or comparable to the cells from the collagen free group at N/P ratios ranging from 2 to 12 and DNA dosages ranging from 2.5 to 15.0 g/cm 2 ; whereas the transfection efficiency of the GAC (Fig. 3) was comparable to PEI/DNA complexes in the collagen free group at all investigated N/P ratios and DNA dosages ranging from 5.0 to 15.0 g/cm 2 . The optimal parameters of GACs corresponding to the highest cell viability and transfection efficiency were 7.5 gDNA/cm 2 , N/P ϭ 2 and 10.0 gDNA/cm 2 , N/P ϭ 4, respectively. Considering both of the cell viability and transfection efficiency, we selected the N/P ratio of 4 and DNA dosage of 10 g DNA/cm 2 as the optimal parameters for GAC which kept cell viability above 75% and transfection efficiency around 5.8 ϫ 10 5 RLU/mg protein.
Localized gene delivery is an expected property of gene-activated substrate. Localized gene delivery from GAC was evaluated by (PEI/pcDNA3.1-LacZ/collagen)-GAC and (PEI/pEGFP-N3-SDF-1␣/collagen)-GAC, respectively. As shown in Fig. 5, the transfected cells were confined to the GAC-coated area. The expression of reporter genes, LacZ and EGFP, was detected and formed a Fig. 2 Cytotoxicity of PEI/DNA complexes at the absence (collagen free) and presence of collagen (GAC). N/P ratios and DNA dosages (expressed as DNA amount in unit area (g/cm 2 )) were varied. Collagen dosage was constant (10 g/cm 2 ). The result was given as average Ϯ standard deviation (N ϭ 6). One way-ANOVA analysis was carried out to determine the optimal parameters corresponding to the highest cell viability for GAC group (*P Ͻ 0.05, ** P Ͻ 0.01). Cells growing on GAC (7.5 gDNA/cm 2 , N/P ϭ 2) have the highest viability comparing with cells on other GACs (P Ͻ 0.05). Fig. 5B and E.

Fig. 7 In vivo co-expression of EGFP and SDF-1␣ induced by GAC (PEI/pEGFP-N3-SDF-1␣/collagen) was evaluated by triple staining of EGFP, SDF-1␣ and nuclei. The GAC was pre-coated on mixed cellulose ester membrane, and then transplanted into the quadriceps of mice. The cryosections were evaluated after 3 days of transplantation (E, F, G, H) by comparing with a negative control which was transplanted with a naked membrane (A, B, C, D). Fluorescence microscopic pictures of (A, E) EGFP staining, (B, F) SDF-1␣ staining and (C, G) nuclei staining and phase-contrast microscopic pictures (D, H).
Inflammation induced by GAC was studied by H&E staining. Figure 9A and B showed that, comparing with the naked membrane, GAC-coated membrane did not induce obvious extra inflammation. There was inflammation still observed after 1 week of transplantation (Fig. 9C). However, after 2 weeks, the inflammation significantly diminished and remained only at the zone surrounding the membrane (Fig. 9D).

Discussion
In this study, we have fabricated a collagen-based gene active substrate to provide the homing signals that promoted stem cell homing and recruitment. The SDF-1␣/PEI complex was immobilized into the collagen scaffold for localized release of homing signal SDF-1␣ protein. Efficient transfection was achieved within the patterned area based on the optimized collagen amount, N/P ratio and DNA dosage. SDF-1␣ secreted by the transfected cells enhanced stem cell recruitment in flow chamber. In vivo, the SDF-1␣ gene activated matrix could recruit CD117 ϩ stem cells after the hindlimb implantation.
Stem cell tissue engineering aimed at generating new tissue to repair damaged tissues or organs by combining biofunctional materials with stem cells. The new tissue could be generated either by direct transplantation of stem cells or stimulating stem cell homing to the injured site. SDF-1␣ is a pivotal signal that guides mature and immature stem/progenitor directional migration towards the high SDF-1␣ gradient and protect the stem cell from apoptosis [28,29]. Bone marrow, with the highest SDF-1␣ level compared with other tissues, harbours a heterogeneous stem and progenitor cells positive for CXCR4 receptor under physiological condition. During regeneration process after injury, the up-regulation of SDF-1␣ level in damage organ stimulates stem cell mobilization from bone marrow to the injured area [8]. However, it has been well documented that SDF-1␣ is easily proteolytically degraded in very short time by matrix metalloproteinases and other proteinases, which are up-regulated after inflammation and hypoxia insult [18][19][20]. Therefore, it is important to have the active SDF-1␣ sustainedly released with a controlled manner. In this study, we successfully demonstrated that SDF-1␣ plasmid release could be sustained as long as 60 days. Furthermore, the manner of the stem cell homing to the SDF-1␣ active area was found with a refined spatial localization. Interestingly, our study showed that homed CD117 ϩ cells may infuse with COS7 cells in an extremely low frequency. It suggested that endogenous somatic cells could acquire stem cell markers through simple fusion events. This result could provide an alternative interpretation for the evidence that engraftments of marked bone marrow stem cells (BMSCs) apparently displayed remarkable lineage plasticity following engraftment [3,30].
For successful stem cell-based therapy, one major obstacle is the low cell engraftment. Stem cell microenvironment especially extracellular matrix (ECM) is a determined factor for stem cell local activity, such as adhesion, differentiation and proliferation. Many functional molecules of ECM and their interaction with stem cells were crucial to regulate stem cell survival, self-renewal and maintaining of stemness. Tissue engineered matrice for the delivery and support of transplanted cells is of great interest recently [21]. To this end, a wide range of natural and synthetic extracellular matrix with good biocompatibility, have been selected for stem cell tissue engineering purposes. However, synthetic materials could be rigid, elicit a mechanical or frictional irritation, limit cell mobility and vascularization, and have been inferior to extracellular matrix scaffolds that allow regeneration to occur; whereas collagen scaffold is a promising gene delivery system that offers good cytocompatibility and known structural physical and chemical properties. In addition, it offers a wide range of advantages for stem cell homing and migration. First, collagen gels provide a three-dimensional microenvironment in which stem cells can grow naturally. Second, collagen is a natural material, which minimizes the foreign body inflammatory response to the surrounding tissue and favours stem cell engraftment. Third, the degradation rate of collagen can be easily controlled with noncytotoxic crosslinking agents such as carbodiimide [31]. Furthermore, collagen is also chemotactic to mesenchymal stem cells [32], fibroblast [33] and other cell types [34]. In present study, SDF-1␣ plasmid condensed by PEI was readily incorporated into collagen matrix. We found that GAC significantly promote the cell proliferation. In vivo, no evidence of inflammation was found to be associated with GAC implantation, suggesting the GAC implantation would not increase the inflammation-induced matrix metalloproteinases and other proteinases, consequently the local released SDF-1␣ from transfected cells on the GAC could home the stem cells to the GAC for stem cell adhesion and proliferation on the surface of the matrices. All the findings demonstrated that GAC is an outstanding platform for stem cell tissue engineering.
In summary, gene-activated collagen substrate in this study may open up an important prospect to create a microenvironment for local and sustained stem cell recruitment. This gene-activated substrate for stem cell recruitment may be useful for regenerating a variety of tissue types that could also apply by combining with other chemokine genes. This gene delivery system may be therapeutically effective or serve as a model system to study the molecular interplay between cytokines and/or adhesion molecules involved in stem cell recruitment.